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Diagnostics of Animal Viral Infectious Diseases

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A special issue of Pathogens (ISSN 2076-0817). This special issue belongs to the section "Viral Pathogens".

Deadline for manuscript submissions: 1 June 2024 | Viewed by 11486

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Special Issue Editors

Dr. Virginia Friedrichs

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Guest Editor

Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Suedufer 10, Insel Riems, 17493 Greifswald, Germany
Interests: virology; immunology; diagnostics

Dr. Tessa Carrau

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Guest Editor

Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Suedufer 10, Insel Riems, 17493 Greifswald, Germany
Interests: virology; entomology; diagnostics

Special Issue Information

Dear Colleagues,

Animal viral diseases play a major role in animal health and can lead to suffering, death, and economic losses worldwide, threatening global health as well as food security. Their correct and early diagnosis allows local and national authorities to rapidly react and control the outbreak as well as spread of diseases. High sensitivity and specificity are required features of any diagnostic tool. To this end, it is of the utmost importance to establish and validate novel diagnostic strategies which, in the long run, will allow better treatment and prevention of animal viral diseases.

The diagnosis of viral infectious diseases can be performed by the direct and/or indirect detection of infectious agents. Usually, direct detection methods seek the presence of a virus. However, there are plenty of methods: diagnoses via polymerase chain reaction (PCR), isothermal amplification, or loop-mediated isothermal amplification (LAMP) are widely established in many laboratories. Direct diagnostic assays can either be based on the amplification and detection of viral nucleic acids or on the isolation and/or replication of the causative agent, hence offering relatively accurate disease confirmation. Indirect diagnostic methods observe the effects of a virus in an infected animal or tissue, such as cell death or the production of antibodies by an infected individual.

I would like to invite colleagues investigating or validating novel direct or indirect diagnostic approaches for virus infection detection. Any of those working within the areas of genetics, virology, genomics, immunology, detection, and public health development are welcome to submit their manuscripts to this Special Issue in the form of original research and reviews.

Dr. Virginia Friedrichs
Dr. Tessa Carrau
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Pathogens is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

diagnostics
laboratory diagnosis
performance
sensitivity
specificity
virology

Published Papers (7 papers)

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Research

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10 pages, 217 KiB

Open AccessArticle

Development of a Targeted NGS Assay for the Detection of Respiratory Pathogens including SARS-CoV-2 in Felines

by Jobin J. Kattoor, Mothomang Mlalazi-Oyinloye, Sarah M. Nemser and Rebecca P. Wilkes

Pathogens 2024, 13(4), 335; https://doi.org/10.3390/pathogens13040335 - 17 Apr 2024

Viewed by 866

Abstract

Acute respiratory diseases in felines can be attributed to a diverse range of pathogens. The recent emergence of novel viruses, particularly SARS-CoV-2 and its variants, has also been associated with respiratory ailments in cats and other pets, underscoring the need for a highly sensitive diagnostic assay capable of concurrently detecting multiple respiratory pathogens. In this study, we developed a targeted next generation sequencing panel using Ion Torrent Ampliseq technology to detect multiple respiratory pathogens, including recent SARS-CoV-2 variants and Feline herpesvirus-1, Feline calicivirus, Bordetella bronchiseptica, Mycoplasmopsis (previously Mycoplasma) felis, and Chlamydia felis. A PCR amplification-based library preparation, employing primers designed for pathogen target regions, was synthesized and divided into two pools, followed by sequencing and assembly to a repertoire of target pathogen genomes. Analytical sensitivity was assessed based on Ct values from real-time PCR for the corresponding pathogens, indicating an equivalent detection limit. Most of the pathogens under study were positively identified to a limit of approximately Ct 36, whereas for Feline herpesvirus-1 and SARS-CoV-2, positive reads were observed in samples with a Ct of 37. Based on a limited number of samples, the diagnostic sensitivity values for the SARS-CoV-2, Feline herpesvirus-1, and M. felis samples were 100% with no false negative results. The diagnostic specificity of SARS-CoV-2, Feline herpesvirus-1, Feline calicivirus, and C. felis were 100%. Importantly, none of the target primers exhibited non-specific amplification, ensuring the absence of false positive results for other pathogens within the study. Additionally, the assay’s specificity was validated by cross-referencing the raw sequencing data with established databases like BLAST, affirming the high specificity of the targeted Next-Generation Sequencing (tNGS) assay. Variations in the sequencing reads of different pathogens were observed when subjected to diverse extraction methods. Rigorous assessment of the assay’s reliability involved reproducibility across testing personnel and repeated runs. The developed assay’s clinical applicability was tested using samples submitted to the diagnostic laboratory from cat shelters and suspected cases. The developed targeted next-generation sequencing methodology empowers the detection of multiple respiratory pathogens manifesting similar clinical symptoms while offering confirmation of results through genome sequencing. Full article

(This article belongs to the Special Issue Diagnostics of Animal Viral Infectious Diseases)

14 pages, 702 KiB

Open AccessArticle

BoLA-DRB3 Polymorphism Associated with Bovine Leukemia Virus Infection and Proviral Load in Holstein Cattle in Egypt

by Rania Hamada, Samy Metwally, Ryosuke Matsuura, Liushiqi Borjigin, Chieh-Wen Lo, Alsagher O. Ali, Adel E. A. Mohamed, Satoshi Wada and Yoko Aida

Pathogens 2023, 12(12), 1451; https://doi.org/10.3390/pathogens12121451 - 14 Dec 2023

Viewed by 1589

Abstract

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis, the most prevalent neoplastic disease of cattle worldwide. The immune response to BLV and disease susceptibility and resistance in cattle are strongly correlated with the bovine leukocyte antigen (BoLA)-DRB3 allelic polymorphism. BLV infection continues to spread in Egypt, in part because the relationships between BLV infection, proviral load in Egypt, and BoLA-DRB3 polymorphism are unknown. Here, we identified 18 previously reported alleles in 121 Holstein cows using a polymerase chain reaction sequence-based typing method. Furthermore, BoLA-DRB3 gene polymorphisms in these animals were investigated for their influence on viral infection. BoLA-DRB3*015:01 and BoLA-DRB3*010:01 were identified as susceptible and resistant alleles, respectively, for BLV infection in the tested Holsteins. In addition, BoLA-DRB3*012:01 was associated with low PVL in previous reports but high PVL in Holstein cattle in Egypt. This study is the first to demonstrate that the BoLA-DRB3 polymorphism confers resistance and susceptibility to PVL and infections of BLV in Holstein cattle in Egypt. Our results can be useful for the disease control and eradication of BLV through genetic selection. Full article

(This article belongs to the Special Issue Diagnostics of Animal Viral Infectious Diseases)

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15 pages, 1636 KiB

Open AccessEditor’s ChoiceArticle

Characterization of mRNA Signature in Milk Small Extracellular Vesicles from Cattle Infected with Bovine Leukemia Virus

by Md. Matiur Rahman, Hinata Ishikawa, Marika Yamauchi, Shigeo Takashima, Yuji O. Kamatari, Kaori Shimizu, Ayaka Okada and Yasuo Inoshima

Pathogens 2023, 12(10), 1239; https://doi.org/10.3390/pathogens12101239 - 13 Oct 2023

Cited by 1 | Viewed by 2304

Abstract

This study aimed to characterize the mRNA signature of milk small extracellular vesicles (sEVs) from BLV-infected cattle. A total of 23 mRNAs, which showed greater abundance in milk sEVs from BLV-infected cattle compared to those from BLV-uninfected (control) cattle, were identified through microarray analyses conducted in our previous study. To assess the significance of these differences in mRNA abundance, milk was collected from six control cattle and twenty-six cattle infected with BLV. The infected cattle were categorized into two distinct groups based on their proviral loads: a group of eight cattle with low proviral loads (LPVL), characterized by <10,000 copies per 10⁵ white blood cells (WBC), and a group of eighteen cattle with high proviral loads (HPVL), marked by ≥10,000 copies per 10⁵ WBC. The qPCR analysis quantified 7 out of 23 mRNAs, including BoLA, CALB1, IL33, ITGB2, MYOF, TGFBR1, and TMEM156, in the milk sEVs from control cattle, LPVL cattle, and HPVL cattle. Significantly, the average relative expression of CALB1 mRNA in milk sEVs was higher in LPVL cattle compared to HPVL cattle and control cattle (p < 0.05), while it was relatively lower in HPVL cattle compared to LPVL cattle and control cattle (p > 0.05). Likewise, the average relative expression of TMEM156 mRNA in milk sEVs was significantly higher in LPVL cattle compared to HPVL cattle (p < 0.05), and relatively lower in HPVL cattle compared to LPVL cattle and control cattle (p > 0.05). The results indicate distinct patterns of CALB1 and TMEM156 mRNA levels in milk sEVs, with higher levels observed in LPVL cattle and lower levels in HPVL cattle. The current study could provide essential information to comprehend the complexities during the progression of BLV infection and direct the exploration of mRNA biomarkers for monitoring the clinical stage of BLV infection. Full article

(This article belongs to the Special Issue Diagnostics of Animal Viral Infectious Diseases)

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16 pages, 3401 KiB

Open AccessArticle

Designing and Validation of a Droplet Digital PCR Procedure for Diagnosis and Accurate Quantification of Nervous Necrosis Virus in the Mediterranean Area

by Sandra Souto, José G. Olveira, Carmen López-Vázquez, Isabel Bandín and Carlos P. Dopazo

Pathogens 2023, 12(9), 1155; https://doi.org/10.3390/pathogens12091155 - 12 Sep 2023

Viewed by 1147

Abstract

The viral nervous necrosis virus (VNNV) is the causative agent of an important disease affecting fish species cultured worldwide. Early and accurate diagnosis is, at present, the most effective control and prevention tool, and molecular techniques have been strongly introduced and accepted by official organizations. Among those, real-time quantitative polymerase chain reaction (rt-qPCR) is nowadays displacing other molecular techniques. However, another PCR-based technology, droplet digital PCR (ddPCR), is on the increase. It has many advantages over qPCR, such as higher sensitivity and more reliability of the quantification. Therefore, we decided to design and validate a protocol for the diagnosis and quantification of SJ and RG type VNNV using reverse transcription-ddPCR (RT-ddPCR). We obtained an extremely low limit of detection, 10- to 100-fold lower than with RT-qPCR. Quantification by RT-ddPCR, with a dynamic range of 6.8–6.8 × 10⁴ (SJ type) or 1.04 × 10¹–1.04 × 10⁵ (RG type) cps/rctn, was more reliable than with RT-qPCR. The procedure was tested and validated in field samples, providing high clinical sensitivity and negative predictive values. In conclusion, we propose this method to substitute RT-qPCR protocols because it exceeds the expectations of qPCR in the diagnosis and quantification of VNNV. Full article

(This article belongs to the Special Issue Diagnostics of Animal Viral Infectious Diseases)

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14 pages, 7213 KiB

Open AccessArticle

Simple and Rapid Colorimetric Detection of Canine Parainfluenza Virus 5 (Orthorubulavirus mammalis) Using a Reverse-Transcription Loop-Mediated Isothermal Amplification Assay

by Jong-Min Kim, Hye-Ryung Kim, Ji-Su Baek, Oh-Kyu Kwon, Hae-Eun Kang, Yeun-Kyung Shin and Choi-Kyu Park

Pathogens 2023, 12(7), 921; https://doi.org/10.3390/pathogens12070921 - 08 Jul 2023

Viewed by 1212

Abstract

Despite its many advantages, a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay has yet to be developed for canine parainfluenza virus 5 (CPIV5). In this study, a visual RT-LAMP (vRT-LAMP) assay was developed for the rapid detection of CPIV5 in clinical samples. At a constant reaction temperature of 62 °C, the assay was completed within 40 min, and the results could be directly detected with the naked eye using a hydroxynaphthol blue (HNB) metal indicator without any additional detection apparatuses. The assay specifically amplified CPIV5 RNA with a limit of detection of 10 RNA copies/reaction, which was 10-fold more sensitive than the previously reported conventional reverse-transcription polymerase chain reaction (cRT-PCR) assay and was comparable to the previously reported real-time RT-PCR (qRT-PCR) assay. In a clinical evaluation using 267 nasopharyngeal swab samples collected from hospitalized dogs with respiratory symptoms, the CPIV5 detection rate using the vRT-LAMP assay was 5.24% (14/267), which was higher than that of the cRT-PCR assay (4.49%, 12/267) and consistent with that of the qRT-PCR assay, demonstrating 100% concordance with a kappa coefficient value (95% confidence interval) of 1 (1.00–1.00). The discrepancies in the results of the assays were confirmed to be attributed to the low sensitivity of the cRT-PCR assay. Owing to the advantages of a high specificity, rapidity, and simplicity, the developed vRT-LAMP assay using an HNB metal indicator will be a valuable diagnostic tool for the detection of CPIV5 in canine clinical samples, even in resource-limited laboratories. Full article

(This article belongs to the Special Issue Diagnostics of Animal Viral Infectious Diseases)

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11 pages, 2105 KiB

Open AccessArticle

Improving African Swine Fever Surveillance Using Fluorescent Rapid Tests

by Cristina Aira, Alejandro Monedero, Sonia Hernández-Antón, Juan Martínez-Cano, Ana Camuñas, Nadia Casado, Raquel Nieto, Carmina Gallardo, Marga García-Durán, Paloma Rueda and Alba Fresco-Taboada

Pathogens 2023, 12(6), 811; https://doi.org/10.3390/pathogens12060811 - 07 Jun 2023

Cited by 2 | Viewed by 1164

Abstract

African swine fever (ASF) is a viral disease of swine with a huge impact due to its high mortality. Lately, the disease has actively spread around the world, affecting new areas from which it had been eradicated long ago. To date, ASF control is carried out by the implementation of strict biosecurity measures such as the early identification of infected animals. In this work, two fluorescent rapid tests were developed to improve the sensitivity of point-of-care diagnosis of ASF. For antigen (Ag) detection in blood, a double-antibody sandwich fluorescent lateral flow assay (LFA) was developed, employing a newly developed recombinant antibody to the VP72 of the virus. To complement the diagnosis, a double-recognition fluorescent LFA was developed using the VP72 for the detection of specific antibodies (Ab) in sera or blood. Both assays statistically improved the detection of the disease when compared to the commercial colorimetric assays INgezim^® ASFV CROM Ag and INgezim^® PPA CROM Anticuerpo, respectively, with higher statistical significance between 11 and 39 days post-infection. From the observation of results, it can be concluded that the combination of both Ag-LFA and Ab-LFA assays would facilitate the identification of infected animals, regardless of post-infection time. Full article

(This article belongs to the Special Issue Diagnostics of Animal Viral Infectious Diseases)

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11 pages, 3802 KiB

Open AccessCase Report

Feline Parvovirus Lethal Outbreak in a Group of Adult Cohabiting Domestic Cats

by Maria Irene Pacini, Mario Forzan, Giovanni Franzo, Claudia Maria Tucciarone, Milena Fornai, Fabrizio Bertelloni, Micaela Sgorbini, Carlo Cantile and Maurizio Mazzei

Pathogens 2023, 12(6), 822; https://doi.org/10.3390/pathogens12060822 - 11 Jun 2023

Cited by 1 | Viewed by 2515

Abstract

Feline panleukopenia is a highly contagious and often fatal disease in cats. The virus, known as feline panleukopenia virus (FPV), primarily affects kittens and unvaccinated cats. It is transmitted through contact with infected cats or their bodily fluids, as well as contaminated objects and environments. The diagnosis of FPV infection can be confirmed through a combination of clinical signs, blood tests, and fecal testing. Prevention through vaccination is recommended for all cats. This case report describes an outbreak of feline panleukopenia in a group of unvaccinated domestic cats that resulted in acute mortality. The lesions were evaluated using histopathology, and the specific viral strain was characterized using molecular techniques. The clinical course of the outbreak was peracute, with a hemorrhagic pattern and 100% of lethality. The observed clinical-pathological pattern was unusual; nevertheless, molecular studies did not highlight peculiar genomic features of the parvovirus isolate. The outbreak affected 3 out of 12 cats in a very short time. However, the prompt application of biosecurity measures and vaccination resulted in an effective interruption of virus spread. In conclusion, we could assume that the virus found the ideal conditions to infect and replicate at high titers, resulting in a particularly aggressive outbreak. Full article

(This article belongs to the Special Issue Diagnostics of Animal Viral Infectious Diseases)

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