Viruses, Volume 10, Issue 7 (July 2018) – 51 articles

Due to the emergence of non-endemic mosquito vectors and the recent outbreaks of mosquito-borne diseases, mosquito-borne pathogens are considered an increasing risk to public and animal health in Europe. To obtain a status quo regarding mosquito-borne viruses and their vectors in Germany, 97,648 mosquitoes collected from 2011 to 2016 throughout the country were screened for arboviruses. Mosquitoes were identified to species, pooled in groups of up to 50 individuals according to sampling location and date, and screened with different PCR assays for Flavi-, Alpha- and Orthobunyavirus RNA. Two pools tested positive for Usutu virus-RNA, two for Sindbis virus-RNA, and 24 for Batai virus-RNA. The pools consisted of Culex pipiens s.l., Culex modestus, Culex torrentium, Culiseta sp., Aedes vexans, Anopheles daciae, and Anopheles messeae mosquitoes and could be assigned to nine different collection sites, with seven of them located in northeastern Germany. Phylogenetic analyses of the viral RNA sequences showed relationships with strains of the viruses previously demonstrated in Germany. These findings confirm continuing mosquito-borne zoonotic arbovirus circulation even though only a rather small percentage of the screened samples tested positive. With respect to sampling sites and periods, virus circulation seems to be particularly intense in floodplains and after flooding events when mosquitoes develop in excessive numbers and where they have numerous avian hosts available to feed on. Full article

Ixodes scapularis ticks harbor a variety of microorganisms, including eukaryotes, bacteria and viruses. Some of these can be transmitted to and cause disease in humans and other vertebrates. Others are not pathogenic, but may impact the ability of the tick to harbor and transmit pathogens. A growing number of studies have examined the influence of bacteria on tick vector competence but the influence of the tick virome remains less clear, despite a surge in the discovery of tick-associated viruses. In this study, we performed shotgun RNA sequencing on 112 individual adult I. scapularis collected in Wisconsin, USA. We characterized the abundance, prevalence and co-infection rates of viruses, bacteria and eukaryotic microorganisms. We identified pairs of tick-infecting microorganisms whose observed co-infection rates were higher or lower than would be expected, or whose RNA levels were positively correlated in co-infected ticks. Many of these co-occurrence and correlation relationships involved two bunyaviruses, South Bay virus and blacklegged tick phlebovirus-1. These viruses were also the most prevalent microorganisms in the ticks we sampled, and had the highest average RNA levels. Evidence of associations between microbes included a positive correlation between RNA levels of South Bay virus and Borrelia burgdorferi, the Lyme disease agent. These findings contribute to the rationale for experimental studies on the impact of viruses on tick biology and vector competence. Full article

The mechanisms by which bacteriophage T4 converts the metabolism of its E. coli host to one dedicated to progeny phage production was the subject of decades of intense research in many labs from the 1950s through the 1980s. Presently, a wide range of phages are starting to be used therapeutically and in many other applications, and also the range of phage sequence data available is skyrocketing. It is thus important to re-explore the extensive available data about the intricacies of the T4 infection process as summarized here, expand it to looking much more broadly at other genera of phages, and explore phage infections using newly-available modern techniques and a range of appropriate environmental conditions. Full article

The number of novel bacteriophage sequences has expanded significantly as a result of many metagenomic studies of phage populations in diverse environments. Most of these novel sequences bear little or no homology to existing databases (referred to as the “viral dark matter”). Also, these sequences are primarily derived from DNA-encoded bacteriophages (phages) with few RNA phages included. Despite the rapid advancements in high-throughput sequencing, few studies enrich for RNA viruses, i.e., target viral rather than cellular fraction and/or RNA rather than DNA via a reverse transcriptase step, in an attempt to capture the RNA viruses present in a microbial communities. It is timely to compile existing and relevant information about RNA phages to provide an insight into many of their important biological features, which should aid in sequence-based discovery and in their subsequent annotation. Without comprehensive studies, the biological significance of RNA phages has been largely ignored. Future bacteriophage studies should be adapted to ensure they are properly represented in phageomic studies. Full article

Little cherry virus 1 (LChV1, Velarivirus, Closteroviridae) is a widespread pathogen of sweet or sour cherry and other Prunus species, which exhibits high genetic diversity and lacks a putative efficient transmission vector. Thus far, four distinct phylogenetic clusters of LChV1 have been described, including isolates from different Prunus species. The recent application of high throughput sequencing (HTS) technologies in fruit tree virology has facilitated the acquisition of new viral genomes and the study of virus diversity. In the present work, several new LChV1 isolates from different countries were fully sequenced using different HTS approaches. Our results reveal the presence of further genetic diversity within the LChV1 species. Interestingly, mixed infections of the same sweet cherry tree with different LChV1 variants were identified for the first time. Taken together, the high intra-host and intra-species diversities of LChV1 might affect its pathogenicity and have clear implications for its accurate diagnostics. Full article

In situ hybridization (ISH) is a technique to determine potential correlations between viruses and lesions. The aim of the study was to compare ISH techniques for the detection of various viruses in different tissues. Tested RNA viruses include atypical porcine pestivirus (APPV) in the cerebellum of pigs, equine and bovine hepacivirus (EqHV, BovHepV) in the liver of horses and cattle, respectively, and Schmallenberg virus (SBV) in the cerebrum of goats. Examined DNA viruses comprise canine bocavirus 2 (CBoV-2) in the intestine of dogs, porcine bocavirus (PBoV) in the spinal cord of pigs and porcine circovirus 2 (PCV-2) in cerebrum, lymph node, and lung of pigs. ISH with self-designed digoxigenin-labelled RNA probes revealed a positive signal for SBV, CBoV-2, and PCV-2, whereas it was lacking for APPV, BovHepV, EqHV, and PBoV. Commercially produced digoxigenin-labelled DNA probes detected CBoV-2 and PCV-2, but failed to detect PBoV. ISH with a commercially available fluorescent ISH (FISH)-RNA probe mix identified nucleic acids of all tested viruses. The detection rate and the cell-associated positive area using the FISH-RNA probe mix was highest compared to the results using other probes and protocols, representing a major benefit of this method. Nevertheless, there are differences in costs and procedure time. Full article

Human cytomegalovirus (HCMV) is a β-herpes virus that is a significant pathogen within immune compromised populations. HCMV morbidity is induced through viral dissemination and inflammation. Typically, viral dissemination is thought to follow Fenner’s hypothesis where virus replicates at the site of infection, followed by replication in the draining lymph nodes, and eventually replicating within blood filtering organs. Although CMVs somewhat follow Fenner’s hypothesis, they deviate from it by spreading primarily through innate immune cells as opposed to cell-free virus. Also, in vivo CMVs infect new cells via cell-to-cell spread and disseminate directly to secondary organs through novel mechanisms. We review the historic and recent literature pointing to CMV’s direct dissemination to secondary organs and the genes that it has evolved for increasing its ability to disseminate. We also highlight aspects of CMV infection for studying viral dissemination when using in vivo animal models. Full article

Porcine respiratory disease is a multifactorial disease that can be influenced by a number of different microorganisms, as well as by non-infectious factors such as the management and environment of the animals. It is generally believed that the interaction between different infectious agents plays an important role in regard to respiratory diseases. Therefore, we used high-throughput sequencing combined with viral metagenomics to characterise the viral community of tonsil samples from pigs coming from a conventional herd with lesions in the respiratory tract at slaughter. In parallel, samples from specific pathogen-free pigs were also analysed. This study showed a variable co-infection rate in the different pigs. The differences were not seen at the group level but in individual pigs. Some viruses such as adenoviruses and certain picornaviruses could be found in most pigs, while others such as different parvoviruses and anelloviruses were only identified in a few pigs. In addition, the complete coding region of porcine parvovirus 7 was obtained, as were the complete genomes of two teschoviruses. The results from this study will aid in elucidating which viruses are circulating in both healthy pigs and in pigs associated with respiratory illness. This knowledge is needed for future investigations into the role of viral-viral interactions in relation to disease development. Full article

Crimean-Congo hemorrhagic fever virus is one the most important and wide spread tick-borne viruses. Very little is known about the transmission from the tick and the early aspects of pathogenesis. Here, we generate human cutaneous antigen presenting cells—dermal dendritic cells and Langerhans cells—from umbilical cord progenitor cells. In order to mimic the environment created during tick feeding, tick salivary gland extract was generated from semi-engorged Hyalomma marginatum ticks. Our findings indicate that human dermal dendritic cells and Langerhans cells are susceptible and permissive to Crimean-Congo hemorrhagic fever virus infection, however, to different degrees. Infection leads to cell activation and cytokine/chemokine secretion, although these responses vary between the different cell types. Hyalomma marginatum salivary gland extract had minimal effect on cell responses, with some synergy with viral infection with respect to cytokine secretion. However, salivary gland extract appeared to inhibit antigen presenting cells (APCs) migration. Based on the findings here we hypothesize that human dermal dendritic cells and Langerhans cells serve as early target cells. Rather affecting Crimean-Congo hemorrhagic fever virus replication, tick saliva likely immunomodulates and inhibits migration of these APCs from the feeding site. Full article

Dental caries is a common disease caused by oral bacteria. Streptococcus mutans and Streptococcus sobrinus are the primary cariogenic microbes that often survive as biofilms on teeth. In this study, we evaluated the activity of ClyR, a well-known chimeric lysin with extended streptococcal host range, against common Gram-positive oral microbes and its anticaries efficacy in rat models. ClyR demonstrated high lytic activity against S. mutans MT8148 and S. sobrinus ATCC6715, with minor activity against Streptococcus sanguinis, Streptococcus oralis, and Streptococcus salivarius, which are considered as harmless commensal oral bacteria. Confocal laser scanning microscopy showed that the number of viable cells in 72-h aged S. mutans and S. sobrinus biofilms are significantly (p < 0.05) decreased after treatment with 50 µg/mL ClyR for 5 min. Furthermore, continuous administration of ClyR for 40 days (5 µg/day) significantly (p < 0.05) reduced the severity of caries in rat models infected with a single or a mixed bacteria of S. mutans and S. sobrinus. Therefore, ClyR could be a promising agent or additive for the prevention and treatment of dental caries. Full article

Differential diagnosis of the Zika virus (ZIKV) is hampered by cross-reactivity with other flaviviruses, mainly dengue viruses. The aim of this study was to compare two commercial methods for detecting ZIKV immunoglobulin M (IgM), an indirect immunofluorescence (IIF) and an enzyme immunoassay (ELISA), using the non-structural (NS) 1 protein as an antigen, both from EuroImmun, Germany. In total, 255 serum samples were analyzed, 203 of which showed laboratory markers of ZIKV infections (PCR-positive in serum and/or in urine and/or positive or indeterminate specific IgM). When tested with IIF, 163 samples were IgM-positive, while 13 samples were indeterminate and 78 were negative. When IIF-positive samples were tested using ELISA, we found 61 positive results, 14 indeterminate results, and 88 negative results. Among the indeterminate cases tested with IIF, ELISA analysis found two positive, two indeterminate, and nine negative results. Finally, 74 of the 78 IIF-negative samples proved also to be negative using ELISA. For the calculations, all indeterminate results were considered to be positive. The agreement, sensitivity, and specificity between ELISA and IIF were 60.2%, 44.9%, and 94.9%, respectively. Overall, 101 samples showed discrepant results; these samples were finally classified on the basis of other ZIKV diagnostic approaches (PCR-positive in serum and/or in urine, IgG determinations using IIF or ELISA, and ZIKV Plaque Reduction Neutralization test—positive), when available. A final classification of 228 samples was possible; 126 of them were positive and 102 were negative. The corresponding values of agreement, sensitivity, and specificity of IIF were 86.0%, 96.8%, and 72.5%, respectively. The corresponding figures for ELISA were 81.1%, 65.9%, and 100%, respectively. The ELISA and IIF methods are both adequate approaches for detecting ZIKV-specific IgM. However, considering their respective weaknesses (low sensitivity in ELISA and low specificity in IIF), serological results must be considered jointly with other laboratory results. Full article

Felis catus gammaherpesvirus 1 (FcaGHV1) is a widely endemic infection of domestic cats. Current epidemiological data identify domestic cats as the sole natural host for FcaGHV1. The Tsushima leopard cat (TLC; Prionailurus bengalensis euptilurus) is a critically endangered species that lives only on Tsushima Island, Nagasaki, Japan. Nested PCR was used to test the blood or spleen of 89 TLCs for FcaGHV1 DNA; three (3.37%; 95% CI, 0.70–9.54) were positive. For TLC management purposes, we also screened domestic cats and the virus was detected in 13.02% (95% CI, 8.83–18.27) of 215 cats. Regarding phylogeny, the partial sequences of FcaGHV1 from domestic cats and TLCs formed one cluster, indicating that similar strains circulate in both populations. In domestic cats, we found no significant difference in FcaGHV1 detection in feline immunodeficiency virus-infected (p = 0.080) or feline leukemia virus-infected (p = 0.163) cats, but males were significantly more likely to be FcaGHV1 positive (odds ratio, 5.86; 95% CI, 2.27–15.14) than females. The higher frequency of FcaGHV1 detection in domestic cats than TLCs, and the location of the viral DNA sequences from both cats within the same genetic cluster suggests that virus transmission from domestic cats to TLCs is likely. Full article

The antibiotic-resistant bacterium Paenibacillus larvae is the causative agent of American foulbrood (AFB), currently the most destructive bacterial disease in honeybees. Phages that infect P. larvae were isolated as early as the 1950s, but it is only in recent years that P. larvae phage genomes have been sequenced and annotated. In this study we analyze the genomes of all 48 currently sequenced P. larvae phage genomes and classify them into four clusters and a singleton. The majority of P. larvae phage genomes are in the 38–45 kbp range and use the cohesive ends (cos) DNA-packaging strategy, while a minority have genomes in the 50–55 kbp range that use the direct terminal repeat (DTR) DNA-packaging strategy. The DTR phages form a distinct cluster, while the cos phages form three clusters and a singleton. Putative functions were identified for about half of all phage proteins. Structural and assembly proteins are located at the front of the genome and tend to be conserved within clusters, whereas regulatory and replication proteins are located in the middle and rear of the genome and are not conserved, even within clusters. All P. larvae phage genomes contain a conserved N-acetylmuramoyl-l-alanine amidase that serves as an endolysin. Full article

Emergence of human immunodeficiency virus type 1 (HIV-1) drug resistance arises from mutation fixation in the viral genome during antiretroviral therapy. Primary mutations directly confer antiviral drug resistance, while secondary mutations arise that do not confer drug resistance. The A62V amino acid substitution in HIV-1 reverse transcriptase (RT) was observed to be associated with multi-drug resistance, but is not known to be a resistance-conferring mutation. In particular, A62V was observed in various multi-dideoxynucleoside resistant (MDR) mutation complexes, including the Q151M complex (i.e., A62V, V75I, F77L, F116Y, and Q151M), and the T69SSS insertion complex, which has a serine–serine insertion between amino acid positions 69 and 70 (i.e., M41L, A62V, T69SSS, K70R, and T215Y). However, what selective advantage is conferred to the virus remains unresolved. In this study, we hypothesized that A62V could influence replication fidelity and viral fitness with viruses harboring the Q151M and T69SSS MDR mutation complexes. A single-cycle replication assay and a dual-competition fitness assay were used to assess viral mutant frequency and viral fitness, respectively. A62V was found to increase the observed lower mutant frequency identified with each of the viruses harboring the MDR mutation complexes in the single-cycle assay. Furthermore, A62V was observed to improve viral fitness of replication-competent MDR viruses. Taken together, these observations indicate an adaptive role of A62V in virus replication fidelity and viral fitness, which would likely enhance virus persistence during drug-selective pressure. Full article

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (MTB) and transmitted through inhalation of aerosolized droplets. Eighty-five percent of new TB cases occur in resource-limited countries in Asia and Africa and fewer than 40% of TB cases are diagnosed due to the lack of accurate and easy-to-use diagnostic assays. Currently, diagnosis relies on the demonstration of the bacterium in clinical specimens by serial sputum smear microscopy and culture. These methods lack sensitivity, are time consuming, expensive, and require trained personnel. An alternative approach is to develop an efficient immunoassay to detect antibodies reactive to MTB antigens in bodily fluids, such as serum. Sarcoidosis and TB have clinical and pathological similarities and sarcoidosis tissue has yielded MTB components. Using sarcoidosis tissue, we developed a T7 phage cDNA library and constructed a microarray platform. We immunoscreened our microarray platform with sera from healthy (n = 45), smear positive TB (n = 24), and sarcoidosis (n = 107) subjects. Using a student t-test, we identified 192 clones significantly differentially expressed between the three groups at a False Discovery Rate (FDR) <0.01. Among those clones, we selected the top ten most significant clones and validated them on independent test set. The area under receiver operating characteristics (ROC) for the top 10 significant clones was 1 with a sensitivity of 1 and a specificity of 1. Sequence analyses of informative phage inserts recognized as antigens by active TB sera may identify immunogenic antigens that could be used to develop therapeutic or prophylactic vaccines, as well as identify molecular targets for therapy. Full article

Bacteriophages have been used as molecular tools in fundamental biology investigations for decades. Beyond this, however, they play a crucial role in the eco-evolutionary dynamics of bacterial communities through their demographic impact and the source of genetic information they represent. The increasing interest in describing ecological and evolutionary aspects of bacteria–phage interactions has led to major insights into their fundamental characteristics, including arms race dynamics and acquired bacterial immunity. Here, we review knowledge on the phages of the myxobacteria with a major focus on phages infecting Myxococcus xanthus, a bacterial model system widely used to study developmental biology and social evolution. In particular, we focus upon the isolation of myxophages from natural sources and describe the morphology and life cycle parameters, as well as the molecular genetics and genomics of the major groups of myxophages. Finally, we propose several interesting research directions which focus on the interplay between myxobacterial host sociality and bacteria–phage interactions. Full article

In a globalized world, the threat of emerging pathogens plays an increasing role, especially if their zoonotic potential is unknown. In this study, a novel respirovirus, family Paramyxoviridae, was isolated from a Sri Lankan Giant squirrel (Ratufa macroura), which originated in Sri Lanka and deceased with severe pneumonia in a German zoo. The full-genome characterization of this novel virus, tentatively named Giant squirrel respirovirus (GSqRV), revealed similarities to murine (71%), as well as human respiroviruses (68%) with unique features, for example, a different genome length and a putative additional accessory protein. Congruently, phylogenetic analyses showed a solitary position of GSqRV between known murine and human respiroviruses, implicating a putative zoonotic potential. A tailored real-time reverse transcription-polymerase chain reaction (RT-qPCR) for specific detection of GSqRV confirmed a very high viral load in the lung, and, to a lesser extent, in the brain of the deceased animal. A pilot study on indigenous and exotic squirrels did not reveal additional cases in Germany. Therefore, further research is essential to assess the geographic distribution, host range, and zoonotic potential of this novel viral pathogen. Full article

While amphibians around the globe are facing catastrophic declines, in part because of infections with pathogens such as the Frog Virus 3 (FV3) ranavirus; the mechanisms governing amphibian susceptibility and resistance to such pathogens remain poorly understood. The type I and type III interferon (IFN) cytokines represent a cornerstone of vertebrate antiviral immunity, while our recent work indicates that tadpoles and adult frogs of the amphibian Xenopus laevis may differ in their IFN responses to FV3. In this respect, it is notable that anuran (frogs and toads) tadpoles are significantly more susceptible to FV3 than adult frogs, and thus, gaining greater insight into the differences in the tadpole and adult frog antiviral immunity would be invaluable. Accordingly, we examined the FV3-elicited expression of a panel of type I and type III IFN genes in the skin (site of FV3 infection) and kidney (principal FV3 target) tissues and isolated cells of X. laevis tadpoles and adult frogs. We also examined the consequence of tadpole and adult frog skin and kidney cell stimulation with hallmark pathogen-associated molecular patterns (PAMPs) on the IFN responses of these cells. Together, our findings indicate that tadpoles and adult frogs mount drastically distinct IFN responses to FV3 as well as to viral and non-viral PAMPs, while these expression differences do not appear to be the result of a distinct pattern recognition receptor expression by tadpoles and adults. Full article

Interferon (IFN)-induced 2′-5′-oligoadenylate synthetase (OAS) proteins exhibit an extensive and efficient antiviral effect against flavivirus infection in mammals and birds. Only the 2′-5′-oligoadenylate synthetase-like (OASL) gene has been identified thus far in birds, except for ostrich, which has both OAS1 and OASL genes. In this study, we first investigated the antiviral activity of goose OASL (goOASL) protein against a duck-origin Tembusu virus (DTMUV) in duck embryo fibroblast cells (DEFs). To investigate the relationship of conserved amino acids that are related to OAS enzyme activity and ubiquitin-like (UBL) domains with the antiviral activity of goOASL, a series of mutant goOASL plasmids was constructed, including goOASL-S64C/D76E/D78E/D144T, goOASL∆UBLs and goOASL∆UBLs-S64C/D76E/D78E/D144T. Interestingly, all these mutant proteins significantly inhibited the replication of DTMUV in DEFs in a dose-dependent manner. Immunofluorescence analysis showed that the goOASL, goOASL-S64C/D76E/D78E/D144T, goOASL∆UBLs and goOASL∆UBLs-S64C/D76E/D78E/D144T proteins were located not only in the cytoplasm where DTMUV replicates but also in the nucleus of DEFs. However, the goOASL and goOASL mutant proteins were mainly colocalized with DTMUV in the cytoplasm of infected cells. Our data indicated that goOASL could significantly inhibit DTMUV replication in vitro, while the active-site residues S64, D76, D78 and D144, which were associated with OAS enzyme activity, the UBL domains were not required for the antiviral activity of goOASL protein. Full article

Densovirus genome replication and capsid assembly take place in the nucleus of the infected cells. However, the mechanisms underlying such processes as the delivery of virus proteins to the nucleus and the export of progeny virus from the nucleus remain elusive. It is evident that nuclear transport signals should be involved in these processes. We performed an in silico search for the putative nuclear localization signal (NLS) and nuclear export signal (NES) motifs in the capsid proteins of the Blattella germanica Densovirus 1 (BgDV1) densovirus. A high probability NLS motif was found in the common C-terminal of capsid proteins together with a NES motif in the unique N-terminal of VP2. We also performed a global search for the nuclear traffic signals in the densoviruses belonging to five Densovirinae genera, which revealed high diversity in the patterns of NLSs and NESs. Using a heterologous system, the HeLa mammalian cell line expressing GFP-fused BgDV1 capsid proteins, we demonstrated that both signals are functionally active. We suggest that the NLS shared by all three BgDV1 capsid proteins drives the trafficking of the newly-synthesized proteins into the nucleus, while the NES may play a role in the export of the newly-assembled BgDV1 particles into the cytoplasm through nuclear pore complexes. Full article

Five isolates of a new member of the family Closteroviridae, tentatively named blackcurrant leafroll-associated virus 1 (BcLRaV-1), were identified in the currant. The 17-kb-long genome codes for 10 putative proteins. The replication-associated polyprotein has several functional domains, including papain-like proteases, methyltransferase, Zemlya, helicase, and RNA-dependent RNA polymerase. Additional open reading frames code for a small protein predicted to integrate into the host cell wall, a heat-shock protein 70 homolog, a heat-shock protein 90 homolog, two coat proteins, and three proteins of unknown functions. Phylogenetic analysis showed that BcLRaV-1 is related to members of the genus Closterovirus, whereas recombination analysis provided evidence of intraspecies recombination. Full article

The Zika virus (ZIKV) has recently attracted major research interest as infection was unexpectedly associated with neurological manifestations in developing foetuses and with Guillain-Barré syndrome in infected adults. Understanding the underlying molecular mechanisms requires reverse genetic systems, which allow manipulation of infectious cDNA clones at will. In the case of flaviviruses, to which ZIKV belongs, several reports have indicated that the construction of full-length cDNA clones is difficult due to toxicity during plasmid amplification in Escherichia coli. Toxicity of flaviviral cDNAs has been linked to the activity of cryptic prokaryotic promoters within the region encoding the structural proteins leading to spurious transcription and expression of toxic viral proteins. Here, we employ an approach based on in silico prediction and mutational silencing of putative promoters to generate full-length cDNA clones of the historical MR766 strain and the contemporary French Polynesian strain H/PF/2013 of ZIKV. While for both strains construction of full-length cDNA clones has failed in the past, we show that our approach generates cDNA clones that are stable on single bacterial plasmids and give rise to infectious viruses with properties similar to those generated by other more complex assembly strategies. Further, we generate luciferase and fluorescent reporter viruses as well as sub-genomic replicons that are fully functional and suitable for various research and drug screening applications. Taken together, this study confirms that in silico prediction and silencing of cryptic prokaryotic promoters is an efficient strategy to generate full-length cDNA clones of flaviviruses and reports novel tools that will facilitate research on ZIKV biology and development of antiviral strategies. Full article

Several medically important flaviviruses that are transmitted by mosquitoes have been shown to bind to the C-type lectin fold that is present in either vertebrate or invertebrate proteins. While in some cases this interaction is part of a neutralizing anti-viral immune response, many reports have implicated this as critical for successful virus entry. Despite the establishment of mosquito C-type lectin domain containing proteins (CTLDcps) as known host factors in assisting the infectious process for flaviviruses, little is known about the structural characteristics of these proteins and their relationships to each other. In this report, we describe the manual annotation and structural characterization of 52 Aedes aegypti CTLDcps. Using existing RNAseq data, we establish that these genes can be subdivided into two classes: those highly conserved with expression primarily in development (embryo/early larvae) and those with no clear orthologs with expression primarily in late larvae/pupae or adults. The latter group contained all CTLDcps that are regulated by the Toll/Imd immune pathways, all known microbiome-regulating CTLDcps, and almost all CTLDcps that are implicated as flavivirus host factors in A. aegypti. Finally, we attempt to synthesize results from multiple conflicting gene expression profiling experiments in terms of how flavivirus infection changes steady-state levels of mRNA encoding CTLDcps. Full article

The Baculoviridae, a family of insect-specific large DNA viruses, is widely used in both biotechnology and biological control. Its applied value stems from millions of years of evolution influenced by interactions with their hosts and the environment. To understand how ecological interactions have shaped baculovirus diversification, we reconstructed a robust molecular phylogeny using 217 complete genomes and ~580 isolates for which at least one of four lepidopteran core genes was available. We then used a phylogenetic-concept-based approach (mPTP) to delimit 165 baculovirus species, including 38 species derived from new genetic data. Phylogenetic optimization of ecological characters revealed a general pattern of host conservatism punctuated by occasional shifts between closely related hosts and major shifts between lepidopteran superfamilies. Moreover, we found significant phylogenetic conservatism between baculoviruses and the type of plant growth (woody or herbaceous) associated with their insect hosts. In addition, we found that colonization of new ecological niches sometimes led to viral radiation. These macroevolutionary patterns show that besides selection during the infection process, baculovirus diversification was influenced by tritrophic interactions, explained by their persistence on plants and interactions in the midgut during horizontal transmission. This complete eco-evolutionary framework highlights the potential innovations that could still be harnessed from the diversity of baculoviruses. Full article

The baculovirus-insect cell expression system has been widely used for heterologous protein expression and virus-like particles (VLPs) expression. In this study, we established a new method for antiviral screening targeting to glycoprotein E of flaviviruses based on the baculovirus expression system. ZIKV is a mosquito-borne flavivirus and has posed great threat to the public health. It has been reported that ZIKV infection was associated with microcephaly and serious neurological complications. Our study showed that either ZIKV E or prME protein expressed in insect cells can form VLPs and induce membrane fusion between insect cells. Therefore, the E protein, which is responsible for receptor binding, attachment, and virus fusion during viral entry, achieved proper folding and retained its fusogenic ability in VLPs when expressed in this system. The syncytia in insect cells were significantly reduced by the anti-ZIKV-E specific polyclonal antibody in a dose-dependent manner. AMS, a thiol-conjugating reagent, was also shown to have an inhibitory effect on the E protein induced syncytia and inhibited ZIKV infection by blocking viral entry. Indeed the phenomenon of syncytial formation induced by E protein expressed VLPs in insect cells is common among flaviviruses, including Japanese encephalitis virus (JEV), Dengue virus type 2 (DENV-2), and tick-borne encephalitis virus (TBEV). This inhibition effect on syncytial formation can be developed as a novel, safe, and simple antiviral screening approach for inhibitory antibodies, peptides, or small molecules targeting to E protein of ZIKV and other flaviviruses. Full article

Foot-and-mouth disease (FMD) is a highly contagious disease that results in enormous economic loses worldwide. Although the protection provided by vaccination is limited during early infection, it is recognized as the best method to prevent FMD outbreaks. Furthermore, the mechanism of host early responses against foot-and-mouth disease virus (FMDV) infection remains unclear. In our study, a pig kidney cell line (PK-15) was used as a cell model to reveal the mechanism of early pig responses to FMDV infection. Four non-treated control and four FMDV-treated PK-15 cells were sequenced with RNA-seq technology, and the differentially expressed genes (DEGs) were analyzed. The results showed that 1212 DEGs were in the FMDV-infected PK-15 cells, including 914 up-regulated and 298 down-regulated genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were significantly enriched in the tumor necrosis factor (TNF), cytokine-cytokine receptor interaction, NOD-like receptor, toll-like receptor, NF-κB, and the chemokine signaling pathways. To verify the results of the DEGs, 30 immune-related DEGs (19 up-regulated and 11 down-regulated) were selected for Quantitative Reverse Transcriptase polymerase chain reaction (RT-qPCR) verification. The results showed that RT-qPCR-measured genes exhibited a similar pattern as the RNA-seq analyses. Based on bioinformatics analysis, during FMDV early infection, we found that a series of cytokines, such as interleukins (IL6), chemokines (CXCL2, CCL20 and CCL4), and transcription factors (ZFP36, FOS, NFKBIA, ZBTB3, ZNF503, ZNF283, dymeclin (DYM), and orthodenticle homeobox 1 (OTX1)) were involved in the battle between FMDV and the host. Combined with their features and functions, we propose inflammation as the main early mechanism by which the host responds to FMDV infection. These data provide an additional panel of candidate genes for deciphering the mechanisms of a host’s early response against FMDV infection. Full article

Chronic HBV + HDV infection is associated with greater risk of liver fibrosis, earlier hepatic decompensation, and liver cirrhosis hepatocellular carcinoma compared to HBV mono-infection. However, to-date no direct anti-HDV drugs are available in clinical practice. Here, we identified conserved and variable regions in HBsAg and HDAg domains in HBV + HDV infection, a critical finding for the design of innovative therapeutic agents. The extent of amino-acid variability was measured by Shannon-Entropy (Sn) in HBsAg genotype-d sequences from 31 HBV + HDV infected and 62 HBV mono-infected patients (comparable for demographics and virological-parameters), and in 47 HDAg genotype-1 sequences. Positions with Sn = 0 were defined as conserved. The percentage of conserved HBsAg-positions was significantly higher in HBV + HDV infection than HBV mono-infection (p = 0.001). Results were confirmed after stratification for HBeAg-status and patients’ age. A Sn = 0 at specific positions in the C-terminus HBsAg were correlated with higher HDV-RNA, suggesting that conservation of these positions can preserve HDV-fitness. Conversely, HDAg was characterized by a lower percentage of conserved-residues than HBsAg (p < 0.001), indicating higher functional plasticity. Furthermore, specific HDAg-mutations were significantly correlated with higher HDV-RNA, suggesting a role in conferring HDV replicative-advantage. Among HDAg-domains, only the virus-assembly signal exhibited a high genetic conservation (75% of conserved-residues). In conclusion, HDV can constrain HBsAg genetic evolution to preserve its fitness. The identification of conserved regions in HDAg poses the basis for designing innovative targets against HDV-infection. Full article

Tick-borne viral diseases continue to emerge in the United States, as clearly evident from the increase in Powassan encephalitis virus, Heartland virus, and Bourbon virus infections. Tick-borne flaviviruses (TBFVs) are transmitted to the mammalian host along with the infected tick saliva during blood-feeding. Successful tick feeding is facilitated by a complex repertoire of pharmacologically active salivary proteins/factors in tick saliva. These salivary factors create an immunologically privileged micro-environment in the host’s skin that influences virus transmission and pathogenesis. In this review, we will highlight tick determinants of TBFV transmission with a special emphasis on tick–virus–host interactions at the cutaneous interface. Full article

The Tembusu virus (TMUV) is an avian pathogenic flavivirus that causes a highly contagious disease and catastrophic losses to the poultry industry. The myxovirus resistance protein (Mx) of innate immune effectors is a key antiviral “workhorse” of the interferon (IFN) system. Although mammalian Mx resistance against myxovirus and retrovirus was witnessed for decades, whether or not bird Mx has anti-flavivirus activity remains unknown. In this study, we found that the transcription of goose Mx (goMx) was obviously driven by TMUV infection, both in vivo and in vitro, and that the titers and copies of TMUV were significantly reduced by goMx overexpression. In both primary (goose embryo fibroblasts, GEFs) and passaged cells (baby hamster kidney cells, BHK21, and human fetal kidney cells, HEK 293T), it was shown that goMx was mainly located in the cytoplasm, and sporadically distributed in the nucleus. The intracellular localization of this protein is attributed to the predicted bipartite nuclear localization signal (NLS; 30 residues: the 441st–471st amino acids of goMx). Intuitively, it seems that the cells with a higher level of goMx expression tend to have lower TMUV loads in the cytoplasm, as determined by an immunofluorescence assay. To further explore the antiviral determinants, a panel of variants was constructed. Two amino acids at the 125th (Lys) and 145th (Thr) positions in GTP-binding elements, not in the L4 loop (40 residues: the 532nd–572nd amino acids of goMx), were vital for the antiviral function of goMx against TMUV in vitro. These findings will contribute to our understanding of the functional significance of the antiviral system in aquatic birds, and the development of goMx could be a valuable therapeutic agent against TMUV. Full article

Herpes simplex virus (HSV) causes numerous mild-to-serious human diseases, including mucocutaneous herpes infections and life-threatening herpes encephalitis. Moreover, herpes viral lesions can be complicated by inflammation and secondary bacterial infections. The development of resistance to antiviral drugs along with the undesirable side effects of these drugs are relevant argue for the development of new anti-HSV drugs with diverse mechanisms of action. Eucalyptus extracts have been used for decades to combat various infectious diseases. We isolated and studied 12 pure compounds and one mixture of two constitutional isomers from the leaves and twigs of E. globulus. The structures were identified by spectroscopic methods (NMR, HR-MS, IR) and all of them were tested for antiherpetic activity against the replication of antigen types HSV-1 and HSV-2. Tereticornate A (12) (IC₅₀: 0.96 μg/mL; selectivity index CC₅₀/IC₅₀: 218.8) showed the strongest activity in the anti-HSV-1 assay, even greater than acyclovir (IC₅₀: 1.92 μg/mL; selectivity index CC₅₀/IC₅₀: 109.4), a standard antiviral drug. Cypellocarpin C (5) (EC₅₀: 0.73 μg/mL; selectivity index CC₅₀/EC₅₀: 287.7) showed the most potent anti-HSV-2 activity, also more intensive than acyclovir (EC₅₀: 1.75 μg/mL; selectivity index CC₅₀/EC₅₀: 120.0). The antimicrobial activity of the isolated compounds was also evaluated against the bacteria Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Pseudomonas aeruginosa and the yeast Candida albicans. The anti-inflammatory potential was examined using LPS-stimulated THP-1-XBlue™-MD2-CD14 and THP-1 macrophages and focusing on the influences of the NF-κB/AP-1 activity and the secretion of pro-inflammatory cytokines IL-1β and TNF-α. Full article