Editorial Board Members’ Collection Series: Molecular Epidemiology, Diagnostics and Management of Viral Pathogens

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

Deadline for manuscript submissions: 31 October 2024 | Viewed by 23758

Special Issue Editors


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Guest Editor
Unité des Virus Emergents, Aix Marseille Université, IRD 190, INSERM U1207, Hôpitaux Universitaires de Marseille-APHM, 13005 Marseille, France
Interests: arthropod-borne viruses; vector-borne and zoonotic viral diseases; viruses of medical importance; diagnostics; epidemiology; virus discovery; taxonomy
Special Issues, Collections and Topics in MDPI journals

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Guest Editor
Virology Unit, Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise “G. Caporale”, Teramo, Italy
Interests: diagnosis of viral infectious diseases via innovative molecular methods; coronaviruses; morbilliviruses; reverse genetics; swine influenza viruses; next generations sequencing; arbovirus; orbiviruses; West Nile virus; viral diagnostics; virus discovery; virus evolution; pathogenesis studies
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

We are pleased to announce this Collection titled “Editorial Board Members’ Collection Series: Molecular Epidemiology, Diagnostics and Management of Viral Pathogens”, which will collect papers invited by the Editorial Board Members.

The aim of this Collection is to provide a venue for networking and communication between the journal Pathogens and scholars in the field of viral pathogens’ epidemiology, diagnostics, and management. All papers will be published in open access following peer review.

Prof. Dr. Remi N. Charrel
Dr. Alessio Lorusso
Guest Editors

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Published Papers (12 papers)

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Research

14 pages, 817 KiB  
Article
High-Sensitivity RT-LAMP for Molecular Detection of O’nyong-nyong (Alphavirus onyong)
by David Faísca-Silva, Gonçalo Seixas, Mónica Nunes and Ricardo Parreira
Pathogens 2024, 13(10), 892; https://doi.org/10.3390/pathogens13100892 (registering DOI) - 11 Oct 2024
Abstract
Mosquitoes serve as vectors for many arthropod-borne viruses (arboviruses) that are responsible for millions of human infections and thousands of deaths each year. Among these arboviruses, O’nyong-nyong virus (ONNV) is an African alphavirus mainly transmitted by Anopheles mosquitoes. ONNV can be detected through [...] Read more.
Mosquitoes serve as vectors for many arthropod-borne viruses (arboviruses) that are responsible for millions of human infections and thousands of deaths each year. Among these arboviruses, O’nyong-nyong virus (ONNV) is an African alphavirus mainly transmitted by Anopheles mosquitoes. ONNV can be detected through serological or molecular tests, the first showing cross-reactivity to co-circulating alphaviruses and requiring technically demanding confirmation, while the latter, usually based on real-time PCR, are costly and demand specific equipment. Isothermal amplification approaches, such as Loop-Mediated Isothermal Amplification (LAMP), should therefore provide a cost-effective, sensitive, and specific alternative for virus detection, suitable for the resource-limited regions where ONNV circulates up to the present time. Here, we describe the development and optimization of a rapid and highly sensitive (10 pfu/reaction) RT-LAMP assay for ONNV detection. Additionally, we demonstrate that it is possible to bypass the RNA extraction step, reducing sample handling time and costs. The final RT-LAMPONNV is a promising field detection tool for ONNV, enabling a better understanding of its impact and serving as a point-of-care diagnostic method. Full article
12 pages, 1063 KiB  
Article
Identification of New Single Nucleotide Polymorphisms Potentially Related to Small Ruminant Lentivirus Infection Susceptibility in Goats Based on Data Selected from High-Throughput Sequencing
by Magdalena Materniak-Kornas, Katarzyna Ropka-Molik, Katarzyna Piórkowska, Joanna Kowalik, Tomasz Szmatoła, Jacek Sikora, Aldona Kawęcka and Jacek Kuźmak
Pathogens 2024, 13(10), 830; https://doi.org/10.3390/pathogens13100830 - 25 Sep 2024
Abstract
Small ruminant lentivirus (SRLV) infections are spread in the flocks of sheep and goats all over the world, causing economic loss. Although only a fraction of infected animals develop disease symptoms, all of them may shed the virus, causing uncontrolled spread of the [...] Read more.
Small ruminant lentivirus (SRLV) infections are spread in the flocks of sheep and goats all over the world, causing economic loss. Although only a fraction of infected animals develop disease symptoms, all of them may shed the virus, causing uncontrolled spread of the infection. Antibodies against the virus can be detected in the blood of infected animals and are the main marker of infection. Additionally, in most infected animals, proviral DNA can also be detected, but at different levels. Due to the lack of treatment or vaccines, the most effective strategy to prevent SRLV infections are control programmes introduced by several countries based on the elimination of seropositive individuals from the flock. An alternative approach, which has currently become the rationale, is an identification of host factors which may predispose certain individuals or breeds to resistance or susceptibility to small ruminant lentivirus infection. In our work, attention was paid to goats of the Carpathian breed infected with SRLV. Available RNA-seq results from the blood of 12 goats with a determined level of SRLV proviral load were used to analyse single nucleotide polymorphisms (SNPs) by the variant calling method. Six SNPs within five genes (POU2AF1, BCAT2, TMEM154, PARP14, UBASH3A) were selected for genotyping to determine their association with the level of small ruminant lentivirus proviral DNA in a group of 60 goats. Interestingly, in seronegative individuals, only the TT genotype of the PARP14 gene was observed, while the TMEM154 CC genotype was found only in seropositive goats. Both genes may be considered potential markers for resistance/susceptibility to SRLV infection. In contrast, polymorphisms identified in POU2AF1 and UBASH3A genes seemed to be deleterious for respective protein functions; therefore, these genes are less likely to be recognised as resistance/susceptibility markers of SRLV infection. Full article
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10 pages, 4360 KiB  
Communication
Preservation of scRNA-Seq Libraries Using Existing Inactivation Protocols
by Gail L. Sturdevant, Kimberly D. Meade-White, Sonja M. Best and Emily Speranza
Pathogens 2024, 13(2), 167; https://doi.org/10.3390/pathogens13020167 - 13 Feb 2024
Cited by 1 | Viewed by 1653
Abstract
Single-cell RNA sequencing has soared in popularity in recent years. The ability to deeply profile the states of individual cells during the course of disease or infection has helped to expand our knowledge of coordinated responses. However, significant challenges arise when performing this [...] Read more.
Single-cell RNA sequencing has soared in popularity in recent years. The ability to deeply profile the states of individual cells during the course of disease or infection has helped to expand our knowledge of coordinated responses. However, significant challenges arise when performing this analysis in high containment settings such as biosafety level 3 (BSL-3), BSL-3+ and BSL-4. Working in containment is necessary for many important pathogens, such as Ebola virus, Marburg virus, Lassa virus, Nipah and Hendra viruses. Since standard operating procedures (SOPs) for inactivation are extensive and may compromise sample integrity, we tested whether the removal of single-cell sequencing libraries from containment laboratories using existing inactivation protocols for nucleic acid extraction (Trizol, RLT buffer, or AVL buffer) was feasible. We have demonstrated that the inactivation does not affect sample quality and can work with existing methods for inactivation. Full article
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21 pages, 2534 KiB  
Article
Five Species of Wild Freshwater Sport Fish in Wisconsin, USA, Reveal Highly Diverse Viromes
by Charlotte E. Ford, Christopher D. Dunn, Eric M. Leis, Whitney A. Thiel and Tony L. Goldberg
Pathogens 2024, 13(2), 150; https://doi.org/10.3390/pathogens13020150 - 7 Feb 2024
Cited by 1 | Viewed by 1868
Abstract
Studies of marine fish have revealed distant relatives of viruses important to global fish and animal health, but few such studies exist for freshwater fish. To investigate whether freshwater fish also host such viruses, we characterized the viromes of five wild species of [...] Read more.
Studies of marine fish have revealed distant relatives of viruses important to global fish and animal health, but few such studies exist for freshwater fish. To investigate whether freshwater fish also host such viruses, we characterized the viromes of five wild species of freshwater fish in Wisconsin, USA: bluegill (Lepomis macrochirus), brown trout (Salmo trutta), lake sturgeon (Acipenser fulvescens), northern pike (Esox lucius), and walleye (Sander vitreus). We analyzed 103 blood serum samples collected during a state-wide survey from 2016 to 2020 and used a metagenomic approach for virus detection to identify known and previously uncharacterized virus sequences. We then characterized viruses phylogenetically and quantified prevalence, richness, and relative abundance for each virus. Within these viromes, we identified 19 viruses from 11 viral families: Amnoonviridae, Circoviridae, Coronaviridae, Hepadnaviridae, Peribunyaviridae, Picobirnaviridae, Picornaviridae, Matonaviridae, Narnaviridae, Nudnaviridae, and Spinareoviridae, 17 of which were previously undescribed. Among these viruses was the first fish-associated coronavirus from the Gammacoronavirus genus, which was present in 11/15 (73%) of S. vitreus. These results demonstrate that, similar to marine fish, freshwater fish also harbor diverse relatives of viruses important to the health of fish and other animals, although it currently remains unknown what effect, if any, the viruses we identified may have on fish health. Full article
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12 pages, 1340 KiB  
Article
Comprehensive Analysis of HIV-1 Integrase Resistance-Related Mutations in African Countries
by Francesco Branda, Marta Giovanetti, Leonardo Sernicola, Stefania Farcomeni, Massimo Ciccozzi and Alessandra Borsetti
Pathogens 2024, 13(2), 102; https://doi.org/10.3390/pathogens13020102 - 24 Jan 2024
Viewed by 1535
Abstract
The growing emergence of non-nucleoside reverse transcriptase inhibitor (NNRTI) HIV drug resistance in sub-Saharan Africa (SSA) led to the World Health Organization (WHO) recommending, in 2018, a transition to dolutegravir (DTG) as a first-line antiretroviral therapy (ART) in SSA. The broad HIV-1 genetic [...] Read more.
The growing emergence of non-nucleoside reverse transcriptase inhibitor (NNRTI) HIV drug resistance in sub-Saharan Africa (SSA) led to the World Health Organization (WHO) recommending, in 2018, a transition to dolutegravir (DTG) as a first-line antiretroviral therapy (ART) in SSA. The broad HIV-1 genetic diversity in SSA could shape DTG effectiveness and the pattern of drug resistance mutations (DRMs) in this region. This study evaluated HIV-1 integrase (IN) DRMs and conserved regions among published groups M, N, O, and P HIV-1 sequences spanning forty years of the HIV epidemic during the transition of DTG-based ART. Overall, we found low levels of integrase strand transfer inhibitor (INSTI)-DRMs (<1%) across HIV groups between the years 1983 and 2023; however, it was unexpected to detect DRMs at statistically significantly higher frequencies in pre-INSTI (1983–2007) than in the INSTI (2008–2023) era. The variability of accessory INSTI-DRMs depended on the HIV subtypes, with implications for susceptibility to DTG. Our findings provide new perspectives on the molecular epidemiology and drug resistance profiles of INSTIs in SSA, emphasizing the need for ongoing surveillance and customized treatment approaches to address the continent’s varied HIV subtypes and changing resistance patterns. Full article
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17 pages, 4501 KiB  
Article
Multivalent Epigraph Hemagglutinin Vaccine Protects against Influenza B Virus in Mice
by Erika Petro-Turnquist, Brigette Corder Kampfe, Amber Gadeken, Matthew J. Pekarek and Eric A. Weaver
Pathogens 2024, 13(2), 97; https://doi.org/10.3390/pathogens13020097 - 23 Jan 2024
Cited by 2 | Viewed by 1734
Abstract
Influenza B virus is a respiratory pathogen that contributes to seasonal epidemics, accounts for approximately 25% of global influenza infections, and can induce severe disease in young children. While vaccination is the most commonly used method of preventing influenza infections, current vaccines only [...] Read more.
Influenza B virus is a respiratory pathogen that contributes to seasonal epidemics, accounts for approximately 25% of global influenza infections, and can induce severe disease in young children. While vaccination is the most commonly used method of preventing influenza infections, current vaccines only induce strain-specific responses and have suboptimal efficacy when mismatched from circulating strains. Further, two influenza B virus lineages have been described, B/Yamagata-like and B/Victoria-like, and the limited cross-reactivity between the two lineages provides an additional barrier in developing a universal influenza B virus vaccine. Here, we report a novel multivalent vaccine using computationally designed Epigraph hemagglutinin proteins targeting both the B/Yamagata-like and B/Victoria-like lineages. When compared to the quadrivalent commercial vaccine, the Epigraph vaccine demonstrated increased breadth of neutralizing antibody and T cell responses. After lethal heterologous influenza B virus challenge, mice immunized with the Epigraph vaccine were completely protected against both weight loss and mortality. The superior cross-reactive immunity conferred by the Epigraph vaccine immunogens supports their continued investigation as a universal influenza B virus vaccine. Full article
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13 pages, 1704 KiB  
Article
Complement Suppresses the Initial Type 1 Interferon Response to Ocular Herpes Simplex Virus Type 1 Infection in Mice
by Daniel J. J. Carr, Adrian Filiberti and Grzegorz B. Gmyrek
Pathogens 2024, 13(1), 74; https://doi.org/10.3390/pathogens13010074 - 13 Jan 2024
Viewed by 1829
Abstract
The complement system (CS) contributes to the initial containment of viral and bacterial pathogens and clearance of dying cells in circulation. We previously reported mice deficient in complement component 3 (C3KO mice) were more sensitive than wild-type (WT) mice to ocular HSV-1 infection, [...] Read more.
The complement system (CS) contributes to the initial containment of viral and bacterial pathogens and clearance of dying cells in circulation. We previously reported mice deficient in complement component 3 (C3KO mice) were more sensitive than wild-type (WT) mice to ocular HSV-1 infection, as measured by a reduction in cumulative survival and elevated viral titers in the nervous system but not the cornea between days three and seven post infection (pi). The present study was undertaken to determine if complement deficiency impacted virus replication and associated changes in inflammation at earlier time points in the cornea. C3KO mice were found to possess significantly (p < 0.05) less infectious virus in the cornea at 24 h pi that corresponded with a decrease in HSV-1 lytic gene expression at 12 and 24 h pi compared to WT animals. Flow cytometry acquisition found no differences in the myeloid cell populations residing in the cornea including total macrophage and neutrophil populations at 24 h pi with minimal infiltrating cell populations detected at the 12 h pi time point. Analysis of cytokine and chemokine content in the cornea measured at 12 and 24 h pi revealed that only CCL3 (MIP-1α) was found to be different between WT and C3KO mice with >2-fold increased levels (p < 0.05, ANOVA and Tukey’s post hoc t-test) in the cornea of WT mice at 12 h pi. C3KO mouse resistance to HSV-1 infection at the early time points correlated with a significant increase in type I interferon (IFN) gene expression including IFN-α1 and IFN-β and downstream effector genes including tetherin and RNase L (p < 0.05, Mann–Whitney rank order test). These results suggest early activation of the CS interferes with the induction of the type I IFN response and leads to a transient increase in virus replication following corneal HSV-1 infection. Full article
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17 pages, 4577 KiB  
Article
Heat Inactivation of Nipah Virus for Downstream Single-Cell RNA Sequencing Does Not Interfere with Sample Quality
by Adam J. Hume, Judith Olejnik, Mitchell R. White, Jessie Huang, Jacquelyn Turcinovic, Baylee Heiden, Pushpinder S. Bawa, Christopher J. Williams, Nickolas G. Gorham, Yuriy O. Alekseyev, John H. Connor, Darrell N. Kotton and Elke Mühlberger
Pathogens 2024, 13(1), 62; https://doi.org/10.3390/pathogens13010062 - 9 Jan 2024
Cited by 1 | Viewed by 2674
Abstract
Single-cell RNA sequencing (scRNA-seq) technologies are instrumental to improving our understanding of virus–host interactions in cell culture infection studies and complex biological systems because they allow separating the transcriptional signatures of infected versus non-infected bystander cells. A drawback of using biosafety level (BSL) [...] Read more.
Single-cell RNA sequencing (scRNA-seq) technologies are instrumental to improving our understanding of virus–host interactions in cell culture infection studies and complex biological systems because they allow separating the transcriptional signatures of infected versus non-infected bystander cells. A drawback of using biosafety level (BSL) 4 pathogens is that protocols are typically developed without consideration of virus inactivation during the procedure. To ensure complete inactivation of virus-containing samples for downstream analyses, an adaptation of the workflow is needed. Focusing on a commercially available microfluidic partitioning scRNA-seq platform to prepare samples for scRNA-seq, we tested various chemical and physical components of the platform for their ability to inactivate Nipah virus (NiV), a BSL-4 pathogen that belongs to the group of nonsegmented negative-sense RNA viruses. The only step of the standard protocol that led to NiV inactivation was a 5 min incubation at 85 °C. To comply with the more stringent biosafety requirements for BSL-4-derived samples, we included an additional heat step after cDNA synthesis. This step alone was sufficient to inactivate NiV-containing samples, adding to the necessary inactivation redundancy. Importantly, the additional heat step did not affect sample quality or downstream scRNA-seq results. Full article
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9 pages, 1120 KiB  
Communication
An ARMS-Multiplex PCR Targeting SARS-CoV-2 Omicron Sub-Variants
by Petros Bozidis, Eleni Petridi and Konstantina Gartzonika
Pathogens 2023, 12(8), 1017; https://doi.org/10.3390/pathogens12081017 - 6 Aug 2023
Cited by 1 | Viewed by 1210
Abstract
As of November 2021, the SARS-CoV-2 Omicron variant had made its appearance, gradually replacing the predominant Delta variant. Since its emergence, the Omicron variant has been continuously evolving through more than 500 strains, most of which belong to five sub-variants known as BA.1, [...] Read more.
As of November 2021, the SARS-CoV-2 Omicron variant had made its appearance, gradually replacing the predominant Delta variant. Since its emergence, the Omicron variant has been continuously evolving through more than 500 strains, most of which belong to five sub-variants known as BA.1, BA.2, BA.3, BA.4, and BA.5. The aim of this study was to develop a multiplex polymerase chain reaction (PCR) that will be able to distinguish the basic sub-variants of Omicron in a rapid and specific way. Full genome sequences of Omicron strains with high frequency and wide geographical distribution were retrieved by the NCBI Virus and ENA databases. These sequences were compared to each other in order to locate single nucleotide polymorphisms common to all strains of the same sub-variant. These polymorphisms should also be capable of distinguishing Omicron sub-variants not only from each other but from previously circulating variants of SARS-CoV-2 as well. Thus, specific primers targeting characteristic polymorphisms of the four Omicron main branches BA.1, BA.2, BA.4, and BA.5 were designed according to the principles of the amplification refractory mutation system (ARMS) and with the ability to react under multiplex PCR conditions. According to our results, the ARMS-multiplex PCR could successfully distinguish all Omicron sub-variants that carry the corresponding mutations. Full article
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22 pages, 13797 KiB  
Article
Art of the Kill: Designing and Testing Viral Inactivation Procedures for Highly Pathogenic Negative Sense RNA Viruses
by Judith Olejnik, Adam J. Hume, Stephen J. Ross, Whitney A. Scoon, Scott Seitz, Mitchell R. White, Ben Slutzky, Nadezhda E. Yun and Elke Mühlberger
Pathogens 2023, 12(7), 952; https://doi.org/10.3390/pathogens12070952 - 19 Jul 2023
Cited by 5 | Viewed by 3630
Abstract
The study of highly pathogenic viruses handled under BSL-4 conditions and classified as Select Agents frequently involves the transfer of inactivated materials to lower containment levels for downstream analyses. Adhering to Select Agent and BSL-4 safety regulations requires validation or verification of the [...] Read more.
The study of highly pathogenic viruses handled under BSL-4 conditions and classified as Select Agents frequently involves the transfer of inactivated materials to lower containment levels for downstream analyses. Adhering to Select Agent and BSL-4 safety regulations requires validation or verification of the inactivation procedures, which comes with an array of challenges for each method. This includes the use of cytotoxic reagents for chemical inactivation and defining the precise inactivation parameters for physical inactivation. Here, we provide a workflow for various inactivation methods using Ebola, Nipah, and Lassa viruses as our examples. We choose three distinct inactivation methods (TRIzol/TRIzol LS, aldehyde fixation using different fixatives, and heat) to highlight the challenges of each method and provide possible solutions. We show that, whereas published chemical inactivation methods are highly reliable, the parameters for heat inactivation must be clearly defined to ensure complete inactivation. In addition to the inactivation data, we also provide examples and templates for the documentation required for approval and use of inactivation SOPs, including an inactivation report, the procedure sections of developed SOPs, and an electronic inactivation certificate that accompanies inactivated samples. The provided information can be used as a roadmap for similar studies at high and maximum containment laboratories. Full article
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14 pages, 5257 KiB  
Article
Establishment of an Inactivation Method for Ebola Virus and SARS-CoV-2 Suitable for Downstream Sequencing of Low Cell Numbers
by Judith Olejnik, Juliette Leon, Daniel Michelson, Kaitavjeet Chowdhary, Silvia Galvan-Pena, Christophe Benoist, Elke Mühlberger and Adam J. Hume
Pathogens 2023, 12(2), 342; https://doi.org/10.3390/pathogens12020342 - 17 Feb 2023
Cited by 4 | Viewed by 1959
Abstract
Technologies that facilitate the bulk sequencing of small numbers of cells as well as single-cell RNA sequencing (scRNA-seq) have aided greatly in the study of viruses as these analyses can be used to differentiate responses from infected versus bystander cells in complex systems, [...] Read more.
Technologies that facilitate the bulk sequencing of small numbers of cells as well as single-cell RNA sequencing (scRNA-seq) have aided greatly in the study of viruses as these analyses can be used to differentiate responses from infected versus bystander cells in complex systems, including in organoid or animal studies. While protocols for these analyses are typically developed with biosafety level 2 (BSL-2) considerations in mind, such analyses are equally useful for the study of viruses that require higher biosafety containment levels. Many of these workstreams, however, are not directly compatible with the more stringent biosafety regulations of BSL-3 and BSL-4 laboratories ensuring virus inactivation and must therefore be modified. Here we show that TCL buffer (Qiagen), which was developed for bulk sequencing of small numbers of cells and also facilitates scRNA-seq, inactivates both Ebola virus (EBOV) and SARS-CoV-2, BSL-4 and BSL-3 viruses, respectively. We show that additional heat treatment, necessary for the more stringent biosafety concerns for BSL-4-derived samples, was additionally sufficient to inactivate EBOV-containing samples. Critically, this heat treatment had minimal effects on extracted RNA quality and downstream sequencing results. Full article
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12 pages, 1043 KiB  
Article
Genome Characterization and Spaciotemporal Dispersal Analysis of Bagaza Virus Detected in Portugal, 2021
by Marta Falcão, Margarida Barros, Margarida D. Duarte, Fábio Abade dos Santos, Teresa Fagulha, Margarida Henriques, Fernanda Ramos, Ana Duarte, Tiago Luís, Ricardo Parreira and Sílvia C. Barros
Pathogens 2023, 12(2), 150; https://doi.org/10.3390/pathogens12020150 - 17 Jan 2023
Cited by 4 | Viewed by 2434
Abstract
In September 2021, Bagaza virus (BAGV), a member of the Ntaya group from the Flavivirus genus, was detected for the first time in Portugal, in the heart and the brain of a red-legged partridge found dead in a hunting ground in Serpa (Alentejo [...] Read more.
In September 2021, Bagaza virus (BAGV), a member of the Ntaya group from the Flavivirus genus, was detected for the first time in Portugal, in the heart and the brain of a red-legged partridge found dead in a hunting ground in Serpa (Alentejo region; southern Portugal). Here we report the genomic characterization of the full-length sequence of the BAGV detected (BAGV/PT/2021), including phylogenetic reconstructions and spaciotemporal analyses. Phylogenies inferred from nucleotide sequence alignments, complemented with the analysis of amino acid alignments, indicated that the BAGV strain from Portugal is closely related to BAGV strains previously detected in Spain, suggesting a common ancestor that seems to have arrived in the Iberia Peninsula in the late 1990s to early 2000s. In addition, our findings support previous observations that BAGV and Israel turkey meningoencephalitis virus (ITV) belong to the same viral species. Full article
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