**Phylogenetic, Evolutionary and Structural Analysis of Canine Parvovirus (CPV-2) Antigenic Variants Circulating in Colombia**

## **Sebastián Giraldo-Ramirez, Santiago Rendon-Marin and Julián Ruiz-Saenz \***

Grupo de Investigación en Ciencias Animales—GRICA, Facultad de Medicina Veterinaria y Zootecnia, Universidad Cooperativa de Colombia, Sede Bucaramanga 680005, Colombia; sebastian.giraldor2@udea.edu.co (S.G.-R.); santiago.rendonm@udea.edu.co (S.R.-M.) **\*** Correspondence: julianruizsaenz@gmail.com or julian.ruizs@campusucc.edu.co

Received: 10 April 2020; Accepted: 24 April 2020; Published: 30 April 2020

**Abstract:** Canine parvovirus (CPV-2) is the causative agent of haemorrhagic gastroenteritis in canids. Three antigenic variants—CPV-2a, CPV-2b and CPV-2c—have been described, which are determined by variations at residue 426 of the VP2 capsid protein. In Colombia, the CPV-2a and CPV-2b antigenic variants have previously been reported through partial VP2 sequencing. Mutations at residues Asn428Asp and Ala514Ser of variant CPV-2a were detected, implying the appearance of a possible new CPV-2a variant in Colombia. The purpose of the present study was to characterise the full VP2 capsid protein in samples from Antioquia, Colombia. We conducted a cross-sectional study with 56 stool samples from dogs showing clinical symptoms of parvoviral disease. Following DNA extraction from the samples, VP2 amplification was performed using PCR and positive samples were sequenced. Sequence and phylogenetic analyses were performed by comparison with the VP2 gene sequences of the different CPV-2 worldwide. VP2 was amplified in 51.8% of the analysed samples. Sequencing and sequence alignment showed that 93.1% of the amplified samples belonged to the new CPV-2a antigenic variant previously. Analysing the amino acid sequences revealed that all CPV-2a contain Ala297Asn mutations, which are related to the South America I clade, and the Ala514Ser mutation, which allows characterization as a new CPV-2a sub-variant. The Colombian CPV-2b variant presented Phe267Tyr, Tyr324Ile and Thr440Ala, which are related to the Asia-I clade variants. The CPV-2c was not detected in the samples. In conclusion, two antigenic CPV-2 variants of two geographically distant origins are circulating in Colombia. It is crucial to continue characterising CPV-2 to elucidate the molecular dynamics of the virus and to detect new CPV-2 variants that could be becoming highly prevalent in the region.

**Keywords:** antigenicity; sequencing; virus

## **1. Introduction**

One of the main infectious agents that affect the canine population is the canine parvovirus type 2 (CPV-2), which causes acute haemorrhagic diarrhoea, primarily in puppies [1]. CPV-2 is a single-stranded DNA virus that belongs to the genus *Protoparvovirus*[2], with a genome of approximately 5200 nucleotides that contain two open reading frames (ORFs). The ORF 3 codes for non-structural proteins NS1 and NS2 are important in controlling viral replication and assembly. The ORF 5 codes for structural proteins are termed viral protein 1 (VP1) and 2 (VP2). VP2 is the major component of the viral capsid, which has an icosahedral structure of 60 subunits with a T = 1 symmetry, comprising approximately 4–5 copies of VP1 and 54–55 copies of VP2 [3].

VP2 subunits interact with transferrin receptors (TfRs) present on the outer surface of the cell membrane [4]. Subsequently, the absorption to the host cell is facilitated by clathrin-mediated endocytosis [5]. The VP2 capsid protein plays a crucial role because its mutations determine the antigenic changes that originate in the different antigenic CPV-2 variants [4].

It is believed that CPV-2 is derived from the feline panleukopenia virus (FPLV) [6], where specific mutations at Lys80Arg, Lys93Asn, Val103Ala, Asp323Asn, Asn564Ser and Ala568Gly capsid protein VP2 residues facilitated a change of host, thereby allowing the virus to infect canines and losing the ability to infect felines [7,8]. These mutations originated in the CPV-2 variant, firstly reported in the 1970s, and spread to countries in Europe, America, Asia and Oceania by 1978 [6].

By 1982, CPV-2 was replaced by a variant of the virus that genetically and antigenically differed [9]. This variant was called CPV-2a and differs from CPV-2 in 6 amino acids: Met87Leu, Ile101Thr, Ser297Ala, Ala300Gly, Asp305Tyr and Val555Ile residues [10].

Residue 426 of VP2 is located in the outermost part of the threefold axis, where 3 VP2 subunits converge. It is the site of greatest antigenicity of the virus [11]; therefore, the amino acid variation causing the antigenic changes that led to the origin of the CPV-2b antigenic variants (Asn426Asp) was reported in 1984 [10] and that for CPV-2c (Asp426Glu) was reported in Italy in 2001 [12]. Unlike CPV-2, CPV-2a, CPV-2b and CPV-2c variants infect canines as well as regaining the ability to infect felines and other wild carnivores [8,13]

In 2017, the antigenic CPV-2a and CPV-2b variants were reported via the study of a partial region of VP2 in Colombia [14]. The CPV-2c variant was not detected in Colombia, despite being the most prevalent antigenic variant in South America [15] and despite Colombia sharing borders with countries such as Peru, Ecuador and Brazil, where the variant has previously been reported [16]. Additionally, two mutations were observed in the Colombian CPV-2a variants (Asn428Asp and Ala514Ser) that suggest the emergence of a new CPV-2a variant unique in Colombia [14].

The aim of the present study was to characterise the coding region of the entire VP2 capsid protein of the circulating parvovirus variants in Antioquia (north-western Colombia) for determining the total amino acid variations in VP2 and to obtain information regarding the molecular evolution of the CPV-2 in Colombia.

## **2. Materials and Methods**

## *2.1. Patient Selection and Sampling*

A cross-sectional study was conducted with a convenience sampling of canine patients attending different veterinary clinics of the department of Antioquia and reporting clinical symptoms compatible with canine parvovirus, such as: haemorrhagic diarrhoea, dehydration, vomiting, loss of appetite and weakness. The samples were collected from February 2018 to March 2019. Gender, breed, age, and vaccination status of each patient was registered. No previous confirmation nor positive faecal antigen ELISA were required for any patient. Diagnosis was based on typical clinical signs. Faecal samples of each animal were collected (approximately 5 g) and stored under freezing conditions (−80 ◦C) until further use; prior authorisation was obtained from the owner of the animals that met the inclusion criteria. This study was approved by the Bioethics Committee of the Universidad Cooperativa de Colombia (project INV1473–bioethics Acta 002-2016). An informed consent was obtained from all owners.

## *2.2. DNA Extraction and Quantification*

Viral DNA extraction from the collected stool samples was performed using QIAamp DNA fast stool mini kit (Qiagen®, Hilden, Germany), in accordance with the manufacturer's instructions. DNA obtained was quantified using 1 μL of the product with NanoDrop 2000 (Thermo Fisher Scientific®, Waltham, MA, USA).

## *2.3. VP2 Amplification Using PCR*

To identify CPV-2-positive samples, PCR amplification of VP2 was conducted using the conventional method. For each PCR reaction, 25 <sup>μ</sup>L of DreamTaqTM PCR Master Mix (2×) (Thermo Fisher Scientific®) was used, with 4 μL of the Forward Ext1F primer (5- -ATGAGTGATGGAGCAGTTCA-3- ) and 4 μL of the Ext3R Reverse primer (5- -AGGTGCTAGTTGAGATTTTTCATATAC-3- ) described by [17] and 17 μL of a mixture of DNA and molecular grade water, reaching an amount of 500 ng DNA for each reaction. Molecular grade water was used as a negative control for amplifications. PCR protocol used was as follows: an initial denaturation cycle at 94 ◦C for 5 min, 35 denaturation cycles at 94 ◦C for 30 s, alignment at 50 ◦C for 45 s, extension at 72 ◦C for 1 min and a final extension cycle at 72 ◦C for 5 min.

PCR amplification results were visualised using 1.5% horizontal agarose gel electrophoresis. Gels were stained with the Invitrogen ™ SYBR®Safe DNA Gel Stain (Thermo Fisher Scientific®). In each well, 4.2 μL of each sample obtained after amplification and 0.8 μL of the 6× DNA loading buffer were used, and the GeneRuler™ 100-bp DNA Plus Ladder (Thermo Fisher Scientific®) was used as a molecular weight marker. Gels were developed in the ultraviolet light Gel Doc™ XR+ imaging system (Bio-Rad, Molecular imager®, USA) and were visualised using ImageLab™ software.

#### *2.4. Sequencing and Sequence Analysis*

Samples positive for VP2 amplification after electrophoresis visualisation were purified and sequenced atMacrogen Inc. (Seoul, Korea) using Forward Ext1F and Reverse Ext 3R primers, alongwith a set ofinternal sequencing primers to amplify the entire VP2 [18]—F1: 5- -AGATAGTAATAATACTATGCCATTT-3- , F2: 5- -ACAGGAGAAACACCTGAGAGATTTA-3- , R1: 5- -TGGTTGGTTTCCATGGATA-3- , and R2: 5- -TTTTGAATCCAATCTCCTTCTGGAT-3- . The resulting electropherograms from the sequencing were analysed using Chormas™ v. 2.6 software. Contig generation, resulting from the overlapping of the sequences amplified by the primers, was performed on the SeqMan Pro platform with Lasergene™. After constructing the complete nucleotide sequences for each sample, alignment was performed using the ClustalW method, following which these sequences were compared with the DNA sequences of CPV-2a, CPV-2b and CPV-2c obtained from GenBank. All analyses were performed in the MEGA™ 7.0 software for Windows®.

#### *2.5. Phylogenetic Analysis*

For the phylogenetic analysis, we calculated the best nucleotide substitution model for the dataset generated with the sequences of FPLV, CPV-2, CPV-2a, CPV-2b and CPV-2c obtained from GenBank. Phylogenetic analysis was inferred using distance-based (neighbor-joining) and character based (maximum likelihood—Bayesian) approaches implemented in MEGA™ 7.0 and Mr Bayes™ software for Windows®. The nucleotide replacement model selected was Tamura-3 parameter with Gamma distributed rate and invariant sites (T92+G+I) and then the Markov chain Monte Carlo (MCMC) Bayesian analysis. We ran the MCMC searches for 1,000,000 generations. The TRACER™ v1.7.1 software was used to confirm all the parameters generated in the Bayesian analysis. The effective sample size was up to 200. The FigTree v1.4.3 software was used to display the consensus phylogenetic tree generated after Bayesian analysis.

#### *2.6. Evolutionary Analysis*

The construction of the phylogenetic evolutionary tree required the generation of a dataset containing the sequences obtained from GenBank and the Colombian CPV-2a and CPV-2b samples. The evolutionary rates, time to the most recent common ancestor (tMRCA) and geographic movements of CPV-2 were performed using the BEAST v1.8.4 software package. The phylogenetic evolutionary tree was generated according to the Hasegawa, Kishino and Yano nucleotide substitution model + gamma distribution + invariable sites (HKY + G + I) and a strict molecular clock. The Bayesian stochastic search variable selection was used to determine links between sequences. The length of

the Markov chain Monte Carlo chain was 15 million. All parameters generated in the analysis were confirmed by verifying the effective sample size of >200 using the TRACER v1.7.1 software. With TreeAnnotator v1.8.4 software, 10% of the steps (1.5 million burn-in) were eliminated to obtain the tree with the most credible clades. FigTree v1.4.3 software was used to display the generated tree.

## *2.7. Structural Analysis*

The VP2 tertiary structure construction and surface analysis was performed using the sequences of sample 1 (CPV-2a) and sample 50 (CPV-2b) as references. Homology modeling of the CPV-2a and CPV-2b sequences under study was generated using MODELLER software based on PDB: 1C8D structure. The three-dimensional models of the VP2 tertiary structure were created using the PyMOL™ software. VP2 surface analysis was performed using the RIVEM (Radial Interpretation of Viral Electron density Maps) software that facilitates the generation of a 'road map' of the viral surface and determination of the locations of the amino acids that constitute the analysed structure [19].

## *2.8. Analysis of Selection Pressure*

The relationship of non-synonymous (dN) to synonymous (dS) substitutions was calculated using ML phylogenetic reconstruction and the general reversible nucleotide substitution model available through the web program Datamonkey. To detect non-neutral selection, the Fast, Unconstrained Bayesian AppRoximation (FUBAR) was implemented in the Datamonkey program. The values dN/dS > 1, dN/dS = 1, and dN/dS <1 were used to define positive selection (adaptive molecular evolution), neutral mutations, and negative selection (purification selection), respectively. Also, we implemented FEL (Fixed Effects Likelihood) which uses a ML approach to infer nonsynonymous (dN) and synonymous (dS) substitution rates on a per-site basis for a given coding alignment and corresponding phylogeny and permits the identification of positive selection at specific sites along particular clades.

## *2.9. Statistical Analyses*

A descriptive analysis of the collected data was performed representing the qualitative and quantitative variables in tables and graphs.

## **3. Results**

For this study, a total of 56 faecal matter samples were collected from dogs that presented clinical symptoms compatible with canine parvovirus, from different veterinary centres located in the department of Antioquia, Colombia.

PCR amplification of VP2 showed that a total of 29 samples (51.8%) were CPV-2-positive. Of these samples, a total of 41.4% (*n* = 12) belonged to females and 58.6% (*n* = 17) to males. Approximately 55.1% of the positive samples belonged to mixed-breed animals. According to the data provided at the time of sample collection, when visiting the veterinary centres with symptoms compatible with those of parvovirus infection, 20.7% (*n* = 6) of the animals had undergone a complete vaccination schedule as required by age, whereas 44.8% (*n* = 13) did not comply with the vaccination schedule. The remaining 34.5% (*n* = 10) of the animals presented incomplete vaccination schedules (Table 1).

**Table 1.** Information on Canine Parvovirus (CPV-2)-positive samples included in the present study.



**Table 1.** *Cont*.

According to age distribution, 15 positive samples (51.7%) belonged to animals aged 1–3 months, which was the most prevalent age group for the virus, whereas only 1 sample (3.4%) belonged to an animal aged >1 year (Figure 1).

**Figure 1.** Distribution of CPV-2-positive samples by animal's age (in months).

#### *3.1. Sequence Analysis*

An almost full sequence of 1711 nucleotides of the VP2 gene was achieved. Sequencing analysis showed that the antigenic variants present in the study are CPV-2a and CPV-2b. The CPV-2a variant (Asn426) was present in 27 from the positive samples (93.1%) and was the most prevalent variant in the study, whereas the CPV-2b variant (Asp426) was detected in two samples (6.9%). No evidence was obtained regarding the presence of the CPV-2c variant (Glu426) in any sample (Table 1).

Sequence analysis revealed specific mutations with respect to the reference sequences of CPV-2a, CPV-2b and CPV-2c obtained from GenBank. The Colombian CPV-2a variants showed mutations at

the amino acid residues Ala297Asn, Tyr324Ile and Ala514Ser, with the latter being reported for the first time in Colombia in 2017 [14]. Regarding the CPV-2b variants in the present study, the amino acid variations detected were Phe267Tyr and Thr440Ala. Similar to the CPV-2a variants, the CPV-2b sequenced from the sampling showed the Tyr324Ile mutation; however, a single Ala514Ser mutation was not detected in these samples (Table 2).

**Table 2.** Amino acid variations in the samples identified as CPV-2a and CPV-2b in relation to reference variants (in bold).


## *3.2. Phylogenetic Analysis*

The phylogenetic relationships based on the nucleotide alignment of VP2 Sequences inferred by distance (neighbor joining) and character approaches (maximum likelihood and Bayesian inference) resulted in trees with a similar topology. The phylogenetic tree generated by Bayesian inference included the CPV-2 positive samples representing the antigenic variants detected in the study (CPV-2a and CPV-2b). GenBank accession numbers for Colombian nucleotide sequences are: MT152347 to MT152375. Additionally, these samples exhibited differences in the pairwise distances. Representative samples of FLPV, CPV-2, CPV2a, CPV-2b and CPV-2c from different countries reported in GenBank were used for phylogenetic evolutionary tree construction. The Colombian CPV-2a antigenic variant constituted a monophyletic clade that substantially differs from the European CPV-2a antigenic variants as well as from Uruguayan variants and only shared distribution with an Ecuadorian CPV-2a variant (MG264075). The two samples of the study from the antigenic CPV-2b variant were located within a clade that includes the Uruguayan CPV-2a variants and Asian CPV-2b variants (Figure 2).

**Figure 2.** Phylogenetic Bayesian analysis of CPV-2 sequences. The phylogenetic analysis was performed using the nucleotide sequences of VP2 of the Colombian CPV-2a and CPV 2b variants and other sequences belonging to the three antigenic CPV-2a, CPV-2b and CPV-2c variants from different countries, as well as feline panleukopenia virus (FPLV) and original CPV-2 variants. The sequences are identified with the accession number, country, and date of collection. Tree was constructed with Bayes and every clade was supported by Bayesian posterior probabilities (BPP). The tree was rooted with the sequence of FPLV (EU221281.1). Red lines indicate the sequences belonging to the Colombian CPV-2a variant and the blue line indicates that belonging to the Ecuadorian CPV-2a variant related to Colombian samples. Green lines indicate the Colombian CPV-2b variant.

In 2017, by using partial VP-2 sequences, the presence of "new" CPV-2 variants in Colombia was shown [14]. To confirm this, we performed maximum likelihood phylogenetic analysis using VP-2 partial sequences, including those that showed the change Ala514Ser in CPV-2a and the sequences belonging to CPV-2b (Supplementary Figure S1). The analysis reveals that the CPV-2a variants of both studies have strong similar sequences. The CPV-2a sequences from 2017 and current analysis showed minor nucleotide differences; however, they turn out to be synonymous variations that do not represent amino acid changes, and for this reason they are all in the same clade. Regarding the CPV-2b sequences, it can also be seen that they are very similar and are located in a single well differentiated clade. Clade 2b sequences show variations at the amino acid level. CPV-2b 2019 sequences (M50 and M55) are located on a separate branch. This subdivision is due to the fact that only the CPV-2b 2019 sequences present the Thr440Ala mutation, one of the mutations reported in the present work (Supplementary Figure S1).

## *3.3. Evolutionary Analysis*

For the evolutionary analysis, the same sequences of samples used in the phylogenetic analysis and a dataset of the sequences of CPV-2a, CPV-2b and CPV-2c variants from different countries were used. The estimated tMRCA for the phylogenetic evolutionary tree was generated 40 years ago (1979), based on the most recent analysed sequence (2019), with a 95% highest probability density and a range of 38–44 years. The analysis revealed that the Colombian CPV-2a and CPV-2b variants have their common ancestor in sequences in Italy (Figure 3), from which a branch was identified that originated a clade with South American sequences (Uruguay, Argentina and Brazil) derived ca.1990; the Colombian CPV-2a variant is located in this clade. Another branch deriving from the sequences from Italy originated a clade with sequences from Asia (China and India) and Uruguay around 1994, and the CPV-2b variant is located in this clade.

**Figure 3.** Phylogenetic evolutionary tree of Colombian CPV-2 variants. The tree was generated using CPV-2a, CPV-2b and CPV-2c variants from different countries. The sequences are identified with the accession number, country, and date of collection. The timeline shows the moment of evolutionary divergence of the sequences of each country. The common ancestor for both the Colombian CPV-2a and CPV-2b variants, in red, are sequences of Italian origin, represented in yellow. The Italian variants originated a clade with South American variants in 1990. In 2006, there is a divergence in the Argentine variants that originated the Brazilian variants and the Colombian CPV-2a variant. In 1994, a divergent branch appears that originated the Asian variants. In this clade, there are Chinese and Indian variants. Additionally, Uruguayan sequences related to the Chinese variants are found. In 2012, there is a divergence from the Chinese origin variants that originated the ColombianCPV-2bvariant.

## *3.4. Structural Analysis*

The three-dimensional reconstruction of the VP2 tertiary structure reveals the spatial location of the mutations detected by sequencing for the Colombian CPV-2a and CPV-2b variants. The CPV-2a variant exhibited the Ala297Asn, Tyr324Ile and Ala514Ser mutations, with all mutations being detected in the exposed VP2 regions. The amino acids 297Asn and 324Ile were located in the loop 3, whereas 514Ser was located in a less prominent region. The mutations found in the Colombian CPV-2a sequences are located at the region that divides the barrel and the depression of the 2-fold axis, an important site for the interaction between VP2 and the TfR receptor. The CPV-2b variant exhibited the Phe267Tyr, Tyr324Ile and Thr440Ala mutations. In the three-dimensional VP2 reconstruction, 267Tyr has been found to be located in an unexposed area in the internal structure of the protein. By contrast, 324Ile and 440Ala were detected in more prominent regions of the protein (Supplementary Figure S2).

## *3.5. Sites under Positive Selection*

The positive selection sites were evaluated using the FUBAR and FEL methods. In CPV-2a variant, we found two sites under positive selection: 297 and 324 with a posterior probability of 0.9 and a Bayes factor of 48.8 and 39.4, respectively. In CPV-2b variant we found two positive selection sites 324 and 440 with a posterior probability of 0.9 and a Bayes factor of 39.4 and 53.9, respectively. As previously reported, both variants showed that the 426 amino acid also had a positive selection (Supplementary Table S1). By FEL, we also analysed the positive selection between clades and we found that Ala514Ser in the Colombian new CPV-2a had strong positive selection (*p* = 0.033).

#### **4. Discussion**

Despite the wide distribution of vaccination strategies for CPV-2 and the knowledge obtained from its evolution, CPV-2 remains a viral pathogen that has the greatest of impacts on animal health [20]. In our study, 51.7% of the samples were CPV-2-positive demonstrating that canine parvovirus is the causative agent for most cases of haemorrhagic gastroenteritis in canines [21]. The new CPV-2a variant was detected in 93.1% of the samples, indicating that this new CPV-2a variant previously reported [14] has gradually become the most prevalent virus in Colombia and this site is under positive selection.

Although the youngest animals—aged between 1 and 3 months—represent the population most affected by CPV-2 infection, consistent with the usual presentation of this infection [21], the animal population aged between 7 and 12 months showed a high rate of CPV-2-positive cases. However, animals belonging to the group of mature puppies (aged 7–12 months) showed an incomplete vaccination schedule or absence of any vaccination history (Table 1), rendering them susceptible to CPV-2 because they lack acquired immunity. Only one animal in the sample was aged >12 months; however, this animal had no vaccination history. Despite this unconventional finding, this serves as a starting point to demonstrate the importance and infectious potential of CPV-2 in immunologically mature animals, even in animals that have received a complete vaccination schedule, indicating that the mutational ability of the virus can result in immune response evasion [22].

Amino acid sequence analysis revealed important changes in the Colombian CPV-2b (Table 2). The Phe267Tyr change has been reported since the early 2000s in Asian CPV-2b variants [23] and in the Uruguayan CPV-2a antigenic variant [24]. Since its detection, this mutation has consistently appeared in the sequences reported in different studies and has become predominant in the CPV-2 population since 2014, suggesting that this mutation has a positive selection. Although this amino acid was not detected in an exposed area of antigenic change site, the fixation of this mutation reflects an evolutionary advantage for CPV-2 that has not yet been elucidated [6,15].

The Tyr324Ile mutation observed in our CPV-2b variant (Table 2) could be related to the ability of the virus to infect different hosts. The residue 324 is adjacent to the residue 323 and, in combination with the amino acid residue 93 of VP2, these residues reportedly exert effects on the host change of the CPV-2 because they are involved in the binding of the virus with TfR [4,25,26]. This amino acid is found in the loop 3 [15], a moderately exposed VP2 region, which is a part of the 'shoulder' of the structure that forms the threefold axis, a site of greater antigenic importance [11]. This mutation was first reported in Asia [27,28], and similar to residue 267, there exists a relationship with Uruguayan variants [24], where this mutation has been reported.

Another mutation observed in our samples regarding the reference variants was Thr440Ala (Table 2). This residue is found in the loop 4 in the most prominent region of the viral capsid—the threefold axis [29]. Therefore, mutations in this region could greatly impact antigenic changes that have implications on the host immune response [24]. Considering that this mutation has undergone a strong positive selection, identifying this mutation in different populations is possible as they underwent an independent evolution [30]. This mutation was first reported in 1993, and it has consistently occurred in the viral populations of CPV-2 since 2005 [15].

It is evident that our findings regarding the Colombian CPV-2b variant are related to the Uruguayan CPV-2a variant. In both cases, these mutations are observed in the same amino acid residues (267, 324 and 440), although they differ in 426. According to the phylogenetic analysis, both the Colombian CPV-2b and Uruguayan CPV-2a variants are present in the same clade as the Asian CPV-2b variants (Figure 2). Based on the study by Grecco et al. [16], the Uruguayan CPV-2a variant originated from the Asian variants, and they named this group the Asia I clade. This clade originated in Asia in the late 80s and arrived in South America between 2009 and 2010, when the dissemination of a divergent CPV-2a variant was reported in Uruguay, where the predominant population at that time was CPV-2c [24].

In the phylogenetic evolutionary analysis (Figure 3), the Colombian CPV-2b variant, although appearing in the same clade as the Uruguayan CPV-2a variant, has a direct relationship with the Chinese variants rather than with the Uruguayan ones. This is possibly attributable to the Uruguayan variants being CPV-2a, whereas in Asia, CPV-2b variants have been reported to have the same mutations as the Colombian CPV-2b sequences [31]. This relationship with the Asiatic variants has been well described recently in Italy, where a case of transcontinental spread of CPV-2c variant was identified with mutation Tyr324Ile and Phe267Tyr characteristically from Asiatic variants [32]. Further, in recent years a local spread of Asiatic-like CPV-2c variant in Italy has been reported [33]. Just as in Uruguay, this spread in Italy demonstrates the intercontinental dissemination of Asiatic CPV-2 variants being able to reach Colombia in a similar way. Structural analysis of CPV-2b has shown that the 440Ala mutation is located in a zone of antibody neutralization. Similarly, it is contiguous to the capsid interaction domain with the TfR cell receptor [34].

Regarding the Colombian CPV-2a, an Ala297Asn mutation was shown that has been reported in South American countries, such as Brazil, Uruguay and Argentina [16]. Residue 297 is located at medium exposure zone in the structure of the viral capsid [11] at a site of lower antigenicity and is not located in the most prominent place in the structure [6]. The Colombian CPV-2a-positive and CPV-2b samples contain the Tyr324Ile mutation in the present study, indicating that equivalent changes can occur in different antigenic variants.

The amino acid residue 514 showed the Ala → Ser mutation, which was first reported in Colombia [14] in CPV-2a variants and occurred in 66% of the samples evaluated. However, in our study, 100% of the samples belonging to the new antigenic CPV-2a variant were reported. Surface structure analysis of the virus revealed the spatial position of these mutations. Amino acids 297, 324 and 514 are located in a central region of VP2 (between the depression of the two-fold axis and the canyon). This area is critical for the interaction between the virus and the TfR receptor. The mutations in 297 and 324 are immediately adjacent to the region determined for the coupling of the receptor on the surface of the virus [35]. The mutation in 514 is distant from the virus-receptor interaction zone (Supplementary Figure S2); however, it is located at the antibody neutralization areas [34]. It is possible that these mutations favour the union between the virus with TfR receptor or may represent a change in the structure of VP2 that allows avoidance of neutralization by antibodies (514Ser) which, added to the fact of being under positive selection, can help explain the reason for this mutation becoming predominant in Antioquia by replacing the viruses that lack these mutations. Interestingly, these same mutations have been reported in Ecuador [36] in a single sample belonging to the CPV-2a antigenic variant, which is closely related to the Colombian CPV-2a variant, highlighting the need to continue the genotypic surveillance of CPV-2 variants circulating in the region due to the possible appearance of new genotypic variants with different possible pathogenic or antigenic potentials.

In agreement with these amino acid variations in the CPV-2a variants of the present study and based on phylogenetic analysis, it is possible that both the Colombian and Ecuadorian CPV-2a samples belong to the clade called South America I. This clade has the peculiarity of containing both South American CPV-2a and CPV-2b variants with the Ala297Asn mutation [16]. The phylogenetic tree presented in Figure 2 shows a well differentiated large clade where all our CPV-2a samples are grouped along with the Ecuadorian CPV-2a. These samples share an identical amino acid sequence, although they differ in some nucleotide level changes that are synonymous variations.

Similarly, in our phylogenetic evolutionary tree construction, Brazilian CPV-2b variants, which contains the Ala297Asn mutation and is present within the clade known as South America I, are most closely related to CPV-2a variants. Although the mutation in 297 groups our CPV-2a variants with variants belonging to the South America I clade, the unique mutations in 324 and 514 differentiate our samples from the others belonging to this clade. This suggests that the Colombian CPV-2 sequences configure a Colombian CPV-2a sub-variant within the CPV-2a variants belonging to the South American clade I, as postulated by Duque-García et al. [14].

To understand the evolutionary importance of the mutations found in CPV-2a and CPV-2b variants, an analysis was performed to determine the positive selection sites (Supplementary Table S1). Our results confirmed for CPV-2b, that sites 324 and 440 have a strong positive selection, as previously reported in Asian CPV-2 variants [37,38], which are related to the Colombian CPV-2b variant. In the case of CPV-2a, the positive selection sites were 297 and 324. Both sites are adjacent to the virus-receptor interaction domain [34], which demonstrates the importance of these residues in the evolutionary adaptation of the virus and therefore how these mutations have been established in viral populations. Also, by FEL analysis we confirmed that 514Ser in Colombian CPV-2a have strong positive selection, highlighting the importance of our results and the need for active genomic surveillance programs that help to early detect new CPV-2 variants that could be becoming highly prevalent in the region.

According to the phylogenetic analyses performed in the present study, it can be inferred that there are two antigenic variants (CPV-2a and CPV-2b) with different origins currently circulating in Antioquia, Colombia. However, the Colombian CPV-2a variants contain the Tyr324Ile mutation, which is characteristic of the variants belonging to the Asia I clade [16]. To clarify the relationship and origin of the two variants found in Colombia, the phylogenetic evolutionary analysis was performed. Our analysis revealed that two variants have different origins or present specific differential mutations while sharing a common origin.

The phylogenetic evolutionary analysis revealed that the Colombian CPV-2b variant shows a direct relationship with Asian variants that comprise the Asia I clade. This clade arrives in South America, initially in Uruguay in 2009, followed by Colombia in 2012. According to these results, the CPV-2b variant in Antioquia arrived in Colombia in a manner similar to that in Uruguay, i.e., it arrived directly from Asia and not as a migratory process within the continent from Uruguay to Colombia (Figure 3). On the contrary, the Colombian CPV-2a variant was related to variants of the South American clade I, which originated from the European variants that subsequently underwent evolutionary and migratory processes into the interior of the continent to finally configure a clade with characteristics of variants present only in South America [16]. According to the evolutionary analysis, the CPV-2a variant arrived in Colombia between 2005 and 2006.

As previously reported [16], our evolutionary analysis shows that the most ancestral sequences of CPV-2 are the viral sequences reported in the United States at the end of the 1970s. The subsequent arrive of CPV-2 to Europe can be observed, mainly represented by Italian sequences, which give rise to the clade South America-I, in which the Colombian CPV-2a variant is located (Figure 3). Additionally, the Colombian CPV-2b variant iw related to variants of Asian origin, in a similar way to the Uruguayan

variants. The evolutionary rate in VP-2 was shown to be similar to that reported by others [16]. However, the resulting trees diverge in their topology without modifying the common ancestry of the clades. These differences in topology in comparison to previously published results may be due to the addition of new and updated sequences to the dataset. It is clear that variations in spatiotemporal sampling added different bias to the analysis, as has been previously reported [39].

Both the CPV-2a and the CPV-2b variant found in our study present the 324Ile mutation (Table 2; Supplementary Figure S2). This change has been previously reported in cats infected with CPV-2a [38], evidencing that it is an important amino acid in determining host range. Additionally, it has been recently shown that this amino acid residue is close to the virus-receptor interaction domain and that few structural changes are required in CPV-2 to be able to adapt and interact with TfR receptors from different species [34]. This result supports the fact that this mutation has a strong positive selection and is present in all our samples (CPV-2a and CPV-2b). It is possible that other carnivorous species different to canines could be involved in determining the changes in VP2 supporting the adaptation of the virus to carry out an efficient replicative cycle.

## **5. Conclusions**

The antigenic variants, CPV-2a and CPV-2b, circulating in Antioquia, Colombia, originated in the South America I clade and Asia I clade, respectively. The mutations detected in CPV-2a variant have gradually undergone positive selection that appears to favour the virus–receptor interaction, rendering this Colombian CPV-2a sub-variant the most predominant in the region. The antigenic implications of the 440Ala mutation in CPV-2b and those related to the virus–receptor interaction should be elucidated in future investigations, with special emphasis on the proximity to sites of virus-receptor and virus-antibody interactions; results that will allow us to understand the functional impact of these genomic changes on the biology, immunology and pathogenesis of CPV-2.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1999-4915/12/5/500/s1, Supplementary Figure S1. Partial VP-2 Maximum likelihood phylogenetic analysis of Colombian CPV-2 sequences (Bootstrap 1000—Tamura 3 parameter model). Current sequences denote as Colombia 2019. Supplementary Figure S2. Three-dimensional reconstruction and structural analysis of the VP2 surface of the Colombian CPV-2a and CPV-2b variants. Supplementary Table S1. Sites under positive selection in Colombian CPV-2a and CPV-2b variants.

**Funding:** This study was supported by CONADI—Universidad Cooperativa de Colombia, by a grant to JRS. The funding source had no role in study design, analysis, and interpretation of data, writing of the report and in the decision to submit the article for publication.

**Acknowledgments:** The authors are grateful to the veterinary clinics and hospitals that participated in the sampling. Also, authors want to thank to July Duque-Valencia for her helpful suggestions in phylogenetic analysis.

**Conflicts of Interest:** The authors of this work do not have any personal or financial conflict that may improperly influence the content of this document.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Molecular Investigation of Canine Parvovirus-2 (CPV-2) Outbreak in Nevis Island: Analysis of the Nearly Complete Genomes of CPV-2 Strains from the Caribbean Region**

**Kerry Gainor 1,†, April Bowen 2,†, Pompei Bolfa 1, Andrea Peda 3, Yashpal S. Malik <sup>4</sup> and Souvik Ghosh 1,\***


**Abstract:** To date, there is a dearth of information on canine parvovirus-2 (CPV-2) from the Caribbean region. During August–October 2020, the veterinary clinic on the Caribbean island of Nevis reported 64 household dogs with CPV-2-like clinical signs (hemorrhagic/non-hemorrhagic diarrhea and vomiting), of which 27 animals died. Rectal swabs/fecal samples were obtained from 43 dogs. A total of 39 of the 43 dogs tested positive for CPV-2 antigen and/or DNA, while 4 samples, negative for CPV-2 antigen, were not available for PCR. Among the 21 untested dogs, 15 had CPV-2 positive littermates. Analysis of the complete VP2 sequences of 32 strains identified new CPV-2a (CPV-2a with Ser297Ala in VP2) as the predominant CPV-2 on Nevis Island. Two nonsynonymous mutations, one rare (Asp373Asn) and the other uncommon (Ala262Thr), were observed in a few VP2 sequences. It was intriguing that new CPV-2a was associated with an outbreak of gastroenteritis on Nevis while found at low frequencies in sporadic cases of diarrhea on the neighboring island of St. Kitts. The nearly complete CPV-2 genomes (4 CPV-2 strains from St. Kitts and Nevis (SKN)) were reported for the first time from the Caribbean region. Eleven substitutions were found among the SKN genomes, which included nine synonymous substitutions, five of which have been rarely reported, and the two nonsynonymous substitutions. Phylogenetically, the SKN CPV-2 sequences formed a distinct cluster, with CPV-2b/USA/1998 strains constituting the nearest cluster. Our findings suggested that new CPV-2a is endemic in the region, with the potential to cause severe outbreaks, warranting further studies across the Caribbean Islands. Analysis of the SKN CPV-2 genomes corroborated the hypothesis that recurrent parallel evolution and reversion might play important roles in the evolution of CPV-2.

**Keywords:** canine parvovirus; Caribbean region; new CPV-2a; outbreak; endemic; nearly complete genomes; virus evolution

## **1. Introduction**

Canine parvovirus-2 (CPV-2), members of the genus *Protoparvovirus* within the family *Parvoviridae*, are highly contagious enteric pathogens of household dogs, often causing fatal hemorrhagic gastroenteritis in puppies [1–4]. Morphologically, CPV-2 are small, nonenveloped viruses containing a single-stranded, negative-sense DNA genome (~5200 bp in size) [2,5]. The CPV-2 genome possesses at least two major open reading frames (ORFs), designated as ORF1 and ORF2. The ORF1 encodes two nonstructural (NS1 and NS2)

**Citation:** Gainor, K.; Bowen, A.; Bolfa, P.; Peda, A.; Malik, Y.S.; Ghosh, S. Molecular Investigation of Canine Parvovirus-2 (CPV-2) Outbreak in Nevis Island: Analysis of the Nearly Complete Genomes of CPV-2 Strains from the Caribbean Region. *Viruses* **2021**, *13*, 1083. https://doi.org/ 10.3390/v13061083

Academic Editor: Giorgio Gallinella

Received: 10 May 2021 Accepted: 3 June 2021 Published: 6 June 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

proteins, while ORF2 codes for two structural (VP1 and VP2) proteins, translated through alternative splicing of the same viral mRNAs [2,5].

The CPV-2 nonstructural proteins have been shown to be crucial for viral replication, DNA packaging, cytotoxicity, and pathogenesis [6–8]. On the other hand, the structural proteins form the viral capsid, of which VP2 is the major component [9,10]. The VP2 protein plays important roles in determining host range, tissue tropisms, and virus-host interactions, and is highly antigenic, forming the basis of the currently licensed CPV-2 vaccines [2,3,11,12]. To date, the majority of the studies on CPV-2 are based on the VP2 encoding gene [2,13,14], while limited information is available on the genetic variations in the nonstructural genes [15,16].

Based on amino acid (aa) differences in the VP2 protein, CPV-2 strains have been classified into four major antigenic variants: CPV-2, CPV-2a, CPV-2b, and CPV-2c [2,13,14]. The earliest CPV-2 strains that emerged in dogs during the late 1970s are referred to as the CPV-2 variant [2,16]. By the end of 1980, CPV-2 was rapidly replaced by a new antigenic variant, designated as the CPV-2a variant [17,18]. The other CPV-2 variants, CPV-2b and CPV-2c, were first reported in 1984 and 2000, respectively [19,20].

The antigenic differences between CPV-2a, CPV-2b, and CPV-2c have been attributed to a single aa mismatch (Asn in CPV-2a, Asp in CPV-2b, and Glu in CPV-2c) at residue 426 of the VP2 protein [2]. However, by phylogenetic analysis of complete/nearly full-length CPV-2 sequences, the CPV-2a, CPV-2b, and CPV-2c variants lacked clear monophyletic segregation, and were grouped together into a single 'CPV-2a clade', which was distinct from the cluster of the earliest CPV-2 strains, referred to as the CPV-2 clade [16]. Other nonsynonymous mutations have also been observed in the CPV-2 variants [2,13,14,16,21]. Notable among these aa changes is the presence of Ser297Ala in VP2 of several CPV-2a and CPV-2b strains, sometimes referred to as new CPV-2a and new CPV-2b, respectively [13]. Currently, the CPV-2a, CPV-2b, and CPV-2c variants and their mutants, such as new CPV-2a and new CPV-2b, are circulating worldwide, with different relative frequencies between countries and between sampling periods [2,13,14,16,21–23].

To date, there is only a single report on the detection and molecular characterization of CPV-2 from the Caribbean region [24]. During February 2015–August 2016, new CPV-2a was detected (25/104 dogs tested CPV-2 positive, 20 samples were sequenced) in sporadic cases of diarrhea in household dogs on St. Kitts Island [24]. In the present study, we report a molecular investigation of a new CPV-2a associated severe outbreak of canine gastroenteritis in the Caribbean island of Nevis that resulted in the death of 27 animals.

Since there are no reports on complete CPV-2 genomes from the Caribbean region, the nearly full-length genomes of 3 CPV-2 strains (representing each of the new CPV-2a mutants with a nonsynonymous substitution in the VP2 gene) detected during the outbreak on Nevis and that of a previously reported CPV-2 strain from St. Kitts were analyzed in the present study.

#### **2. Materials and Methods**

## *2.1. Ethics Statement*

The present study was submitted to the Institutional Animal Care and Use Committee (IACUC) of the Ross University School of Veterinary Medicine (RUSVM), St. Kitts Island. Ethical review and approval were waived for this study by the RUSVM IACUC as the research study was based on leftover samples that were collected for diagnostic purposes at the veterinary clinic on Nevis Island (RUSVM IACUC sample/tissue notification letter number TSU 1.23.21).

#### *2.2. Sampling*

Nevis is a small Caribbean island (total area of 93 km2, human population ~12,000) located in the lesser Antilles, and together with the neighboring island of St. Kitts, constitutes the twin Federation of St. Kitts and Nevis (https://www.paho.org/, accessed on 19 April 2021) (Figure 1A). Although there are no official records on the canine population

in Nevis Island, many of the houses keep dogs as pets, with the island mix breed (a cross between a breed native to the Caribbean Islands and another canine breed) representing the majority of the domestic dogs.

**Figure 1.** (**A**) Geographical location of Nevis Island. (**B**) Map of Nevis Island showing the locations of the 64 dogs (highlighted with pins) with canine parvovirus-2-like (CPV-2-like) clinical signs. Red pin, dogs that tested positive for CPV-2 by the SNAP® Parvo Test (IDEXX, Westbrook, ME, USA) and/or PCR; Yellow pin, untested dogs with CPV-2-like clinical signs that had a CPV-2 positive littermate (by the SNAP® Parvo Test and/or PCR); Blue, untested dogs with CPV-2-like clinical signs. The pins were inserted into the map using the Map Maker software (Maps.co, Mountain View, CA, USA). The maps for Figures 1A and 1B were obtained from https://www.cia.gov/library/publications/the-world-factbook (accessed on 1 April 2021) and https://www.google.com/maps (accessed on 30 March 2021), respectively.

> From 1 August 2020 to 17 October 2020, 64 household dogs with CPV-2-like clinical signs were presented at the veterinary clinic on Nevis Island. A total of 44 rectal swabs/fecal samples were obtained from 43 of the dogs. One dog was sampled twice (5 September 2020 and 5 November 2020).

## *2.3. Screening*

The samples were screened for the presence of CPV-2 antigen using the SNAP® Parvo Test (IDEXX, Westbrook, ME, USA) following the manufacturer's instructions.

#### *2.4. Amplification of CPV-2 Genome*

Viral DNA was extracted using the QIAamp Fast DNA Stool Mini Kit (Qiagen Sciences, Germantown, MD, USA) according to the manufacturer's instructions. Primers used for amplification of the CPV-2 genome and/or obtaining CPV-2 genome sequences were designed in the present study (Supplementary Material S1). The complete VP2 ORF of CPV-2 was amplified by two overlapping PCRs, while three additional overlapping PCRs were employed to amplify the nearly full-length CPV-2 genomes (spanning all the coding regions, corresponding to nucleotide (nt) 272-nt 4585 of reference CPV-2 strain CPV-N (GenBank accession number M19296)) (Supplementary Material S1). PCRs were performed using the Platinum™ Taq DNA Polymerase (Invitrogen™, Thermo Fisher Scientific Corporation, Waltham, MA, USA) following the instructions provided by the manufacturer. Sterile water was used as a negative control during the PCR reactions.

## *2.5. Nucleotide Sequencing*

The PCR amplicons were purified using the Wizard® SV Gel and PCR Clean-Up kit (Promega, Madison, WI, USA) according to the manufacturer's instructions and sequenced in both directions using forward and reverse primers (Supplementary Material S1). Nucleotide sequences were obtained using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA) on an ABI 3730XL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).

## *2.6. Sequence Analysis*

Putative ORFs encoding the CPV-2 NS1 and VP2 proteins were identified using the ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/, accessed on 5 April 2021), while those coding for NS2 and VP1 proteins were determined by alignment of the obtained CPV-2 nt sequences with published CPV-2 coding sequences. The standard BLASTN and BLASTP program (Basic Local Alignment Search Tool, www.ncbi.nlm.nih.gov/blast, accessed on 2 April 2021) was used to perform a homology search for related cognate nt and deduced aa sequences, respectively. Multiple alignments of nt and deduced aa sequences were carried out using the CLUSTALW program (version ddbj, http://clustalw.ddbj.nig.ac. jp/, accessed on 2 April 2021) with default parameters. The nearly complete CPV-2 genome sequences were examined for recombination events using the RDP4 program with default parameters, as described previously [16,25]. Briefly, a CPV-2 sequence was identified as a recombinant if it was supported by two or more detection methods (3Seq, BOOTSCAN, CHIMERA, GENECONV, MAXCHI, RDP, and SISCAN) with a highest acceptable *p*-value of *p* < 0.01 with Bonferroni's correction.

A data set (excluding recombinant sequences) of 210 nearly complete CPV-2 sequences from domestic and wild canids and 6 FPV sequences were created for phylogenetic analysis, based on those reported in previous studies [16,21]. Phylogenetic analysis was performed by the maximum likelihood (ML) method using the MEGA7 software [26], with gamma-distributed rate variation among sites and 1000 bootstrap replicates. Phylogenetic trees were constructed using both the Hasegawa-Kishino-Yano (HKY) and general time-reversible (GTR) substitution models.

## *2.7. GenBank Accession Numbers*

The GenBank accession numbers for the CPV-2 genome sequences determined in this study are MW595661-MW595693 (complete VP2 ORF sequences) and MW616469- MW616472 (nearly full-length CPV-2 genome sequences).

## **3. Results and Discussion**

## *3.1. Molecular Investigation of CPV-2 Outbreak in Nevis Island*

From 1 August 2020 to 17 October 2020, the veterinary clinic on Nevis Island reported 64 household dogs with anorexia, gastroenteritis (with or without blood in feces), lethargy, and vomiting (CPV-2-like clinical signs). A total of 27 (42.2%) of the dogs died. Based on case histories, the outbreak of gastroenteritis appeared to have started at Charlestown, the capital of Nevis, and eventually spread to other parts of the island, with most cases mapped to the more densely populated western coastal region of the island (Figure 1B).

A total of 43 dogs were tested for CPV-2 antigen using the SNAP® Parvo Test (IDEXX, Westbrook, ME, USA), of which 32 samples were available for PCR. A total of 39 (90.6%) of the 43 dogs tested CPV-2 positive by SNAP® Parvo Test (24/43 dogs, 55.8%) and/or PCR (32/32 dogs, 100%) (Table 1), while 4 samples that were negative for CPV-2 antigen could not be tested by PCR. Fifteen of the PCR positive dogs tested negative by the SNAP® Parvo Test (Table 1), which might be attributed to the lower sensitivities of the CPV-2 antigen tests compared to PCR/qPCR assays, even in dogs with CPV-2-like clinical signs, as reported in previous studies [2,27]. We did not observe any differences in clinical severity between dogs that tested negative and positive to the SNAP® Parvo Test. Although samples could not be obtained from 21 dogs with CPV-2-like clinical signs, 15 of the animals had littermates that were positive for CPV-2 antigen and/or DNA. Most of the sick dogs were <6 months of age (89%, 57/64 dogs with CPV-2-like clinical signs) and were either not vaccinated or received incomplete immunization (did not receive all doses of the vaccine) against CPV-2 (95.3%, 61/64 dogs with CPV-2-like clinical signs), corroborating previous observations on the increased risk of CPV-2 infection in unvaccinated puppies [2,4]. Since not all the dogs were tested for CPV-2, and none of the obtained samples were screened for other enteric pathogens, we could not establish whether CPV-2 was the sole etiological agent in the outbreak of gastroenteritis on Nevis Island.

In order to determine the CPV-2 variant/s circulating during the outbreak in Nevis, complete VP2 ORF sequences were obtained from the 32 PCR positive samples. Based on the presence of 297Ala and 426Asn in the putative VP2 proteins [13], all the CPV-2 strains from Nevis were classified as new CPV-2a (Table 2; Supplementary Material S2). In a previous study (2015–2016), new CPV-2a was only identified in sporadic cases of diarrhea on the neighboring island of St. Kitts [24]. These observations suggested that new CPV-2a is endemic and might be predominant in this part of the world. In South America, new CPV-2a, or other mutants of CPV-2a (VP2 Ser297Asn, Phe267Tyr, Tyr324Ile, and Thr440Ala) have been found to coexist with CPV-2b and/or CPV-2c variants at various frequencies, emerging as the major strain in a few studies [13,21,28–33]. In studies from North (Canada and USA) and Central (Mexico) Americas, CPV-2b, or CPV-2c was most prevalent, while a single report from Alaska detected both new CPV-2a and new CPV-2b in an outbreak of canine gastroenteritis [13,34–37]. Taken together, the molecular epidemiology of CPV-2 in the Caribbean region appears to differ from those reported in nearby North, Central, and South American countries, although further studies on the other islands are required to validate this observation.







**Table 2.** *Cont.*

<sup>1</sup> Strain CN27 and strain CN40 were detected in the same dog on 5 September 2020 and 5 November 2020, respectively.

The new CPV-2a strains from Nevis shared absolute deduced VP2 aa identities between themselves and those of new CPV-2a strains reported previously from St. Kitts [24], except for Ala262Thr in 4 CPV-2 strains and Asp373Asn in 3 other CPV-2 strains (Table 2; Supplementary Material S2). Although the significance of the aa at residue 262 of VP2 is not yet known, VP2 Ala262Thr has been described as a novel mutation [38], reported in new CPV-2a and new CPV-2b strains from Western Australia [38], two new CPV-2a strains from India (GenBank accession numbers DQ182624 and KU866399), and a single new CPV-2a strain from China (MH177301). The other nonsynonymous mutation, VP2 Asp373Asn, has been rarely reported in CPV-2 sequences, found in two new CPV-2a strains (one each from Australia (MN259033) and Thailand (GQ379047)), a single CPV-2 strain from a cat in Taiwan (KY010491), and two feline panleukopenia virus strains (FPV) (MH559110 and MK570710). The aa residue at 373 of VP2 is located within the VP2 flexible loop (a surface loop between VP2 residues 359 and 375), which is a pH-sensitive structure that governs binding to divalent ions in FPV and CPV-2 [39,40]. Structural studies on FPV at pH 7.5 have shown that the ion density, adjacent to the flexible loop, is coordinated by Asp 373

and Asp 375, and carbonyl oxygen atoms of Arg 361 and Gly 362 [39]. However, the implication/s of VP2 Asp373Asn remains to be determined. In addition to the two aa mismatches, five synonymous mutations were observed among the VP2 sequences of the new CPV-2a strains from Nevis (Supplementary Material S3).

Following natural CPV-2 infection or immunization with the commercially available modified live virus (MLV) vaccines, dogs have been shown to shed viral DNA for as long as 50 days post-infection [41]. In the present study, one of the dogs was sampled twice (5 September 2020 and 5 November 2020), during hemorrhagic gastroenteritis and after two months, when it was apparently healthy and presented at the clinic for vaccination (Table 1). Both the samples (CN27 and CN40) tested positive for CPV-2 by PCR. Surprisingly, the new CPV-2a strain with VP2 Ala262Thr was identified in the first sample, while the new CPV-2a with VP2 262Ala, detected in the majority of the samples from Nevis and during 2015-2016 in St. Kitts, was detected in the second sample (Table 2). Vaccinated dogs with protective antibody titers have been shown to shed low amounts of CPV-2 field strains in the feces [41,42]. Although the viral DNA was not quantified by qPCR, we observed weak amplification following PCR of the second sample, indicating a low viral load. Therefore, it might be possible that the dog developed immunity following initial infection with the new CPV-2a VP2 Ala262Thr, and eventually was asymptomatically infected with the new CPV-2a VP2 262Ala strain. Alternatively, reversion of VP2 Ala262Thr to the more prevalent new CPV-2a VP2 262Ala might be possible, as reverse mutations have been described in the CPV-2 genome in previous studies [13,16,38,43].

Most of the animals in the present study were not vaccinated or received incomplete vaccination. On the other hand, 3 CPV-2 positive dogs that were completely immunized (vaccine NOBIVAC® CANINE 1-DAPPv, Merck Animal Health, Elkhorn, NE, USA) against the virus suffered from severe clinical disease (Table 1). The vaccine NOBIVAC® CANINE 1-DAPPv contains a CPV-2b variant, while the dogs were infected with new CPV-2a. Although the efficacy of the commercially available CPV-2 MLV vaccines against the different CPV-2 antigenic variants has been debated, they have been shown to be effective in significantly reducing the clinical severity of CPV-2 disease caused by the non-vaccine field variants [3]. Since the dogs were aged ≥ 6 months, it is unlikely that maternal antibodies interfered with the efficacy of the vaccine. Other factors, such as vaccine-related errors (issues with vaccine storage, transport and/or administration) and/or host-related factors (impaired immune status, non-responders, and/or malnutrition), might have contributed to vaccine failure [3].

In a previous study on St. Kitts, new CPV-2a was sporadically detected in 20 diarrheic dogs (25/104 dogs were CPV-2 positive; 5 samples could not be sequenced) during a period of 1 year and 7 months [24]. During and around the duration of the outbreak in Nevis, the veterinary clinic on St. Kitts reported only five sporadic cases of CPV-2 infection (three and two dogs tested CPV-2 positive by the SNAP® Parvo Test and PCR, respectively), of which two dogs died (Supplementary Material S4). Analysis of the complete CPV-2 VP2 sequences from the two dead dogs (strain CK81, GenBank accession number MW616470, and strain CK84, MW616472) revealed 100% deduced aa identities with those of the new CPV-2a reported in our study (Supplementary Material S2). Since the canine breeds, environmental conditions, vaccination trends, husbandry practices, and veterinary care are similar between the two islands, we found it intriguing that new CPV-2a was associated with an outbreak of gastroenteritis on Nevis, while found at low frequencies in sporadic cases of diarrhea on the neighboring island of St. Kitts.

There is limited movement of animals between St. Kitts and Nevis, as the twin islands are connected by ferry service. It might be possible that a new CPV-2a was recently introduced into Nevis from St. Kitts and that the canine population on Nevis Island was naive to infection with the virus, resulting in an outbreak situation. However, sporadic cases of CPV-2 have been previously reported in Nevis (based on old case records at the veterinary clinic in Nevis), although none of the samples were molecularly characterized to identify the CPV-2 variant. Therefore, we could not determine whether a new CPV- 2a was circulating in Nevis before the outbreak. Furthermore, based on the available information, we could not ascertain if the two nonsynonymous mutations (Ala262Thr and Asp373Asn) in the VP2 genes of a few new CPV-2a strains appeared during the outbreak or were already circulating at low frequencies in the island canine population. Nevis has a sizable population of stray dogs, which could have facilitated the spread of the virus across the island.

## *3.2. Analysis of the Nearly Complete Genomes of CPV-2 Strains from St. Kitts and Nevis Islands*

Considering the lack of information on CPV-2 genomes from the Caribbean region, we decided to determine the nearly full-length CPV-2 genome sequences (4269 nt, possessing the entire NS and VP coding regions) of 2 strains representing the new CPV-2a circulating in St. Kitts and Nevis (strain CN10 from the outbreak in Nevis, and strain RVC50 from our previous study in St. Kitts [24]), and one of each of the new CPV-2a with a nonsynonymous mutation in the VP2 gene (strain CN20 with VP2 Ala262Thr and strain CN14 with VP2 Asp373Asn).

The nearly complete genomes of CPV-2 strains from St. Kitts and Nevis (henceforth, collectively referred to as SKN strains) shared nt sequence identities of 99.77–99.93% between themselves (Supplementary Material S5). Absolute deduced NS1 and NS2 aa identities were observed between the SKN strains, while the putative VP1/VP2 proteins of strains CN14 and CN20 differed in an aa residue with those of the other SKN strains (Tables 2 and 3; Supplementary Material S6). By BLASTN analysis, the SKN strains shared ≥ 99% nt sequence identities with several CPV-2a, CPV-2b, and CPV-2c variants, corroborating previous observations that the complete/nearly complete genomes of all CPV-2 variants are ~99% identical in nt sequence [16]. Although the SKN strains were assigned to new CPV-2a, they shared higher nt sequence identities with the nearly complete genomes of CPV-2c (99.46–99.63% with GenBank accession number KX434458) and CPV-2b (99.39–99.58% with EU659121) strains than those of CPV-2a variants (identities of ≤99.32–99.48% with EU659118).

A total of 11 substitutions (5 and 6 within the NS and VP coding region, respectively) were found among the nearly complete SKN CPV-2 genome sequences that included the two nonsynonymous substitutions in the VP coding region (Table 3, Supplementary Materials S5 and S6). The evolution of CPV-2 has been characterized by only a limited number of substitutions that became fixed or widespread during the last 40 years since the emergence of the virus [16]. A previous study identified 38 mutations (15 synonymous and 23 nonsynonymous mutations) that differed in an nt from the earliest CPV-2 strains (the CPV-2 antigenic variant) and were present in >10% of other CPV-2 strains (CPV-2a, CPV-2b, and CPV-2c variants) [16]. In the present study, 35 substitutions (24 synonymous and 11 nonsynonymous substitutions) were observed between the SKN CPV-2 sequences and that of strain CPV12 (representing the earliest CPV-2 strains from the late 1970s, GenBank accession number MN451655), of which 5 synonymous and 1 nonsynonymous substitution have been rarely reported in other CPV-2 strains (<10 CPV-2 sequences) (Table 3, Supplementary Materials S5 and S6). All the four SKN strains retained the five nonsynonymous substitutions in the VP coding region that was characteristic of the global sweep from CPV-2 to CPV-2a during the late 1980s (Table 3, Supplementary Material S5) [16,18].

**Table 3.** Nucleotide (nt) mismatches (highlighted with yellow) between the nearly complete genome sequences (4269 nt, spanning all the coding regions) of canine parvovirus-2 (CPV-2) strain CPV12 (representing the earliest CPV-2 strains from the late 1970s) and CPV-2 strains detected on St. Kitts (strain RVC50) and Nevis (strains CN10, CN14, and CN20) islands. Nonsynonymous mutations that differed in an nt from the CPV12 sequence are shown in red. Alignment of the nearly full-length nt and complete deduced amino acid sequences of the CPV-2 strains from St. Kitts and Nevis with those of strain CPV12 are shown in Supplementary Materials S5 and S6, respectively.


<sup>1</sup> Nucleotide positions are those of the nearly complete genome sequence of strain CPV12 (GenBank accession number MN451655); <sup>2</sup> Present in <10 published CPV-2 sequences, as revealed by BLASTN analysis (https://blast.ncbi.nlm.nih.gov/, accessed on 9 April 2021) and multiple alignments of the dataset of 210 nearly complete CPV-2 sequences; <sup>3</sup> Nucleotide substitutions (and corresponding amino acid changes) that emerged during the global sweep from CPV-2 to CPV-2a [16].

> Since recombinant sequences might influence the outcomes of phylogenetic analysis [16,21], the nearly complete SKN CPV-2 genome sequences were screened for recombination events, as described previously [16,25]. However, no recombination breakpoints were detected in any of the SKN CPV-2 sequences. To rule out biases in clustering patterns, phylogenetic trees were created using both the HKY and GTR substitution models, as the

HKY model was determined as the best-fit model using the 'find best model' function of MEGA7, while the GTR model was employed in a recent, important study on the evolution of CPV-2 [16]. Similar clustering patterns were observed with both the models (Figure 2; Supplementary Materials S7 and S8). Findings from previous studies, such as (i) phylogenetic classification of CPV-2 strains into two major clades: CPV-2 (comprising the earliest CPV-2 strains from the late 1970s) and CPV-2a (consisting of CPV-2a, CPV-2b, and CPV-2c variants) [16], (ii) a lack of phylogenetic resolution within the 'CPV-2a clade', characterized by low bootstrap values for most clusters [15,16], (iii) a lack of monophyletic segregation based on CPV-2 variants (CPV-2a, CPV-2b, and CPV-2c) [15,16], and (iv) presence of Asian and Western (CPV-2 sequences from the Americas and Europe) clades [21,44] were retained in the phylogenetic analysis (Figure 2; Supplementary Materials S7 and S8). Phylogenetically, the new CPV-2a SKN strains were placed with the Western CPV-2 strains and retained the signature aa residues that have been previously described as evolutionarily significant and characteristic to the Western clades (Figure 2; Table 4; Supplementary Materials S7 and S8) [21,44]. Within the Western clade, the SKN strains formed a distinct cluster. The nearest cluster to the SKN strains was that of CPV-2b strains detected in the USA during 1998, followed by the major Western CPV-2c cluster (consisting of primarily South American and Italian strains, designated as Western clade-III in a previous study [44]). However, the clade consisting of the SKN strains and the CPV-2b/USA/1998 strains was supported by a low bootstrap value (bootstrap value of 45 and 47 by the GTR and HKY models, respectively). The clustering patterns were corroborated by nt sequence identities and the geographical proximity of St. Kitts and Nevis to the USA and the South American countries. Except for VP2 Ala262Thr (strain CN20) and VP2 Asp373Asn (strain CN14), only a single nonsynonymous mutation (VP2 residue 426) that constitutes the basis of differentiation of CPV-2 into the CPV-2a, CPV-2b, and CPV-2c antigenic variants was observed between the new CPV-2a SKN strains and CPV-2 strains belonging to the CPV-2b/USA/1998 cluster, or the major Western CPV-2c cluster.


**Table 4.** Evolutionarily relevant amino acid residues in canine parvovirus-2 (CPV-2) protein sequences, as described in previous studies [21,44]. The two nonsynonymous substitutions (VP2 Ala262Thr and VP2 Asp373Asn) observed among the

<sup>1</sup> As described in previous studies [21,44]. Western: CPV-2 strains from Europe and the Americas; <sup>2</sup> Reported in new CPV-2a and new CPV-2b strains from Western Australia [38], 2 new CPV-2a strains from India (GenBank accession numbers DQ182624 and KU866399), and a single new CPV-2a strain from China (MH177301); <sup>3</sup> Found in 2 new CPV-2a strains (one each from Australia (MN259033) and Thailand (GQ379047)), a single CPV-2 strain from a cat in Taiwan (KY010491), and 2 feline panleukopenia virus strains (MH559110 and MK570710).

Kitts/2016 Ile Tyr Glu Glu Leu Val Ala Phe Tyr Asp Asn

**Figure 2.** Phylogenetic analysis of the nearly complete genomes of canine parvovirus-2 (CPV-2) strains from St. Kitts and Nevis with those of other CPV-2 strains. The tree was created using the Hasegawa-Kishino-Yano model with gamma-distributed rate variation among sites and 1000 bootstrap replicates. The name of the strain/CPV-2 antigenic variant/place/year of detection is shown for the CPV-2 strains from St. Kitts and Nevis, while the GenBank accession number/CPV-2 antigenic variant/place/year of detection have been mentioned for the other CPV-2 strains. The two major phylogenetic clades (CPV-2 and CPV-2a) are demarcated with a brown and a light gray bar, respectively. The CPV-2 clade consists of the earliest CPV-2 strains from the late 1970s (the CPV-2 antigenic variants), while the 'CPV-2a clade' is composed of the CPV-2a, CPV-2b, and CPV-2c antigenic variants [16]. Feline panleukopenia virus (FPV) strains were included in the analysis. Sky blue, purple, yellow, pink, orange, green, and red circles indicate that the CPV-2 strain was detected in Asia, Africa, Australia, Europe, North America, South America, and St. Kitts and Nevis, respectively. Scale bar, 0.01 substitutions per nucleotide. Bootstrap values of <70 are not shown. Some of the clusters have been compressed to accommodate the entire tree. The complete phylogenetic tree is shown in Supplementary Material S7. Phylogenetic analysis using the General Time Reversible model is shown in Supplementary Material S8.

Corroborating previous observations [15,16,21,44], phylogenetically, the clustering of the nearly complete SKN CPV-2 genomes with those of other CPV-2 strains did not correspond to the clustering patterns based on VP2 coding sequences (Figure 2; Supplementary Materials S7–S9). The complete VP2 nt sequences of the SKN strains clustered near that of CPV-2a strain CPV-435/USA/2003 (GenBank accession number AY742953), although the clade was supported by a low bootstrap value of 45 (Supplementary Material S9). Since the complete/nearly complete genome sequence of CPV-435/USA/2003 was not available in the GenBank database, we could not include the strain in the analysis of nearly complete CPV-2 genomes.

Taken together, these findings supported previous observations that recurrent parallel evolution and reversion might play important roles in the evolution of CPV-2 and that the recently circulating CPV-2 strains are minor variants of a common 'pan genome' template that emerged after the global sweep from CPV-2 to CPV-2a [15,16].

It is likely that CPV-2 was imported into St. Kitts and Nevis Islands from another country. Since more humans (and pet dogs) travel to St. Kitts from the USA than from any other country, it might be possible the virus was introduced into the twin islands from the USA. On the other hand, St. Kitts and Nevis have a large population of the small Indian mongoose (*Urva auropunctata*) that thrive in wild, rural, and urban habitats [45]. A previous study had reported high rates of detection of parvovirus DNA (58%, n = 99) and antibodies (90%, n = 20) in the Egyptian mongoose (*Herpestes ichneumon*) [46]. Considering the role of wild canids as a potential source of CPV-2 to pet dogs [2,13,17,46,47], it would be interesting to investigate whether the new CPV-2a and/or new CPV-2a mutants (VP2 Ala262Thr and VP2 Asp373Asn) circulating in dogs on St. Kitts and Nevis were derived from the local mongoose population.

### *3.3. Conclusions*

Although the currently licensed CPV-2 vaccines have been extensively used in veterinary practice and shown to confer protection against the different CPV-2 antigenic variants, CPV-2 continues to remain one of the leading causes of mortality and morbidity in domestic dogs [2–4]. To date, there is a dearth of data on CPV-2 from the Caribbean region. Our findings suggested that new CPV-2a might be endemic in this part of the world, with the potential to cause severe outbreaks facilitated by low vaccination rates among the canine population in the region. Recently, the neighboring islands of St. Eustatius and St. Lucia experienced large outbreaks of CPV-2 [48,49]. However, no information was available on the nature of CPV-2 variant/s circulating during the outbreaks. These observations underscore the importance of continuous molecular epidemiological studies on CPV-2 in all the Caribbean Islands alongside creating public awareness on the disease and vaccination.

The nearly complete CPV-2 genomes were reported for the first time from the Caribbean region, providing important insights into the overall genetic makeup of the CPV-2 strains circulating in St. Kitts and Nevis, especially the identification of six substitutions (five synonymous and one nonsynonymous substitution) that have been rarely reported in other CPV-2 sequences. Overall, analysis of the SKN CPV-2 genomes corroborated the hypothesis that recurrent parallel evolution and reversion might play important roles in the evolution of CPV [15,16]. Once again, it was revealed that the analysis of CPV-2 VP2 aa sequences do not reflect the true evolutionary patterns of CPV-2 strains.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/v13061083/s1, Supplementary Material S1: Primers used in the present study, Supplementary Material S2: Multiple alignments of the putative VP2 proteins of canine parvovirus-2 (CPV-2) strains detected in Nevis (designated with prefix CN) with those reported from St. Kitts in 2016 (strain RVC50) and in 2020 (strains CK81 and CK84), Supplementary Material S3: Multiple alignments of the VP2 coding sequences of canine parvovirus-2 (CPV-2) strains detected in Nevis (designated with prefix CN) during 2020 with those reported from the neighboring island of St. Kitts in 2016 (strain RVC50) and in 2020 (strains CK81 and CK84), Supplementary Material S4: Gross (A) and microscopic (B) lesions observed in the small intestine of a dog with hemorrhagic gastroenteritis that eventually died on the island of St. Kitts. Small intestinal scrapings collected during necropsy tested positive for canine parvovirus-2 (CPV-2) by PCR. Analysis of the complete deduced VP2 amino acid sequence identified the CPV-2 strain as new CPV-2a, Supplementary Material S5: Nucleotide (nt) mismatches between the nearly complete genome sequences (4269 nt, spanning all the coding regions) of canine parvovirus-2 (CPV-2) strain CPV12 (representing the earliest CPV-2 strains from the late 1970s) and CPV-2 strains detected on St. Kitts in 2016 (strain RVC50) and Nevis in 2020 (strains CN10, CN14, and CN20), Supplementary Material S6: Multiple alignments of the putative NS1 (A), NS2 (B), VP1 (C) and VP2 (D) proteins of canine parvovirus-2 (CPV-2) strain CPV12 (GenBank accession number MN451655, representing the earliest CPV-2 strains from the late 1970s) and CPV-2 strains detected on St. Kitts in 2016 (strain RVC50) and Nevis in 2020 (strains CN10, CN14, and CN20), Supplementary Material S7: Phylogenetic analysis of the nearly complete genomes of canine parvovirus-2 (CPV-2) strains from St. Kitts and Nevis with those of other CPV-2 strains. The tree was created using the Hasegawa-Kishino-Yano model with gamma-distributed rate variation among sites and 1000 bootstrap replicates, Supplementary Material S8: Phylogenetic analysis of the nearly complete genomes of canine parvovirus-2 (CPV-2) strains from St. Kitts and Nevis with those of other CPV-2 strains. The tree was created using the General Time Reversible model with gammadistributed rate variation among sites and 1000 bootstrap replicates, Supplementary Material S9: Phylogenetic analysis of the complete VP2 coding sequences of canine parvovirus-2 (CPV-2) strains from St. Kitts and Nevis with those of other CPV-2 strains.

**Author Contributions:** Conceptualization, K.G. and S.G.; collected samples, A.B. and P.B.; collected epidemiological data, A.B., A.P., K.G., P.B. and S.G.; secured funding, S.G.; contributed reagents, S.G.; screened samples, A.B., K.G. and S.G.; performed laboratory work, K.G. and S.G.; performed data analysis, K.G. and S.G.; wrote the manuscript, K.G. and S.G.; edited and finalized the manuscript, A.B., A.P., P.B., K.G., S.G. and Y.S.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** The present study was funded by intramural grant # Viruses 41001-21 entitled 'Detection and molecular characterization of viruses in pigs and wildlife in the Caribbean and Central America' from the One Health Center for Zoonoses and Tropical Veterinary Medicine, Ross University School of Veterinary Medicine, St. Kitts and Nevis.

**Institutional Review Board Statement:** The present study was submitted to the Institutional Animal Care and Use Committee (IACUC) of the Ross University School of Veterinary Medicine (RUSVM), St. Kitts Island. Ethical review and approval were waived for this study by the RUSVM IACUC as the research study was based on leftover samples that were collected for diagnostic purposes at the veterinary clinic on Nevis Island (RUSVM IACUC sample/tissue notification letter number TSU 1.23.21).

**Acknowledgments:** We would like to thank the staff members of Nevis Animal Speak, Nevis Island, for helping with the sampling and the division of pathology, Ross University School of Veterinary Medicine, St. Kitts Island, for performing the necropsies on the two dead dogs.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


## *Review* **Battling Neurodegenerative Diseases with Adeno-Associated Virus-Based Approaches**

## **Olja Mijanovi´c 1, Ana Brankovi´c 2, Anton V. Borovjagin 3, Denis V. Butnaru 4, Evgeny A. Bezrukov 5, Roman B. Sukhanov 5, Anastasia Shpichka 4, Peter Timashev 4,6,7,8 and Ilya Ulasov 1,\***


Received: 25 February 2020; Accepted: 15 April 2020; Published: 18 April 2020

**Abstract:** Neurodegenerative diseases (NDDs) are most commonly found in adults and remain essentially incurable. Gene therapy using AAV vectors is a rapidly-growing field of experimental medicine that holds promise for the treatment of NDDs. To date, effective delivery of a therapeutic gene into target cells via AAV has been a major obstacle in the field. Ideally, transgenes should be delivered into the target cells specifically and efficiently, while promiscuous or off-target gene delivery should be minimized to avoid toxicity. In the pursuit of an ideal vehicle for NDD gene therapy, a broad variety of vector systems have been explored. Here we specifically outline the advantages of adeno-associated virus (AAV)-based vector systems for NDD therapy application. In contrast to many reviews on NDDs that can be found in the literature, this review is rather focused on AAV vector selection and their testing in experimental and preclinical NDD models. Preclinical and in vitro data reveal the strong potential of AAV for NDD-related diagnostics and therapeutic strategies.

**Keywords:** AAV; neuro-degenerative disease; gene therapy

## **1. Background**

Neurodegenerative diseases (NDDs) are the most common primary pathologies of the central nervous system (CNS) in adults. They affect the majority of the elderly population and arise either as a consequence of an accumulation of previously acquired genetic modification in the human genome or de novo—i.e., without preceding neurodegenerative pathologies—as a result of an accumulation of neurotoxic proteins in the cytoplasm of neurons (de novo disease). The lack of effective therapies to control NDD made the progression of the disease inevitable [1]. Systemic effects from NDD development are usually associated with infiltration of the brain with peripheral immune cells [2], which contributes to neuroinflammation [3] and neurodegeneration [4]. The cause of systemic and

molecular abnormalities developing throughout the NDD progression remains to be investigated as any developments in the NDD therapy field require a detailed understanding of the molecular mechanism of neurodegeneration process as well as how to intervene with the disease process.

A significant determinant of NDD progression is an accumulation of toxic proteins on a subcellular level [5,6]. Intracellular accumulation of specific abnormally-aggregated proteins in the form of inclusion bodies is associated with different pathologies of most common NDDs: while Alzheimer's destroys memory, Parkinson's and Huntington's diseases affect movement. Previous research has suggested that all three diseases are caused by the death of neurons and other cells in the brain, which is associated with abnormally-folding and aggregating amyloid proteins, different in each disease. For instance, a misfolded amyloid-β and mutated tau, α-synuclein, huntingtin, and ALS-linked trans-activation response (TAR) DNA-binding protein 43 (*TARDBP*) along with FUS are implicated in molecular mechanisms of Alzheimer's, Parkinson's, Huntington's, and amyotrophic lateral sclerosis (ALS), respectively [7]. Those all form insoluble clumps inside brain cells that ultimately rupture the endocytic vesicles. It was suggested that the latter mechanism is conserved and, therefore, common treatment strategies should be possible that would involve boosting a brain cell's ability to degrade protein clumps and damaged vesicles [8]. This observation offers a possibility for therapeutic intervention, as it suggests the involvement of only a few signaling pathways that recently emerged as potential targets for neuronal dysregulation. Among them is autophagy signaling, whose dysfunction permits the accumulation of misfolded proteins in various structural parts of the neural cells [9,10]. All the dysregulation results in excessive inhibition of adenosine triphosphate (ATP)-dependent chaperones and proteolytic machinery [11] that represses neurogenesis [12], stimulates damage via oxidative stress [13], and promotes neuronal cell death. Recent studies suggest that inactivated autophagy-related proteins in damaged neurons [14] represent a suitable target for experimental NDD therapy [15]. Although there is extensive literature on the accumulation of toxic proteins inside neuronal cells as a result of autophagy repression and its pharmacological correction [16], relatively few studies have addressed the possibility of targeting neuronal cells with aberrant signaling [17–21] by using alternate approaches, such as gene therapy. Below, we summarized the benefits of gene therapy application for NDD treatment.

Neurodegenerative diseases are typically caused by numerous genetic abnormalities, whose effect is enhanced by environmental factors [22] and epigenetic events [23,24]. Basic research has identified numerous pathways that contribute to NDD pathogenesis. Based on this rapidly growing knowledge, multiple drugs for NDD therapy have been approved to-date [25–29]. Interestingly, Chinese herbal medicine may also represent a promising alternative for NDD treatment [30]. In the past three decades, the scientific community has made significant efforts in utilizing newly developed molecular technologies to selectively deliver a therapeutic payload into target cells via a nonconventional method, known as "gene therapy" [31,32].

Viral vector-based gene therapy offers a new therapeutic opportunity against an irreversible lethal impact of many inheritable or genetic diseases. For instance, in the case of Spinal Muscular Atrophy type 1 (SMA1), a single dose of intravenously delivered AAV9 vector carrying a transgene encoding the missing SMN protein resulted in longer survival of patients [33]. Another example is the study by Massaro et al., who used a mouse model of an untreatable acute form of infantile neuronopathic Gaucher disease to inject an adeno-associated virus (AAV)-based vector encoding neuronal glucocerebrosidase that abolished neurodegeneration and ameliorated neuroinflammation [34]. In a later study, gene therapy with an AAV vector improved motor skills and increased lifespan of animals in a mouse model of inherited lysosomal storage disorders, known as the neuronal ceroid-lipofuscinoses (NCLs) or more commonly referred to as Batten disease, a form of NCLs with a deficiency in the membrane-bound protein CLN6 (CLN6 disease) [35]. Although it is too early for evaluating the clinical efficacy of the developed AAV gene therapy vectors in human patients, some AAV-based gene therapies demonstrate a remarkable therapeutic effect in preclinical studies. For example, an AAV-based gene

therapy aimed at correcting a rare metabolic aromatic L-amino acid decarboxylase deficiency (AADC) was granted a biologics license application (BLA)-ready status by the FDA in 2018 [36].

## **2. Viral Vector-Based Gene Therapy: E**ffi**ciency and Selectivity of Delivery**

### *2.1. Choosing an Ideal Vector*

Gene therapy for NDDs represents a rapidly developing novel therapeutic approach with a potential capability of restoring normal neuronal functions [37] by intracellular delivery of exogenous genetic material, carrying a disease correcting/therapeutic information in the form of recombinant deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules engineered into a delivery vehicle, known as vector. Once inside a recipient (host) cell, the transferred genetic material utilizes cellular machinery to biosynthesize (translate the encoded information into) therapeutic proteins aimed at replacing their defective counterparts, produced in neurons by mutated endogenous gene, inactivating the mutated gene's product or inserting a foreign transgene with therapeutic properties to battle the disease. Despite the variability in the existing delivery vectors, routes of delivery, and delivery techniques, the intracellular mechanism of the delivered gene's expression remains essentially the same. The transduced or transfected host cells then begin synthesizing protein(s) aimed at repairing a congenital disorder or treat an acquired disease [38]. Depending on the nature of target cells, gene delivery can be classified as either somatic or germline. While the goal of somatic gene delivery is to directly express the transgene in target cells and correct the disease at the individual cell level [39], germline gene transfer is aimed at integrating a therapeutic or correction gene (fully functional wild type copy) into the germline cells, thereby creating inheritable changes, affecting every cell of the next generation. Although more advanced techniques of gene delivery are constantly being developed, the most common approach to correct NDDs involves a direct transfection or transduction (when using virus-based delivery vectors) of isolated patient-derived cells or modification of target cells in situ—i.e., directly in patient's tissue. The former strategy of gene delivery, known as ex vivo gene therapy, involves isolation and in vitro culturing of patient-derived cells for exogenous genetic modification or gene correction and their subsequent injection back into the patient [40]. In contrast, in vivo gene therapy involves the delivery of genetic modifications to target cells directly by systemic [41]—i.e., intraperitoneal (ip) [42,43] or intravenous (iv) [44–46] vector administration. However, large doses of systemically-delivered AAV vectors elicit immune responses affecting transgene expression, treatment longevity and even patient safety. To circumvent those drawbacks, alternate routes of AAV administration to the central nervous system (CNS) have been explored that allowed administration of lower doses of the gene therapy vector directly by into the brain parenchyma or by injection into the cerebrospinal fluid via the intracerebroventricular or intrathecal (cisternal or lumbar) routes. This strategy results in higher transgene expression along with the decreased immune responses. However, the remaining critical concern of the direct CNS-delivery is AAV-associated neuroinflammation, manifested in preclinical studies by the dorsal root ganglion (DRG) and spinal cord pathology with mononuclear cell infiltration [47].

Since the precision of neural cell targeting as well as the expression magnitude of a therapeutic payload are critical characteristics for the efficient repairing of genetic or metabolic abnormalities, the use of viral vector-based gene therapy, well-suited for modulation of those characteristics, offers a significant advantage over the utilization of non-viral vectors.

Due to an essential condition for an effective gene therapy intervention is an efficient and specific delivery of a therapeutic transgene to target cells, an ideal vector should be inherently suitable for or possibly tailored (genetically engineered) to specific target cells of interest. In addition, it should not trigger a robust immune response in the host and be capable of providing a sustained transgene expression for the duration of disease treatment [48]. By these criteria, some virus-based vectors became preferred gene delivery vehicles in contemporary gene therapy applications. Since the natural mechanism of viral cell entry involves attachment to target cell's surface receptor(s), most virus-based gene therapy vectors can be engineered by retargeting the viral particle (capsid) surface to primary receptors abundant on or even specific to target cells by using genetic and other molecular technologies developed in the past two decades. Viral gene therapy vectors specifically designed for NDD applications should be able to efficiently cross the blood–brain barrier (BBB), if delivered by iv infusion [44,49], and support an efficient transgene expression, regardless of aberrant cell signaling caused by the accumulation of toxic proteins or restoration of an autophagic state in neuronal cells [50]. For instance, vectors based on genomes of adeno-associated virus (AAV) [44,49,51–56], lentivirus [57], type 1 herpes simplex virus [58], and Semliki Forest virus (SFV) [59] have been broadly used for neuron targeting. Extensive use of the above viruses in neuroscience is dictated by their natural ability to efficiently and selectively transduce and provide high transgene expression in neurons. Moreover, those viral vectors minimally perturb stress pathways in the infected neural cells [60–62], which makes them attractive tools specifically for the NDD gene therapy applications. When the efficiency and the safety of AAV and lentiviral vectors were compared, AAV demonstrated a significantly higher transgene expression levels and minimal interaction with the innate immune effectors. In contrast, lentiviral transduction resulted in modest transgene expression efficiency and provoked a rapid self-limiting proinflammatory response [63].

In contrast to the majority of viral vectors utilized for gene therapy interventions, AAV-based vectors exhibit the highest potential and success rate in the treatment of several monogenic diseases, as demonstrated by clinical trials [64,65].

#### *2.2. Advantages of AAV-Based Vectors*

What makes AAV an exceptional candidate vector for NDD gene therapy applications is its low pathogenicity in humans: none of the AAV subtypes appear to cause a life-threatening systemic response in humans. This is an important feature since patient safety is the most critical condition for the successful development of gene therapy interventions of NDD and other human diseases. Natural tropism of most AAV serotypes, particularly that of AAV2, towards various human tissue types that include the retina and neural tissues [66], liver [67,68], vascular tissue [69,70], lung [71], and skeletal muscles [72] is the other important criterion making AAV a preferred gene therapy vector for NDD [73,74]. The broad tropism of AAV is owed to the diversity of its natural cell-binding targets, such as primary receptors heparan sulfate proteoglycan (HSPG) [75] and/or *N*-glycans with terminal galactose [48], and several known co-receptors, such as fibroblast growth factor (FGF) receptor 1 [76], platelet-derived growth factor (PDGF) receptor [77], hepatocyte growth factor receptor [78], αVβ<sup>1</sup> and αVβ<sup>5</sup> integrins [79,80], O-linked sialic acid [81] and laminin receptor (LRP/LR), previously found in liver tissue [82], and recently also discovered in neurons [83]. In fact, cell surface receptors have been definitively identified only for some AAV serotypes: HSPG for AAV-3, O-linked sialic acid for AAV4, PDGF for AAV5, and LRP/LR for AAV serotypes 2, 3, 8, and 9 [49]. To date, there have been 11 identified AAV serotypes (AAV1-AAV11). Due to the fact that the transduction of human neural cells with AAV2 serotype is limited, and humans are seropositive with regard to AAV2, the other AAV serotypes have been explored as alternative gene therapy vectors. This allowed achieving more efficient transduction of selective subsets of brain cells in animal NDD models [84].

The versatility of AAV tropism can be attributed to variability in the amino acid (aa) sequence of the AAV structural (capsid) proteins VP1, VP2, and VP3, whose mixture (in the ratio of 1:1:10, respectively) makes icosahedral symmetry capsid of 60 monomers that are involved in cell surface attachment of the viral particles. The variability in the amino-acid (aa) sequence is confined to the 9 surface-exposed (VR-I to VR-IX) domains of the AAV structural/capsid proteins. AAV tropism alteration for tailoring of the vectors to specific gene therapy applications can be achieved by virus pseudotyping—i.e., creating artificial hybrid AAV particles by combining genome from one strain/serotype with capsid proteins from a different strain/serotype [85,86]. AAV capsid engineering is a two-decade-old approach that embodies two distinct strategies: directed evolution and rational design. The former involves shuffling of capsid genes from available serotypes [87], random peptide

insertion into known sites of AAV capsid [88,89] or phage display [90] and ultimately involves a selection process that requires multiple generations of screening to identify functional capsids. Alternatively, a rational design strategy utilizes a structural knowledge to refine capsid structure and/or replace native cell-binding motifs with foreign motifs of desired affinity [90–93]). Recently, a more advanced method of AAV targeting, known as barcoded rational AAV vector evolution (BRAVE), was proposed that encompasses all of the benefits of the rational design approach while maintaining the broad screening diversity permitted by directed evolution, but with only a single generation screening. This new strategy is based on the viral library production approach, where each viral particle displays a protein-derived peptide on the surface, which is linked to a unique barcode in the packaged genome [94].

Naturally, AAV does not produce replication factors and, therefore, is unable to replicate (propagate) without a helper virus (adenovirus, herpes simplex virus (HSV) or vaccinia virus), which provides activating proteins to AAV to enable its replication. Furthermore, in the presence of some helper viruses, such as adenovirus, AAV can even exhibit a lysogenic behavior, which can rarely occur without a helper. Replication deficiency of AAV adds to the biological safety of AAV as yet another benefit for its application as a gene therapy vector.

#### **3. AAV in NDD Experimental Therapy**

## *3.1. In Vitro vs. In Vivo Models*

With regard to NDD gene therapy, an experimental tissue model for the blood-brain barrier (BBB) is of the highest relevance and importance, and the most developed disease models include those for Alzheimer's, Parkinson's, and Huntington's diseases. These and other NDD models are discussed in detail elsewhere [95]. Numerous NDD disease platforms have been created to date by using various approaches [96–99]. This includes several in vitro disease models developed in the last three years, which were successfully used in a few NDD gene therapy studies. One of the crucial criteria in the efficacy assessment of viral vector-based gene therapy of NDD is the ability of delivery vectors to efficiently cross the BBB, as iv administration remains the primary route of vector delivery. In contrast to in vivo (animal) models, in vitro models allow us to easily understand the mechanism of viral trafficking. In this regard, Merkel et al. showed that primary human brain microvascular endothelial cells, as a model of the human BBB, can be effectively utilized as a tool to characterize trans-endothelial movement and transduction kinetics of various AAV serotypes in vitro and even their effect on BBB integrity. Specifically, by using this in vitro BBB model, the authors found that AAV9 penetrates brain microvascular endothelial cell barriers more effectively than AAV2, although it exhibits relatively lower transduction efficiency [100].

A proper design of NDD in vivo models should allow assessing the robustness of therapies in the settings that mimic clinical endpoints. Although information obtained from studying in vitro models is usually capable of providing a sufficient understanding of disease pathogenesis, it fails to characterize the aspect of disease progression. Therefore, the use of in vivo disease models allows investigators to observe and analyze symptomatic manifestation of NDDs alongside with the therapeutic outcomes of their experimental treatment. This also aids in getting a comprehensive insight into the complex interconnection between etiological and symptomatic changes during the progression of NDDs, such as neuronal ceroid lipofuscinosis (NCL or Batten disease) [35], spinal muscular atrophy [101], or Niemann-Pick disease [102], whose development is typically associated with genetic perturbations in multiple genes. Therefore, in contrast to in vitro models, a mouse model of NDD would allow recapitulating onset and progression of the counterpart human disease of a given degree of severity. For instance, the studies involving AAV9-based vectors [103–106] aimed at restoring expression of target genes with genetic abnormalities require testing of the experimental strategies in a preclinical setting and under different routes of vector delivery to affected cells. Hudry et al. [49] were able to evaluate the efficacy of AAV-mediated intracellular delivery of reporter transgene in a CNS disorder. It was found that, depending on the viral capsid composition and the route of vector administration, AAV9 is capable of providing a high level of transduction in mixed astrocyte and neuron culture, although vector biodistribution upon intracerebroventricular injection can vary. As far as the route of injection is concerned, a direct injection of experimental therapeutics into cerebrospinal fluid allows their fast and efficient delivery and spread throughout CNS and spinal cord. Therefore, intrathecal injection is nowadays commonly used in various experimental strategies as an effective route of viral agent delivery and achieving therapeutic effects [107–110].

## *3.2. Selection of Optimal Gene Therapy Strategy*

The choice of a suitable gene therapy strategy for NDD is commonly dictated by the known alterations in signaling pathways associated with a particular NDD. Multiple scientific reports suggest that under normal physiological conditions a microtubule-associated protein tau (MAPT) is sensitive to autophagy-associated ubiquitin-binding protein p62/SQSTM1, which promotes degradation of the misfolded, microtubule-dissociated tau protein. However, in tauopathy, an insoluble form of mutant MAPT becomes resistant to recognition by and binding to p62/SQSTM1 and as a result, fails to degrade inside autophagosomes [6]. This study revealed that the pathology caused by the intracellular formation of neurofibrillary tangles (NFTs) by the hyperphosphorylated form of mutant MAPT, can be suppressed by p62/SQSTM1 overexpression resulting from delivery of AAV 2/9-SQSTM1 expression vector. Earlier, Janda et al. demonstrated that several NDDs are associated with an onset of oxidative stress that inhibits LC3 lipidation and autophagic flux in the affected neurons [111], suggesting multiple reasons for the reduction of basal autophagy and the resulting promotion of NDD. Since the discovery of the relevance of defective autophagy signaling to NDDs, a few studies utilized AAV vectors to overcome the autophagy signaling defects. It was reported that defects in autophagy signaling can be overcome by activation of lysosomal transport via intracellular expression of hydrolase glucocerebrosidase [112] or reduction of tau protein phosphorylation via arginase 1 [113] that eliminates the cargo from vulnerable neurons. In the aggregate, the above-referenced studies strongly suggest that rescuing impairments in the autophagy pathway inside diseased cells is a promising therapeutic strategy for some NDDs. Moreover, aberrant signaling that is attributed to the NDD progression can also be corrected via AAV-based vectors. Recently, it was shown that the expression of triggering receptor expressed on myeloid cells 2 (TREM2) is associated with high risk for AD development. Therefore, AAV-mediated expression of the soluble form of TREM2 (sTREM2), a proteolytic product of TREM2, reduces amyloid plaque load and rescues functional deficits of spatial memory [114]. Overall, the delivery of a therapeutic payload via a modified AAV contributes to the restoration of the disease-affected pathway (Figure 1).

**Figure 1.** Potential applications of gene targeting for therapy of neurodegenerative diseases.

## *3.3. AAV-Based Gene Therapy for Parkinson's Disease*

The movement disorder, known as Parkinson's Disease (PD), the second most common neurodegenerative disorder, occurs largely due to the loss of dopaminergic neurons of the substantia nigra, resulting in striatal depletion of the neurotransmitter dopamine. AAV-based gene therapy vectors could be effectively used for gene therapy of PD through increasing dopamine levels in target cells [115]. Another strategy proposed for gene therapy of PD is based on targeting of neural parenchyma by AAV-based α-synuclein expression vector (AAV-PHP.B-GBA1) delivered via an iv administration, as opposed to commonly used injection in the mouse forebrain. This allowed vector to permeate and diffuse throughout the neural parenchyma, resulting in targeting of both the central and the peripheral nervous systems in a global pattern and recovering animal behavior by reducing synucleinopathy [44].

Gutekunst et al. demonstrated that the delivery of C3-ADP ribosyltransferase (C3) selectively and irreversibly inhibits activation of RhoA GTPase (a key intracellular regulator of axon regrowth

in the mammalian CNS) in neurons or neuronal progenitors and effectively prevents inhibition of axonal regrowth leading to axon regeneration [116]. Furthermore, the use of AAV-mediated C3 delivery allowed the authors to determine a critical C3 concentration promoting neuron outgrowth on chondroitin sulfate substrate. Another example of AAV vector system implementation for gene therapy of PD is the delivery of rat ATP6V0C (a pore-forming stalk c-subunit of the V0 sector of the vacuolar proton ATPase contributing to release of serotonin, acetylcholine, and dopamine by stromal cells) into the substantia nigra of the diseased mice, in which high potassium stimulation increased overflow of the endogenous dopamine (DA) [117,118].

#### *3.4. AAV Vectors and Gene Targeting*

In addition to gene delivery for therapeutic protein expression/overexpression purposes in neural cells, AAV vectors have also been used for gene targeting (knockdown and knockout) applications, which include shRNA and miRNA expression strategies. It becomes increasingly popular to knockdown the expression of cellular factors or enzymes contributing to NDD pathobiology to restore normal physiological conditions in disease-affected neural cells. For instance, an AAV8-based vector has been utilized to modulate the expression of cellular proteins implemented in the pathology of the disease. Lu et al. [119] demonstrated that an elevated level of SM synthase-1 promotes the lysosomal degradation of BACE1. However, due to the dominant-negative nature of some genetic mutations, direct sequestration of aberrant (mutant) proteins with normal function-inhibiting properties or even genetic knock-out of mutant genes (excision/destruction of mutant genes) or their complete replacement with corrected/wild type counterparts becomes a necessary and the most rational strategy.

Mutations in genes that control normal CNS development and metabolism give rise to several genetic (inheritable) NDDs. For instance, Krabbe disease is caused by a genetic abnormality that is associated with mutations in the galactosylceramidase (GALC) gene. The GALC enzyme is contained inside lysosomes, where it hydrolyzes specific galactolipids, including galactosylceramide and psychosine (galactosylsphingosine). While the breakdown of those lipids is part of the normal myelin turnover, accumulation of psychosine metabolite in the absence of galactosylceramidase is toxic for neurons and needs to be prevented by a quick restoration of the galactosylceramidase function. Gene therapy for Krabbe disease can thus be accomplished through the GALC gene correction/replacement strategy in the mouse model [46] as well as in the tissue of human patients.

Mutations in superoxide dismutase 1 gene (SOD1) are known to be one of the causes for amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease caused by progressive loss of upper and lower motor neurons in human CNS. Although responsible for only about 20% of familial (inherited) ALS cases, *SOD1* mutations contribute to the loss of motor neurons during ALS progression via a toxic gain of function mechanism that involves NF-κB-dependent mechanism, resulting in intracellular aggregation of the aberrant protein [120]. A strategy using an AAV9 delivery vector, engineered to express shRNA targeting SOD1 mRNA, was employed by Frakes et al. to reduce (knock-down) SOD1 expression in both astrocytes and motor neurons. The AAV9 vector was able to efficiently penetrate the BBB and increase the survival of the ALS mice [121]. The same vector was used in the AAV9-mediated knockdown of neuritin, which resulted in the reduction of synaptic transmission in the medial prefrontal cortex (mPFC) pyramidal neurons in mice [122]. Selective suppression of mutant huntingtin aggregation and neuronal dysfunction in a rat model of Huntington's disease (HD) by applying AAV5-miHTT-451, induced functional improvements in the HD pathological process without causing an activation of microglia or astrocytes immune response [123].

Despite the remarkable therapeutic efficacy of RNAi-based gene silencing in target cells, most of the shRNA-mediated NDD gene therapy applications only partially reduce the *SOD1* gene expression, since neither a complete knockout of the target mRNA [124–126], nor a sustained gene knockdown can be technically achieved [124,127]. Therefore, the novel gene targeting technologies, based on the recent discovery of transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 nuclease complex (CRISPR-Cas9 nuclease) as components of

bacterial natural "immunity" against viruses, completely revolutionized the genome editing [128] field by dramatically improving gene targeting efficiency and specificity, thereby allowing a highly accurate cleavage of any DNA sequence at any given point [129]. The precision of DNA cleavage mediated by CRISP-Cas9 nucleases has been validated in recent studies aimed at correcting genetic mutations on a single nucleotide level [130]. Wen et al. reported that the deletion of the human insulin growth factor-1 (IGF-1) gene, implicated in hyperpolarization of mitochondrial electrical transmembrane potential, resulted in the accumulation of anti-apoptotic protein factors B-cell lymphoma-extra large (BCL-XL) and B-cell lymphoma 2 (BCL-2). Moreover, the deletion of the *IGF1* gene via an intratracheal injection of an AAV-delivered CRISPR-Cas9 expression system restored mitophagy and reduced protective effect on mitochondria, suggesting a new strategy for treating ALS [131]. A strong therapeutic effect was reported by Raikwar et al. [131] and Gaj et al. [132], who used the CRISPR-Cas9 gene targeting (Figure 1) approach to achieve a reduced expression of the glial maturation factor in glial cells that contributed to the formation of Alzheimer's disease plaques and corrected an autosomal mutation in the *SOD1* gene, responsible for ALS progression, respectively. Most recently, Ekman et al. pointed out that correction of mutant exon 1 of the huntingtin gene (HTT) results in a ~50% decrease of neuronal inclusions and improved motor deficits [133].

Recently, it was suggested that downregulation of some miRNAs may be involved in modulation of apoptotic response and autophagy defects during the NDD progression [134,135]. Miyazaki et al. found that bulbar muscular atrophy (SBMA) is caused by the expansion of the polyglutamine (poly Q) tract in the androgen receptor (AR-poly Q) protein sequence [136]. It was predicted that several miRNAs, such as miR-196a [136], miR-298 or miR-328 [137], might regulate the activity of NDD-related proteins, such as Elav-like family member 2 (CELF2) that enhances the stability of AR mRNA, or beta-amyloid precursor protein-converting enzyme (BACE), respectively. AAV-based delivery and expression of miR-196a promotes decay of the AR mRNA by silencing its stabilizing factor CUGBP, an Elav-like family member 2 (CELF2), and thereby ameliorates the SBMA phenotypes in the mouse model. These studies suggest that AAV vector is a highly efficient tool for the delivery and the expression of recombinant DNA or regulatory, mRNA-silencing miRNAs in monogenic inherited disease applications. Furthermore, in the case of polygenetic NDD diseases, utilization of AAV for targeting a mutant allele to silence the expression of a mutant protein in patients could become a potent therapeutic approach in the future. One of the remaining challenges of such gene silencing strategy that should be addressed in AAV vector design is how to avoid targeting of the wild type or normal (non-mutated) gene copy in the diseased cells.

## **4. Modulation of Interaction Between the CNS and the Environment**

Inflammation is an essential hallmark of various neurodegenerative diseases. An increasing number of clinical studies demonstrate that upon activation microglia and astrocytes produce proinflammatory chemokines, such as tumor necrosis factor alpha (TNFα) and interleukin type 1 (IL1), around amyloid plaques. In most of those cases, administration of interleukin type 2 (IL2) and interleukin type 4 (IL4) has been shown to stimulate an anti-inflammatory response. On the other hand, multiple lines of evidence suggest that the delivery of growth factors via viral vectors can provide significant therapeutic effects. For instance, the nerve growth factor (NGF) was expressed in the brain cells of patients with AD by means of AAV2-based vector delivery in a clinical trial conducted by Tuszynski et al. (Trial Registration: NCT00087789 and NCT00017940). It was found that neurons of the degenerating brain retain the ability to respond to the growth factor administration by axonal sprouting, cell hypertrophy, and activation of functional markers for up to 10 years [138]. Other studies used either lentiviral vector- or AAV-based delivery of a brain-derived neurotrophic factor (BDNF), progranulin (PGRN), or cerebral dopamine neurotrophic factor (CDNF) in mouse models of AD or PD, respectively [71–73]. Delivery of BDNF in transgenic APP mouse model (mutant mice carrying two amyloid precursor protein (APP) mutations associated with early-onset familial Alzheimer's disease) resulted in significant amelioration of cell loss by BDNF and marked improvements in the

hippocampal-dependent behavior (contextual fear conditioning), compared with control-treated APP mice [72]. Elevation of PGRN expression in nigrostriatal neurons upon AAV-mediated PGRN delivery protected nigrostriatal neurons from MPTP toxicity, reduced inflammation and apoptosis, as well as completely preserved locomotor function in the mouse PD model [71]. Striatal delivery of AAV2.CDNF allowed recovery of 6-OHDA-induced behavior deficits and resulted in a significant restoration of tyrosine hydroxylase immunoreactive (TH-ir) neurons in the substantia nigra as well as functional recovery of dopaminergic neurons.

An impressive therapeutic outcome was observed in yet another innovative cell/gene therapy combinational approach utilizing the human umbilical cord blood cells (hUCBCs) transduced with adenoviral (Ad) vectors encoding human vascular endothelial growth factor (VEGF), glial cell-line derived neurotrophic factor (GDNF) [139] and/or neural cell adhesion molecule 1 (NCAM) genes [74–76]. After hUCBCs were transplanted into transgenic amyotrophic lateral sclerosis (model) mice a significant improvement in animals' behavioral performance (open-field and grip-strength tests), as well as an increased life-span were observed. Expression of NCAM-VEGF or NCAM-GDNF was observed in ALS mice 10 weeks after delivering genetically modified hUCBCs, whereas the cell vehicles were detectable for 5 months following the transplantation [140].

Although some NDDs are caused by dysregulation of neuron's synaptic function, AVV-based gene therapy could still offer a therapeutic solution. Specifically, an AAV-mediated delivery of the retinoschisin transgene resulted in the restored structure and function of the photoreceptor cell synapse in the mouse disease models. This restoration of operational synapse in the animal model led to a human clinical trial for gene therapy of X-linked retinoschisis [141]. A recent study established the formation of synaptic connections between NeuroD1-converted neurons with already present neurons. These results indicate possibilities of NeuroD1 AAV-based gene therapy for functional brain repair after ischemic injury through in vivo astrocyte-to-neuron conversion [142].

## **5. Conclusions and Future Directions**

Outcomes of clinical trials for NDDs can be compromised by limited data from the prior animal (preclinical) studies, by suboptimal expression vector's cassette design or by the immunogenicity of either delivery vector itself, or its expression content (a therapeutic transgene). The Clinicaltrials.gov database contains information about 6128 clinical trials worldwide that were aimed at treating various neurodegenerative diseases, including 103 trials using gene therapy approaches 29 of which utilized natural mechanisms of viral replication [143]. At this point, two clinical trials (NCT02418598 and NCT00643890) have been terminated by the organizers (both due to financial reasons) and one (NCT03381729) was suspended by the FDA. The latter study involved 27 patients selected out of 51 with spinal muscular atrophy 1 (SMA1) disorder to receive an injection of AAV9-based vector (6 <sup>×</sup> 1013, 1.2 <sup>×</sup> 1014 and 2.4 <sup>×</sup> 1014 vp per patient) designed to express the SMN protein under the control of hybrid CMV/β-actin promoter (ZOLGENSMA). Currently, the future of ZOLGENSMA use via intrathecal route remains unclear, but the available clinical safety information suggests using caution, when infusing ZOLGENSMA intravenously, and inclusion pre-treatment with corticosteroids, checking blood biochemistry and liver enzymes (https://www.zolgensma.com/how-zolgensma-works). Compared to non-viral vectors (e.g., polyplexes [144]), AAV vectors appear to be highly suitable for NDD gene therapy applications and, therefore, became highly popular for NDD gene therapy studies in the recent years. However, the problem with AAV crossing BBB along with the vector's immunogenicity [47] in human patients remains the main obstacle even for those vectors that are capable of effectively preventing NDD progression.













In this review, we outlined the major basic and clinical research directions in the field of NDD gene therapy using viral vectors, and particularly AAV, for delivery of therapeutic payload aimed at restoration of autophagy and metabolic defects in neurons. Encouragingly, many of the reported studies demonstrate strong and long-lasting therapeutic effects (Table 1). The summarized basic research data appears to be in good agreement with the results of preclinical studies, suggesting strong efficacies of neuronal cell targeting with AAV. In addition, findings from several basic studies suggest existence of a crosstalk between the brain microenvironment and neuronal cell signaling. A combination of AAV-based gene therapy with other therapeutic strategies targeting autophagy signaling components could result in even more favorable outcomes. Such co-therapeutics include traditional and herbal medicines [145]. Therefore, dysfunction in neuronal signaling is closely interconnected with the brain environment, and a combinational intervention using these two disease components could become a promising therapeutic strategy, which may provide a clue for an effective solution for the ultimate NDD cure.

**Author Contributions:** Writing: O.M., A.B., A.S., A.V.B., P.S., and I.U.; Figures: O.M. and I.U.; Editing: A.B., O.M., D.V.B., E.A.B., R.B.S., A.V.B., P.T., A.S., and I.U. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported into frameworks of the state task for ICP RAS 0082-2019-0012 (State registration number AAAA-A20-120021190043-7) and by the Russian Academic Excellence Project "5–100".

**Conflicts of Interest:** Authors declare no conflict of interest.

## **Abbreviations**


**VEGF** Vascular endothelial growth factor

## **References**


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