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Background: Algeria like other North African countries is experiencing recurrent episodes of West Nile Virus (WNV) emergences and new health threats associated with the introduction of Aedes albopictus in 2010 are to be feared. To improve the surveillance of mosquito-borne pathogens, we performed a study using innovative tools based on multiplex molecular methods., Methods: We combined two approaches: a high-throughput chip based on the BioMark Dynamic array system to detect arboviruses in mosquitoes, and a set of immunologic methods (ELISA, microsphere immunoassays (MIA) and virus microneutralization tests (MNT)) for serological surveys in animal hosts. We investigated two distinct regions: a first zone located in the coastal humid region and a second one in the Saharan desert region., Principal Findings: We collected a total of 1,658 mosquitoes belonging to nine different species and found predominantly Culex pipienss. l. (56.5%) and Cx. perexiguus (27.5%). From 180 pools of 10 mosquitoes, we detected four arboviruses: Banna virus (BAV), chikungunya virus (CHIKV), Sindbis virus (SINV), and Usutu virus (USUV). Moreover, we examined 389 blood samples from equids and poultry and found that 52.4% were positive for flavivirus antibodies in ELISA, while 30.8% were positive for WNV and two chickens and two equids were positive for USUV by MNT and MIA respectively., Conclusions: To our knowledge, this is the first report of five arboviruses circulating in Algeria, with three reported for the first time (CHIKV, BAV, and USUV). Our study brings evidence that reinforcing surveillance using more discriminant tools may help in anticipating future emergences and propose adapted control measures., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Boubidi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The Equid alphaherpesvirus type 1 (EHV-1) infection can have devastating economic consequences in the horse industry due to large-scale outbreaks of abortions, perinatal foal mortality, and myeloencephalopathy. The present study analyzed the genome of two isolates obtained from aborted fetuses in Argentina, E/745/99 and E/1297/07. The E745/99 genome shares 98.2% sequence identity with Ab4, a reference EHV-1 strain. The E/1297/07 genome shares 99.8% identity with NY03, a recombinant strain containing part of ORF64 and part of the intergenic region from Equid alphaherpesvirus-4 (EHV-4). The E/1297/07 genome has the same breakpoints as other United States and Japanese recombinants, including NY03. The recombinant regions have varying numbers of tandem repeat sequences and different minor parental sequences (EHV-4), suggesting distinct origins of the recombinant events. These are the first complete genomes of EHV-1 from Argentina and South America available in the Databases., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: West Nile Virus (WNV) is a zoonotic arbovirus worldwide spread. Seasonal WNV outbreaks occur in the Mediterranean basin since the late 1990's with ever-increasing incidence. In Southern Spain WNV is endemic, as disease foci - caused by WNV lineage 1 (WNV-L1) strains - occur every year. On the contrary, WNV-L2 is the dominant lineage in Europe, so most European WNV sequences available belong to this lineage, WNV-L1 sequences being still scarce., Methods: To fill this gap, this study reports the genetic characterisation of 27 newly described WNV-L1 strains, involved in outbreaks affecting wild birds and horses during the last decade in South-Western Spain., Results: All strains except one belong to the Western Mediterranean-1 sub-cluster (WMed-1), related phylogenetically to Italian, French, Portuguese, Moroccan and, remarkably, Senegalese strains. This sub-cluster persisted, spread and evolved into three distinguishable WMed-1 phylogenetic groups that co-circulated, notably, in the same province (Cádiz). They displayed different behaviours: from long-term persistence and rapid spread to neighbouring regions within Spain, to long-distance spread to different countries, including transcontinental spread to Africa. Among the different introductions of WNV in Spain revealed in this study, some of them succeeded to get established, some extinguished from the territory shortly afterwards. Furthermore, Spain's southernmost province, Cádiz, constitutes a hotspot for virus incursion., Conclusion: Southern Spain seems a likely scenario for emergence of exotic pathogens of African origin. Therefore, circulation of diverse WNV-L1 variants in Spain prompts for an extensive surveillance under a One Health approach.

Equid herpesviruses (EHVs) are a group of highly impactful viral pathogens that affect horses, presenting a substantial risk to the global equine industry. Among these, equid herpesvirus-1 (EHV-1) primarily causes respiratory infections. However, its ability to spread to distant organs can lead to severe consequences such as abortion and neurological diseases. These viruses can enter a dormant phase, with minimal activity, and later reactivate to trigger active infections at any time. Recently, there has been a notable rise in the prevalence of a particularly devastating strains of EHV-1 known as equid herpesviral myeloencephalopathy (EHM). In the light of dynamic nature of EHV-1, this review provides a thorough overview of EHV-1 and explores how advances in viral biology affect the pathophysiology of viral infection. The information presented here is crucial for understanding the dynamics of EHV-1 infections and creating practical plans to stop the virus's global spread among equid populations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Equine rhinitis A (ERAV) and B (ERBV) viruses are respiratory pathogens with worldwide distribution. The current study aimed to determine the frequency of infection of ERAV and ERBV among horses and foals at Polish national studs, and to determine genetic variability within the viruses obtained. Virus-specific quantitative RT-PCR assays targeting a 5' untranslated region were used to screen nasal swabs collected from 621 horses at 16 national horse studs from throughout Poland, including 553 healthy horses and 68 horses with respiratory disease. A partial DNA polymerase gene was amplified and sequenced from the qRT-PCR-positive samples. The obtained sequences were analysed using phylogeny and genetic network analysis. None of the nasal swabs were positive for ERAV, whereas ERBV was found in 11/621 (1.78%) samples collected from 10 healthy horses and one foal affected by respiratory disease. Partial DNA polymerase gene sequence variability was correlated with individual horses and studs from which samples were collected when only Polish sequences were analysed, but there was no correlation between country of origin and ERBV sequence when Polish and international sequences were included in the network. The report presents the first detection of ERBV in Poland.

Usutu virus (USUV), an arbovirus from the Flaviviridae family, genus Flavivirus, has recently gained increasing attention because of its potential for emergence. After his discovery in South Africa, USUV spread to other African countries, then emerged in Europe where it was responsible for epizootics. The virus has recently been found in Asia. USUV infection in humans is considered to be most often asymptomatic or to cause mild clinical signs. However, a few cases of neurological complications such as encephalitis or meningo-encephalitis have been reported in both immunocompromised and immunocompetent patients. USUV natural life cycle involves Culex mosquitoes as its main vector, and multiple bird species as natural viral reservoirs or amplifying hosts, humans and horses can be incidental hosts. Phylogenetic studies carried out showed eight lineages, showing an increasing genetic diversity for USUV. This work describes the development and validation of a novel whole-genome amplicon-based sequencing approach to Usutu virus. This study was carried out on different strains from Senegal and Italy. The new approach showed good coverage using samples derived from several vertebrate hosts and may be valuable for Usutu virus genomic surveillance to better understand the dynamics of evolution and transmission of the virus., (© 2024. The Author(s).)

Background: Equid alphaherpesvirus 1 (EHV-1) is a highly contagious respiratory tract pathogen of horses, and infection may be followed by myeloencephalopathy or abortion. Surveillance and early detection have focused on PCR assays using less tolerated nasal swabs. Here, we assess non-invasive non-contact sampling techniques as surveillance tools in naturally equid gammaherpesvirus 2-shedding horses as surrogates for EHV-1., Methods: Horses were individually housed for 10 h periods on 2 consecutive days. Sampling included nasal swabs, nostril wipes, environmental swabs, droplet-catching devices, and air sampling. The latter was completed via two strategies: a combined air sample collected while going from horse to horse and a collective air sample collected at a stationary central point for 6 h. Samples were screened through quantitative PCR and digital PCR., Results: Nine horses on day 1 and 11 horses on day 2 were positive for EHV-1; overall, 90.9% of the nostril wipes, 81.8% of the environmental surfaces, and 90.9% of the droplet-catching devices were found to be positive. Quantitative analysis showed that the mean DNA copies detection per cm 2 of nostril wipe sampled concentration (4.3 × 10 5 per day) was significantly ( p < 0.05) comparable to that of nasal swabs (3.6 × 10 5 per day) followed by environmental swabs (4.3 × 10 5 per day) and droplet catchers (3.5 × 10 3 per day), respectively. Overall, 100% of the air samples collected were positive on both qPCR and dPCR. In individual air samples, a mean concentration of 1.0 × 10 4 copies of DNA were detected in per m 3 air sampled per day, while in the collective air samples, the mean concentration was 1.1 × 10 3 ., Conclusions: Environmental samples look promising in replacing direct contact sampling. Environmental and air sampling could become efficient surveillance tools at equestrian events; however, it needs threshold calculations for minimum detection levels.

Primary cell cultures derived from human embryo lung play a crucial role in virology by aiding virus propagation and vaccine development. These cultures exhibit a notable ability to undergo multiple subcultures, often reaching up to 70 passages. However, finding alternative primary cell cultures with similar longevity and usefulness is challenging. In this study, we introduce a novel primary culture cells derived from equine embryo brain (FEB), which cells exhibited remarkable long-term cultivation potential. The FEB was established and maintained using Sumitomo Nerve-Cell Culture System Comparison studies were conducted with fetal equine kidney cell line (FEK-Tc13) to assess growth rates and subculture longevity. Immunological characterization was performed using neuronal markers to confirm the neural nature of FEB cells. Viral growth assessments were conducted using equine herpesviruses (EHV-1 and EHV-4) to evaluate infectivity and cytopathic effects in FEB cells. PCR analysis and real-time PCR assays were employed to detect viral genomic DNA and transcription activity of EHVs in infected FEB cells. FEB cells demonstrated faster growth rates compared to fetal equine kidney cell line (FEK-Tc13 cells) and exhibited sustained subculture capability exceeding 50 passages. Immunostaining confirmed the glial identity of FEB cells. Both equine herpesviruses 1 and 4 EHV-1 and EHV-4 viruses efficiently replicated in FEB cells, resulting in clear cytopathic effects. PCR analysis detected genomic DNA of EHVs in infected FEB cells, indicating successful viral infection. The establishment of FEB cells with extended subculture capability highlights their potential utility as a model system for studying neural cell biology and viral infections., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Influenza A viruses (IAVs) can overcome species barriers by adaptation of the receptor-binding site of the hemagglutinin (HA). To initiate infection, HAs bind to glycan receptors with terminal sialic acids, which are either N -acetylneuraminic acid (NeuAc) or N -glycolylneuraminic acid (NeuGc); the latter is mainly found in horses and pigs but not in birds and humans. We investigated the influence of previously identified equine NeuGc-adapting mutations (S128T, I130V, A135E, T189A, and K193R) in avian H7 IAVs in vitro and in vivo . We observed that these mutations negatively affected viral replication in chicken cells but not in duck cells and positively affected replication in horse cells. In vivo , the mutations reduced virus virulence and mortality in chickens. Ducks excreted high viral loads longer than chickens, although they appeared clinically healthy. To elucidate why these viruses infected chickens and ducks despite the absence of NeuGc, we re-evaluated the receptor binding of H7 HAs using glycan microarray and flow cytometry studies. This re-evaluation demonstrated that mutated avian H7 HAs also bound to α2,3-linked NeuAc and sialyl-LewisX, which have an additional fucose moiety in their terminal epitope, explaining why infection of ducks and chickens was possible. Interestingly, the α2,3-linked NeuAc and sialyl-LewisX epitopes were only bound when presented on tri-antennary N -glycans, emphasizing the importance of investigating the fine receptor specificities of IAVs. In conclusion, the binding of NeuGc-adapted H7 IAV to tri-antennary N -glycans enables viral replication and shedding by chickens and ducks, potentially facilitating interspecies transmission of equine-adapted H7 IAVs.IMPORTANCEInfluenza A viruses (IAVs) cause millions of deaths and illnesses in birds and mammals each year. The viral surface protein hemagglutinin initiates infection by binding to host cell terminal sialic acids. Hemagglutinin adaptations affect the binding affinity to these sialic acids and the potential host species targeted. While avian and human IAVs tend to bind to N -acetylneuraminic acid (sialic acid), equine H7 viruses prefer binding to N -glycolylneuraminic acid (NeuGc). To better understand the function of NeuGc-specific adaptations in hemagglutinin and to elucidate interspecies transmission potential NeuGc-adapted viruses, we evaluated the effects of NeuGc-specific mutations in avian H7 viruses in chickens and ducks, important economic hosts and reservoir birds, respectively. We also examined the impact on viral replication and found a binding affinity to tri-antennary N -glycans containing different terminal epitopes. These findings are significant as they contribute to the understanding of the role of receptor binding in avian influenza infection., Competing Interests: The authors declare no conflict of interest.

Equine hepacivirus (EqHV, Flaviviridae , hepacivirus) is a small, enveloped RNA virus generally causing sub-clinical hepatitis with occasional fatalities. EqHV is reported in equids worldwide, but for Italy data are limited. To address this, a survey study was set up to estimate prevalence at a national level and among different production categories (equestrian; competition; work and meat; reproduction) and national macro-regions (North, Central, South, and Islands). Data obtained testing 1801 horse serum samples by Real-Time RT PCR were compared within the categories and regions. The NS3 fragment of the PCR-positive samples was sequenced by Sanger protocol for phylogenetic and mutational analysis. The tertiary structure of the NS3 protein was also assessed. The estimated national prevalence was 4.27% [1.97-6.59, 95% CI] and no statistical differences were detected among production categories and macro-regions. The phylogenesis confirmed the distribution in Italy of the three known EqHV subtypes, also suggesting a possible fourth sub-type that, however, requires further confirmation. Mutational profiles that could also affect the NS3 binding affinity to the viral RNA were detected. The present paper demonstrates that EqHV should be included in diagnostic protocols when investigating causes of hepatitis, and in quality control protocols for blood derived products due to its parental transmission.

Importance: Spike-receptor interaction is a critical determinant for the host range of coronaviruses. In this study, we investigated the SARS-CoV-2 WHU01 strain and five WHO-designated SARS-CoV-2 variants of concern (VOCs), including Alpha, Beta, Gamma, Delta, and the early Omicron variant, for their Spike interactions with ACE2 proteins of 18 animal species. First, the receptor-binding domains (RBDs) of Alpha, Beta, Gamma, and Omicron were found to display progressive gain of affinity to mouse ACE2. More interestingly, these RBDs were also found with progressive loss of affinities to multiple ACE2 orthologs. The Omicron RBD showed decreased or complete loss of affinity to eight tested animal ACE2 orthologs, including that of some livestock animals (horse, donkey, and pig), pet animals (dog and cat), and wild animals (pangolin, American pika, and Rhinolophus sinicus bat). These findings shed light on potential host range shift of SARS-CoV-2 VOCs, especially that of the Omicron variant., Competing Interests: The authors declare no conflict of interest.

High-throughput sequences were generated from DNA and cDNA from four Southern white rhinoceros ( Ceratotherium simum simum ) located in the Taronga Western Plain Zoo in Australia. Virome analysis identified reads that were similar to Mus caroli endogenous gammaretrovirus (McERV). Previous analysis of perissodactyl genomes did not recover gammaretroviruses. Our analysis, including the screening of the updated white rhinoceros ( Ceratotherium simum ) and black rhinoceros ( Diceros bicornis ) draft genomes identified high-copy orthologous gammaretroviral ERVs. Screening of Asian rhinoceros, extinct rhinoceros, domestic horse, and tapir genomes did not identify related gammaretroviral sequences in these species. The newly identified proviral sequences were designated SimumERV and DicerosERV for the white and black rhinoceros retroviruses, respectively. Two long terminal repeat (LTR) variants (LTR-A and LTR-B) were identified in the black rhinoceros, with different copy numbers associated with each ( n = 101 and 373, respectively). Only the LTR-A lineage ( n  = 467) was found in the white rhinoceros. The African and Asian rhinoceros lineages diverged approximately 16 million years ago. Divergence age estimation of the identified proviruses suggests that the exogenous retroviral ancestor of the African rhinoceros ERVs colonized their genomes within the last 8 million years, a result consistent with the absence of these gammaretroviruses from Asian rhinoceros and other perissodactyls. The black rhinoceros germ line was colonized by two lineages of closely related retroviruses and white rhinoceros by one. Phylogenetic analysis indicates a close evolutionary relationship with ERVs of rodents including sympatric African rats, suggesting a possible African origin of the identified rhinoceros gammaretroviruses. IMPORTANCE Rhinoceros genomes were thought to be devoid of gammaretroviruses, as has been determined for other perissodactyls (horses, tapirs, and rhinoceros). While this may be true of most rhinoceros, the African white and black rhinoceros genomes have been colonized by evolutionarily young gammaretroviruses (SimumERV and DicerosERV for the white and black rhinoceros, respectively). These high-copy endogenous retroviruses (ERVs) may have expanded in multiple waves. The closest relative of SimumERV and DicerosERV is found in rodents, including African endemic species. Restriction of the ERVs to African rhinoceros suggests an African origin for the rhinoceros gammaretroviruses.

Introduction: Eastern equine encephalitis virus (EEEV) and Venezuelan equine encephalitis virus (VEEV) viruses are zoonotic pathogens affecting humans, particularly equines. These neuroarboviruses compromise the central nervous system and can be fatal in different hosts. Both have significantly influenced Colombia; however, few studies analyse its behaviour, and none develop maps using geographic information systems to characterise it., Objective: To describe the temporal-spatial distribution of those viruses in Colombia between 2008 and 2019., Methods: Retrospective cross-sectional descriptive study, based on weekly reports by municipalities of the ICA, of the surveillance of both arboviruses in equines, in Colombia, from 2008 to 2019. The data were converted into databases in Microsoft Access 365 ® , and multiple epidemiological maps were generated with the Kosmo RC1 ® 3.0 software coupled to shape files of all municipalities in the country., Results: In the study period, 96 cases of EEE and 70 of VEE were reported, with 58% of EEE cases occurring in 2016 and 20% of EEV cases in 2013. The most affected municipalities for EEE corresponded to the department of Casanare: Yopal (20), Aguazul (16), and Tauramena (10). In total, 40 municipalities in the country reported ≥1 case of EEE., Conclusions: The maps allow a quick appreciation of groups of neighbouring municipalities in different departments (1° political division) and regions of the country affected by those viruses, which helps consider the expansion of the disease associated with mobility and transport of equines between other municipalities, also including international borders, such as is the case with Venezuela. In that country, especially for EEV, municipalities in the department of Cesar are bordering and at risk for that arboviral infection. there is a high risk of equine encephalitis outbreaks, especially for VEE. This poses a risk also, for municipalities in the department of Cesar, bordering with Venezuela.

During recent decades, pathogens that originated in bats have become an increasing public health concern. A major challenge is to identify how those pathogens spill over into human populations to generate a pandemic threat 1 . Many correlational studies associate spillover with changes in land use or other anthropogenic stressors 2,3 , although the mechanisms underlying the observed correlations have not been identified 4 . One limitation is the lack of spatially and temporally explicit data on multiple spillovers, and on the connections among spillovers, reservoir host ecology and behaviour and viral dynamics. We present 25 years of data on land-use change, bat behaviour and spillover of Hendra virus from Pteropodid bats to horses in subtropical Australia. These data show that bats are responding to environmental change by persistently adopting behaviours that were previously transient responses to nutritional stress. Interactions between land-use change and climate now lead to persistent bat residency in agricultural areas, where periodic food shortages drive clusters of spillovers. Pulses of winter flowering of trees in remnant forests appeared to prevent spillover. We developed integrative Bayesian network models based on these phenomena that accurately predicted the presence or absence of clusters of spillovers in each of the 25 years. Our long-term study identifies the mechanistic connections between habitat loss, climate and increased spillover risk. It provides a framework for examining causes of bat virus spillover and for developing ecological countermeasures to prevent pandemics., (© 2022. The Author(s).)

While some companion animals have been shown to be susceptible to SARS-CoV-2, their role in the COVID-19 pandemic has remained poorly investigated. Equids are susceptible to SARS-CoV-2 based on the similarity of the human ACE-2 receptor and reports of infection. Clinical disease and prevalence factors associated with SARS-CoV-2 infection in equids have not yet been investigated. The aim of this study was to determine the seroprevalence of SARS-CoV-2 and selected prevalence factors in 1186 equids presented for various conditions to a Veterinary Medical Teaching Hospital over a two-year period. Blood samples were tested for SARS-CoV-2 antibodies using an ELISA targeting the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Further, selected prevalence factors (season, age, breed, sex, presenting complaint) were retrieved from the medical records. No information was available on whether the horses had come into contact with COVID-19-positive individuals. Among the study animals, 42/1186 (3.5%) horses had detectable SARS-CoV-2 antibodies. Amongst the prevalence factors investigated, only seasonality (spring) was associated with a greater frequency of seropositivity to SARS-CoV-2. Horses with medical and surgical complaints were more likely to test seropositive to SARS-CoV-2 compared to horses presented for routine health care procedures, suggesting more frequent and/or longer interactions with individuals with COVID-19. While horses can become infected with SARS-CoV-2 via the occasional spillover from COVID-19 individuals, clinical disease expression remains subclinical, making horses an unlikely contributor to the spread of SARS-CoV-2.

BackgroundWest Nile virus (WNV) and Usutu virus (USUV), two closely related flaviviruses, mainly follow an enzootic cycle involving mosquitoes and birds, but also infect humans and other mammals. Since 2010, their epidemiological situation may have shifted from irregular epidemics to endemicity in several European regions; this requires confirmation, as it could have implications for risk assessment and surveillance strategies.AimTo explore the seroprevalence in animals and humans and potential endemicity of WNV and USUV in Southern France, given a long history of WNV outbreaks and the only severe human USUV case in France in this region.MethodsWe evaluated the prevalence of WNV and USUV in a repeated cross-sectional study by serological and molecular analyses of human, dog, horse, bird and mosquito samples in the Camargue area, including the city of Montpellier, between 2016 and 2020.ResultsWe observed the active transmission of both viruses and higher USUV prevalence in humans, dogs, birds and mosquitoes, while WNV prevalence was higher in horses. In 500 human samples, 15 were positive for USUV and 6 for WNV. Genetic data showed that the same lineages, WNV lineage 1a and USUV lineage Africa 3, were found in mosquitoes in 2015, 2018 and 2020.ConclusionThese findings support existing literature suggesting endemisation in the study region and contribute to a better understanding of USUV and WNV circulation in Southern France. Our study underlines the importance of a One Health approach for the surveillance of these viruses.

The Getah virus (GETV), a mosquito-borne RNA virus, is widely distributed in Oceania and Asia. GETV is not the only pathogenic to horses, pigs, cattle, foxes and boars, but it can also cause fever in humans. Since its first reported case in Chinese mainland in 2017, the number of GETV-affected provinces has increased to seventeen till now. Therefore, we performed an epidemiologic investigation of GETV in the Xinjiang region, located in northwestern China, during the period of 2017-2020. ELISA was used to analyze 3299 serum samples collected from thoroughbred horse, local horse, sheep, goat, cattle, and pigs, with thoroughbred horse (74.8%), local horse (67.3%), goat (11.7%), sheep (10.0%), cattle (25.1%) and pigs (51.1%) being positive for anti-GETV antibodies. Interestingly, the neutralizing antibody titer in horses was much higher than in other species. Four samples from horses and pigs were positive for GETV according to RT-PCR. Furthermore, from the serum of a local horse, we isolated GETV which was designated as strain XJ-2019-07, and determined its complete genome sequence. From the phylogenetic relationships, it belongs to the Group III lineage. This is the first evidence of GETV associated to domestic animals in Xinjiang. Overall, GETV is prevalent in Xinjiang and probably has been for several years. Since no vaccine against GETV is available in China, detection and monitoring strategies should be improved in horses and pigs, especially imported and farmed, in order to prevent economic losses., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest., (Copyright © 2022 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

The equine sarcoid is one of the most common neoplasias in the Equidae family. Despite the association of this tumor with the presence of bovine papillomavirus (BPV), the molecular mechanism of this lesion has not been fully understood. The transgenization of equine adult cutaneous fibroblast cells (ACFCs) was accomplished by nucleofection, followed by detection of molecular modifications using high-throughput NGS transcriptome sequencing. The results of the present study confirm that BPV-E4 - and BPV-E1^E4 -mediated nucleofection strategy significantly affected the transcriptomic alterations, leading to sarcoid-like neoplastic transformation of equine ACFCs. Furthermore, the results of the current investigation might contribute to the creation of in vitro biomedical models suitable for estimating the fates of molecular dedifferentiability and the epigenomic reprogrammability of BPV-E4 and BPV-E4 ^E1 transgenic equine ACFC-derived sarcoid-like cell nuclei in equine somatic cell-cloned embryos. Additionally, these in vitro models seem to be reliable for thoroughly recognizing molecular mechanisms that underlie not only oncogenic alterations in transcriptomic signatures, but also the etiopathogenesis of epidermal and dermal sarcoid-dependent neoplastic transformations in horses and other equids. For those reasons, the aforementioned transgenic models might be useful for devising clinical treatments in horses afflicted with sarcoid-related neoplasia of cutaneous and subcutaneous tissues.

Getah virus (GETV) is a member of the alphavirus genus, and it infects a variety of animal species, including horses, pigs, cattle, and foxes. Human infection with this virus has also been reported. The structure of GETV has not yet been determined. In this study, we report the cryo-EM structure of GETV at a resolution of 3.5 Å. This structure reveals conformational polymorphism of the envelope glycoproteins E1 and E2 at icosahedral 3-fold and quasi-3-fold axes, which is believed to be a necessary organization in forming a curvature surface of virions. In our density map, three extra densities are identified, one of which is believed a "pocket factor"; the other two are located by domain D of E2, and they may maintain the stability of E1/E2 heterodimers. We also identify three N-glycosylations at E1 N141, E2 N200, and E2 N262, which might be associated with receptor binding and membrane fusion. The resolving of the structure of GETV provides new insights into the structure and assembly of alphaviruses and lays a basis for studying the differences of biology and pathogenicity between arthritogenic and encephalitic alphaviruses.

Tailed bacteriophages constitute the bulk of the intestinal viromes of vertebrate animals. However, the relationships between lytic and lysogenic lifestyles of phages in these ecosystems are not always clear and may vary between the species or even between the individuals. The human intestinal (fecal) viromes are dominated mostly by temperate phages, while in horse feces virulent phages are more prevalent. To our knowledge, all the previously reported isolates of horse fecal coliphages are virulent. Temperate coliphage Hf4s was isolated from horse feces, from the indigenous equine Escherichia coli 4s strain. It is a podovirus related to the Lederbergvirus genus (including the well-characterized Salmonella bacteriophage P22). Hf4s recognizes the host O antigen as its primary receptor and possesses a functional O antigen seroconversion cluster that renders the lysogens protected from superinfection by the same bacteriophage and also abolishes the adsorption of some indigenous equine virulent coliphages, such as DT57C, while other phages, such as G7C or phiKT, retain the ability to infect E. coli 4s (Hf4s) lysogens. IMPORTANCE The relationships between virulent and temperate bacteriophages and their impact on high-density symbiotic microbial ecosystems of animals are not always clear and may vary between species or even between individuals. The horse intestinal virome is dominated by virulent phages, and Hf4s is the first temperate equine intestinal coliphage characterized. It recognizes the host O antigen as its primary receptor and possesses a functional O antigen seroconversion cluster that renders the lysogens protected from superinfection by some indigenous equine virulent coliphages, such as DT57C, while other phages, such as G7C or phiKT, retain the ability to infect E. coli 4s (Hf4s) lysogens. These findings raise questions on the significance of bacteriophage-bacteriophage interactions within the ecology of microbial viruses in mammal intestinal ecosystems.

Equine herpesvirus 1 isolates from a 2021 outbreak of neurologic disease in Europe have a mutation, A713G, in open reading frame 11 not detected in 249 other sequences from equine herpesvirus 1 isolates. This single-nucleotide polymorphism could help identify horses infected with the virus strain linked to this outbreak.

Equine penile squamous cell carcinoma (epSCC) is the most frequent tumor of the external male genitalia, representing 67.5% of equine genital cancers. epSCC is associated with papilloma virus (PV) infection and has been recently proposed as a model for human PV-induced squamous cell carcinomas. It has already been suggested that epSCC might undergo epithelial-to-mesenchymal transition (EMT). This work aims to investigate in detail this process and the possible role of PV oncoproteins in epSCC. For this purpose, 18 penile SCCs were retrospectively selected and tested for both EcPV2 presence and oncoproteins (EcPV2 E6 and EcPV2 E7) expression. Moreover, immunohistochemical EMT characterization was carried out by analyzing the main epithelial markers (E-cadherin, β-catenin, and pan-cytokeratin AE3/AE1), the main mesenchymal markers (N-cadherin and vimentin), and the main EMT-related transcription factors (TWIST-1, ZEB-1). PCR analysis was positive for EcPV2 in 16 out of 18 samples. EMT was investigated in epSCC positive for EcPV2. The immunohistochemistry results suggested the presence of EMT processes in the neoplastic cells at the tumor invasive front. Moreover, the significant upregulation of RANKL , together with BCATN1 , LEF1 , and FOSL1 genes, might suggest a canonical Wnt pathway activation, similarly to what is reported in human penile squamous cell carcinomas.

Mosquito-borne West Nile virus (WNV) is the causative agent of West Nile disease in humans, horses, and some bird species. Since the initial introduction of WNV to the United States (US), approximately 30,000 horses have been impacted by West Nile neurologic disease and hundreds of additional horses are infected each year. Research describing the drivers of West Nile disease in horses is greatly needed to better anticipate the spatial and temporal extent of disease risk, improve disease surveillance, and alleviate future economic impacts to the equine industry and private horse owners. To help meet this need, we integrated techniques from spatiotemporal epidemiology, eco-phylogenetics, and distributional ecology to assess West Nile disease risk in horses throughout the contiguous US. Our integrated approach considered horse abundance and virus exposure, vector and host distributions, and a variety of extrinsic climatic, socio-economic, and environmental risk factors. Birds are WNV reservoir hosts, and therefore we quantified avian host community dynamics across the continental US to show intra-annual variability in host phylogenetic structure and demonstrate host phylodiversity as a mechanism for virus amplification in time and virus dilution in space. We identified drought as a potential amplifier of virus transmission and demonstrated the importance of accounting for spatial non-stationarity when quantifying interaction between disease risk and meteorological influences such as temperature and precipitation. Our results delineated the timing and location of several areas at high risk of West Nile disease and can be used to prioritize vaccination programs and optimize virus surveillance and monitoring.

Phleboviruses (genus P hlebovirus , family Phenuiviridae ) are emerging pathogens of humans and animals. Sand-fly-transmitted phleboviruses are found in Europe, Africa, the Middle East, and the Americas, and are responsible for febrile illness and nervous system infections in humans. Rio Grande virus (RGV) is the only reported phlebovirus in the United States. Isolated in Texas from southern plains woodrats, RGV is not known to be pathogenic to humans or domestic animals, but serologic evidence suggests that sheep ( Ovis aries ) and horses ( Equus caballus ) in this region have been infected. Rift Valley fever virus (RVFV), a phlebovirus of Africa, is an important pathogen of wild and domestic ruminants, and can also infect humans with the potential to cause severe disease. The introduction of RVFV into North America could greatly impact U.S. livestock and human health, and the development of vaccines and countermeasures is a focus of both the CDC and USDA. We investigated the potential for serologic reagents used in RVFV diagnostic assays to also detect cells infected with RGV. Western blots and immunocytochemistry assays were used to compare the antibody detection of RGV, RVFV, and two other New World phlebovirus, Punta Toro virus (South and Central America) and Anhanga virus (Brazil). Antigenic cross-reactions were found using published RVFV diagnostic reagents. These findings will help to inform test interpretation to avoid false positive RVFV diagnoses that could lead to public health concerns and economically costly agriculture regulatory responses, including quarantine and trade restrictions.

There is strong evidence that equine parvovirus-hepatitis (EqPV-H) is associated with the onset of Theiler's disease, an acute hepatic necrosis, in horses. However, the impact of this virus on other hepatopathies remains unknown. The objective of this retrospective study was to evaluate the prevalence and quantify the viral loads of EqPV-H in formalin-fixed, paraffin-embedded equine and donkey livers with various histopathologic abnormalities. The pathologies included cirrhosis, circulatory disorders of the liver, toxic and metabolic hepatic diseases as well as neoplastic and inflammatory diseases ( n = 84). Eight normal liver samples were included for comparison as controls. EqPV-H DNA was qualitatively and quantitatively measured by real-time PCR and digital PCR, respectively. The virus was detected in two livers originating from horses diagnosed with abdominal neoplasia and liver metastasis (loads of 5 × 10 3 and 9.5 × 10 3 genome equivalents per million cells). The amount of viral nucleic acids measured indicates chronic infection or persistence of EqPV-H, which might have been facilitated by the neoplastic disease. In summary, this study did not provide evidence for EqPV-H being involved in hepatopathies other than Theiler's disease.

Vaccination is an effective method for the prevention of influenza virus infection. Many manufacturers use embryonated chicken eggs (ECE) for the propagation of vaccine strains. However, the adaptation of viral strains during subsequent passages can lead to additional virus evolution and lower effectiveness of the resulting vaccines. In our study, we analyzed the distribution of single nucleotide variants (SNVs) of equine influenza virus (EIV) during passaging in ECE. Viral RNA from passage 0 (nasal swabs), passage 2 and 5 was sequenced using next generation technology. In total, 50 SNVs with an occurrence frequency above 2% were observed, 29 of which resulted in amino acid changes. The highest variability was found in passage 2, with the most variable segment being IV encoding hemagglutinin (HA). Three variants, HA (W222G), PB2 (A377E) and PA (R531K), had clearly increased frequency with the subsequent passages, becoming dominant. None of the five nonsynonymous HA variants directly affected the major antigenic sites; however, S227P was previously reported to influence the antigenicity of EIV. Our results suggest that although host-specific adaptation was observed in low passages of EIV in ECE, it should not pose a significant risk to influenza vaccine efficacy.

Multiple novel circular replication-associated protein (Rep)-encoding single stranded (CRESS) DNA viruses have been extensively identified in the feces of humans and animals. Here, we first detected CRESS DNA virus (named Horse-CRESS DNA-like virus, HCLV) in two fecal samples from 10 imported thoroughbred (TB) horses in the customs quarantine station in North Xinjiang province, China. Additionally, we found that this virus was not detected in local breeds (LBs) (0/41) and was found only in imported TB horses (2/73). We obtained the whole-genome sequences of four viruses (HCLV ALSK-3-4, ALSK-13-10, CJ-1-2, and CJ-13-1). Unlike Circovirus and Cyclovirus, whose genome sequences have 1700 to 2100 nucleotides (nt), these HCLVs have circular genome with 3503, 3504, 3485, 3491 nt, respectively and five major ORFs. The ORF1 gene encodes the Rep protein in HCLVs. Furthermore, the Rep protein of the four HCLVs share 23.3-84.8%, 21.6-27.4%, 23.7-27.2% amino acid identity with the corresponding reference viruses of Kirkoviruses, genus Circovirus, and genus Cyclovirus, respectively. Moreover, RCR domain, P-loop NTPase domains, and nonanucleotide motif (TAGTATTAC) of the HCLVs are similar to Circovirus and Cyclovirus. Phylogenetic analysis showed that the virus was grouped together with members in Kirkoviruses. These results suggest the HCLV probably entered Xinjiang province via the international trade of horses.

Equine rotavirus group A (ERVA) is one of the most common causes of foal diarrhea. Starting in February 2021, there was an increase in the frequency of severe watery to hemorrhagic diarrhea cases in neonatal foals in Central Kentucky. Diagnostic investigation of fecal samples failed to detect evidence of diarrhea-causing pathogens including ERVA. Based on Illumina -based metagenomic sequencing, we identified a novel equine rotavirus group B (ERVB) in fecal specimens from the affected foals in the absence of any other known enteric pathogens. Interestingly, the protein sequence of all 11 segments had greater than 96% identity with group B rotaviruses previously found in ruminants. Furthermore, phylogenetic analysis demonstrated clustering of the ERVB with group B rotaviruses of caprine and bovine strains from the USA. Subsequent analysis of 33 foal diarrheic samples by RT-qPCR identified 23 rotavirus B-positive cases (69.69%). These observations suggest that the ERVB originated from ruminants and was associated with outbreaks of neonatal foal diarrhea in the 2021 foaling season in Kentucky. Emergence of the ruminant-like group B rotavirus in foals clearly warrants further investigation due to the significant impact of the disease in neonatal foals and its economic impact on the equine industry.

Vesicular stomatitis virus (VSV) is an archetypal member of Mononegavirales which causes important diseases in cattle, horses and pigs. The matrix protein (M) of VSV plays critical roles in the replication, assembly/budding and pathogenesis of VSV. To further investigate the role of M during viral growth, we used a two-hybrid system to screen for host factors that interact with the M protein. Here, NADH: ubiquinone oxidoreductase complex assembly factor 4 (Ndufaf4) was identified as an M-binding partner, and this interaction was confirmed by yeast cotransformation and GST pulldown assays. The globular domain of M was mapped and shown to be critical for the M-Ndufaf4 interaction. Two double mutations (E156A/H157A, D180A/E181A) in M impaired the M-Ndufaf4 interaction. Overexpression of Ndufaf4 inhibited VSV propagation, and knockdown of Ndufaf4 by short hairpin RNA (shRNA) markedly promoted VSV replication. Finally, we also demonstrate that the anti-VSV effect of Ndufaf4 is independent of activation of the type I IFN response. These results indicated that Ndufaf4 might exploit other mechanisms to affect VSV replication. In summary, we identify Ndufaf4 as a potential target for the inhibition of VSV propagation. These results provided further insight into the study of VSV pathogenesis.

Genetic reassortment of influenza A viruses through cross-species transmission contributes to the generation of pandemic influenza viruses. To provide information on the ecology of influenza viruses, we have been conducting a global surveillance of zoonotic influenza and establishing an influenza virus library. Of 4580 influenza virus strains in the library, 3891 have been isolated from over 70 different bird species. The remaining 689 strains were isolated from humans, pigs, horses, seal, whale, and the environment. Phylogenetic analyses of the HA genes of the library isolates demonstrate that the library strains are distributed to all major known clusters of the H1, H2 and H3 subtypes of HA genes that are prevalent in humans. Since past pandemic influenza viruses are most likely genetic reassortants of zoonotic and seasonal influenza viruses, a vast collection of influenza A virus strains from various hosts should be useful for vaccine preparation and diagnosis for future pandemics., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Equine influenza (EI) virus is one of the most economically important pathogens of respiratory diseases of horses worldwide. Despite availability of vaccines for control of EI, the highly contagious nature and variability properties of the virus mean global outbreaks occur. Thus, continuous surveillance programs, including seroprevalence studies of disease in different countries, may contribute to better control of the disease. In this study, the seroprevalence of equine influenza in 850 horses from Brazil was investigated. The serodiagnosis was based on the single radial hemolysis (SRH) assay using influenza A/equine/Richmond/1/2007 (H3N8) antigen. Antibodies against A/equine/Richmond/1/07 (H3N8) were detected in 44.7% (380/850, 95% CI: 41.4-48.1%) of horses. Seroprevalence was significantly lower (p = 0.001) in younger animals (< 5 years, 38.6%) than in "adult" animals (5-14 years, 52.1%). There was also a significant relationship between the year of sampling and seroprevalence (p < 0.0005). The mean SRH antibody value was 42.0 mm 2 (range 4-238.9 mm 2 ), with the majority of horses (95.3%) having an SRH value ≤ 150 mm 2 , which is considered an insufficient level for protection of equine hosts against influenza infections and potential virus shedding. These findings indicate the need to reinforce preventive/control measures against equine influenza in Brazil.

This study compared concurrent and separate primary vaccination against equid alphaherpesviruses 1 and 4, genus Varicellovirus, subfamily Alphaherpesvirinae, family Herpesviridae, and equine influenza A virus, genus Alphainfluenzavirus, family Orthomyxoviridae. Their vernacular names are equine herpesvirus 1 and 4 (EHV1/4) and equine influenza virus (EIV). Infection with these respiratory pathogens is associated with loss of performance, interruption of training schedules, and on occasion, cancellation of equestrian events. Vaccination is highly recommended, and for some activities it is a mandatory requirement of the relevant authority. As there is a dearth of information relating to the impact of concurrent vaccination on the antibody response to EHV and EIV vaccines, they are usually administered separately, often 2 weeks apart. In a previous study of booster vaccination in Thoroughbred racehorses, concurrent vaccination with whole-virus inactivated carbopol-adjuvanted EHV and EIV vaccines did not impact negatively on the antibody response. In this study, investigations were extended to concurrent versus separate primary vaccination of warmblood foals. A field study was conducted to compare the immune response to a carbopol-adjuvanted EHV vaccine and an immune stimulating complex (ISCOM)-adjuvanted EI vaccine administered concurrently and 2 weeks apart. No adverse clinical reactions were observed, the pattern of EI and EHV antibody response was similar for both groups, and there was no evidence that concurrent primary vaccination compromised the humoral response. The results are of relevance to horse owners who wish to decrease veterinary costs, limit handling of young animals, and simplify record keeping by vaccinating concurrently.

Surveillance for the emerging infectious disease Eastern equine encephalitis, and its causative virus in mosquitoes, continued within New York State from 2013 to 2019. There were increases in geographic area and number of consecutive years, with cases in four mammalian species, and virus in 11 mosquito species. The first cases in a goat and in an emu were reported. The first detection of virus in Aedes cinereus was reported. Virus in phylogenetic group NY4 was isolated from a horse and from mosquitoes 6 kilometers and 13 days apart in 2013. Phylogenetic groups NY4 and NY5 were found 15 days apart in two towns 280 kilometers distant in 2013. Within four adjacent counties there was a pattern of overlap, where four had NY5, two adjacent counties had NY6, two adjacent counties had NY7, and one county had NY5, NY6, and NY7, reducible to a Euler diagram. Virus in phylogenetic group NY5, found within an 11-kilometer wide area in New York State, was related to FL4 found in Florida 1,398 kilometers distant. This was consistent with a phylogenetic group originating in Florida, then being moved to a specific location in New York State, by migratory birds in consecutive years 2013 and 2014.

Equine Hepacivirus (EqHV) is a newly discovered equine virus that is classified under the Hepacivirus genus of the Flaviviridae family. There are three sub-types of EqHV worldwide namely; sub-types 1-3. The majority of EqHV sub-type 1 strains were found in China. While different sub-types have been found in Japan and USA, therefore, to investigate whether the other sub-types of EqHV strains were present in China, a total of 60 horse serum samples were collected and screened for EqHV RNA through RT-PCR. The results revealed that 19 serum samples were RNA-positive (19/60) and the EqHV detection rate was 31.67%. One EqHV strain named GD23 was obtained and its near-complete genome sequence was acquired. Analysis of nucleotide p-distance with reference to the entire polyprotein gene revealed that GD23 was classified into sub-type 3. In addition, the phylogenetic analysis demonstrated that GD23 was clustered together with EqHV strains of sub-type 3 in other countries. The present study is the first to identify an EqHV sub-type 3 strain in China.

The California Arbovirus Surveillance Program was initiated over 50 years ago to track endemic encephalitides and was enhanced in 2000 to include West Nile virus (WNV) infections in humans, mosquitoes, sentinel chickens, dead birds and horses. This comprehensive statewide program is a function of strong partnerships among the California Department of Public Health (CDPH), the University of California, and local vector control and public health agencies. This manuscript summarizes WNV surveillance data in California since WNV was first detected in 2003 in southern California. From 2003 through 2018, 6,909 human cases of WNV disease, inclusive of 326 deaths, were reported to CDPH, as well as 730 asymptomatic WNV infections identified during screening of blood and organ donors. Of these, 4,073 (59.0%) were reported as West Nile neuroinvasive disease. California's WNV disease burden comprised 15% of all cases that were reported to the U.S. Centers for Disease Control and Prevention during this time, more than any other state. Additionally, 1,299 equine WNV cases were identified, along with detections of WNV in 23,322 dead birds, 31,695 mosquito pools, and 7,340 sentinel chickens. Annual enzootic detection of WNV typically preceded detection in humans and prompted enhanced intervention to reduce the risk of WNV transmission. Peak WNV activity occurred from July through October in the Central Valley and southern California. Less than five percent of WNV activity occurred in other regions of the state or outside of this time. WNV continues to be a major threat to public and wild avian health in California, particularly in southern California and the Central Valley during summer and early fall months. Local and state public health partners must continue statewide human and mosquito surveillance and facilitate effective mosquito control and bite prevention measures., Competing Interests: The authors have declared that no competing interests exist.

Bovine leukemia virus (BLV) is known as the etiological agent of Enzootic bovine leukosis (EBL), which is the most common neoplastic disease of cattle. While the major route of virus transmission is believed to be iatrogenic, BLV proviral DNA has been identified in biological materials, including nasal secretions, saliva, milk, colostrum, and semen, and in several insect species, including horses flies. However, insects' role in the natural transmission of BLV has not been clearly demonstrated. This study assessed the possible role of midges - Culicoides spp. - in BLV transmission. BLVs were genetically characterized and BLV infection seroprevelance was determined in 224 cattle sampled from 27 different small family herds in five different districts in Hatay province, southern Turkey. Out of the 25 Culicoides spp. pools, one (4.0%; 1/25) was a C.schultzei pool while 2.67% (6/224) of the sampled cattle were positive for BLV nucleic acid. The seroprevalance rates for the sampled herds and all sampled cattle were 7.40% (2/27) and 1.33% (3/224), respectively. According to the phylogenetic analysis, the sequences of the BLVs from the cattle (n = 6) and the one BLV-positive C.schultzei pool clustered on genotype 1 (G1) BLVs. Although these results do not reveal the exact role of Culicoides spp. or other midges flies in BLV transmission, the simultaneous presence of same substitions in BLVs from both cattle and a C.schultzei pool is noteworthy. Further studies on the env gene and other BLV gene regions detected from cattle and C.schultzei pools are ongoing to understand the possible epidemiological relationship between cattle and flies., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Susceptibility to long-term persistent infection with Equine Arteritis Virus (EAV) in stallions is related with EqCXCL16 gene alleles of the host. In our study EqCXCL16 gene alleles were determined for 63 EAV shedders and 126 non-shedders of various horse breeds. In total, 60 (31.7%) out of 189 tested stallions were identified as carriers of susceptible variants of EqCXCL16 by real time PCR and Sanger sequencing. The presence of susceptible genotype was related to horse breed with the highest percentage in Wielkopolska breed, Polish coldblood and Silesian breed horses. Strong correlation between EqCXCL16 susceptible genotypes and EAV shedding in semen (p < .0001) was observed., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Our current understanding of the natural evolution of RNA viruses comes largely from consensus level genetic analyses which ignore the diverse mutant swarms that comprise within-host viral populations. The breadth and composition of viral mutant swarms impact viral fitness and adaptation, and the capacity for swarm plasticity is likely to be particularly important for arthropod-borne viruses (arboviruses) that cycle between taxonomically divergent hosts. Despite this, characterization of the relationship between the selective pressures and genetic signatures of the mutant swarm and consensus sequences is lacking. To clarify this, we analyzed previously generated whole genome, deep-sequencing data from 548 West Nile virus samples isolated from avian tissues or mosquitoes in New York State from 1999-2018. Both consensus level (interhost) and minority level (intrahost) nucleotide and amino acid sequences were analyzed, and diversity at each position was calculated across the genome in order to assess the relationship between minority and consensus sequences for individual genes and hosts. Our results indicate that consensus sequences are an inept representation of the overall genetic diversity. Unique host and gene-specific signatures and selective pressures were identified. These data demonstrate that an accurate and comprehensive understanding of arbovirus evolution and adaptation within and between hosts requires consideration of minority genotypes.

Background: In this report we describe the molecular and pathological characteristics of West Nile virus (WNV) infection that occurred during the summer and fall of 2018 in avian species and equines. WNV is reported in Israel since the 1950s, with occasional outbreaks leading to significant morbidity and mortality in birds, high infection in horses and humans, and sporadic fatalities in humans., Methods: Animal and avian carcasses in a suitable condition were examined by post-mortem analysis. Tissue samples were examined for WNV by RT-qPCR and the viral load was quantified. Samples with sufficient material quality were further analyzed by Endpoint PCR and sequencing, which was used for phylogenetic analysis. Tissue samples from positive animals were used for culturing the virus in Vero and C6/36 cells., Results: WNV RNA was detected in one yellow-legged gull (Larus michahellis), two long-eared owls (Asio otus), two domesticated geese (Anser anser), one pheasant (Phasianus colchicus), four hooded crows (Corvus cornix), three horses and one donkey. Pathological and histopathological findings were characteristic of viral infection. Molecular analysis and viral load quantification showed varying degrees of infection, ranging between 70-1.4 × 10 6 target copies per sample. Phylogenetic analysis of a 906-bp genomic segment showed that all samples belonged to Lineage 1 clade 1a, with the following partition: five samples from 2018 and one sample detected in 2016 were of Cluster 2 Eastern European, two of Cluster 2 Mediterranean and four of Cluster 4. Four of the positive samples was successfully propagated in C6/36 and Vero cell lines for further work., Conclusions: WNV is constantly circulating in wild and domesticated birds and animals in Israel, necessitating constant surveillance in birds and equines. At least three WNV strains were circulating in the suspected birds and animals examined. Quantitative analysis showed that the viral load varies significantly between different organs and tissues of the infected animals.

Equid herpesvirus 1 is one of the most common viral pathogens in the horse population and is associated with respiratory disease, abortion and still-birth, neonatal death and neurological disease. A single point mutation in the DNA polymerase gene (ORF30: A2254G, N752D) has been widely associated with neuropathogenicity of strains, although this association has not been exclusive. This study describes the fortuitous isolation of a strain carrying a new genotype C 2254 (H 752 ) from an outbreak in France that lasted several weeks in 2018 and involved 82 horses, two of which showed neurological signs of disease. The strain was characterised as U L clade 10 using the equid herpesvirus 1 (EHV-1) multi-locus sequence typing (MLST) classification but has not been identified or isolated since 2018. The retrospective screening of EHV-1 strains collected between 2016 and 2018 did not reveal the presence of the C 2254 mutation. When cultured in vitro, the C 2254 EHV-1 strain induced a typical EHV-1 syncytium and cytopathic effect but no significant difference was observed when compared with A 2254 and G 2254 EHV-1 strains. An experimental infection was carried out on four Welsh mountain ponies to confirm the infectious nature of the C 2254 strain. A rapid onset of marked respiratory disease lasting at least 2 weeks, with significant virus shedding and cell-associated viraemia, was observed. Finally, an in vitro antiviral assay using impedance measurement and viral load quantification was performed with three antiviral molecules (ganciclovir (GCV), aciclovir (ACV) and aphidicolin (APD)) on the newly isolated C 2254 strain and two other A/G 2254 field strains. The three strains showed similar sensitivity to ganciclovir and aphidicolin but both C 2254 and A 2254 strains were more sensitive to aciclovir than the G 2254 strain, based on viral load measurement.

Bluetongue virus (BTV) and African horse sickness virus (AHSV) are vector-borne viruses belonging to the Orbivirus genus, which are transmitted between hosts primarily by biting midges of the genus Culicoides . With recent BTV and AHSV outbreaks causing epidemics and important economy losses, there is a pressing need for efficacious drugs to treat and control the spread of these infections. The polyanionic aromatic compound aurintricarboxylic acid (ATA) has been shown to have a broad-spectrum antiviral activity. Here, we evaluated ATA as a potential antiviral compound against Orbivirus infections in both mammalian and insect cells. Notably, ATA was able to prevent the replication of BTV and AHSV in both cell types in a time- and concentration-dependent manner. In addition, we evaluated the effect of ATA in vivo using a mouse model of infection. ATA did not protect mice against a lethal challenge with BTV or AHSV, most probably due to the in vivo effect of ATA on immune system regulation. Overall, these results demonstrate that ATA has inhibitory activity against Orbivirus replication in vitro, but further in vivo analysis will be required before considering it as a potential therapy for future clinical evaluation.

Lentiviral DNA integration favors transcriptionally active chromatin. We previously showed that the interaction of human immunodeficiency virus type 1 (HIV-1) capsid with cleavage and polyadenylation specificity factor 6 (CPSF6) localizes viral preintegration complexes (PICs) to nuclear speckles for integration into transcriptionally active speckle-associated domains (SPADs). In the absence of the capsid-CPSF6 interaction, PICs uncharacteristically accumulate at the nuclear periphery and target heterochromatic lamina-associated domains (LADs) for integration. The integrase-binding protein lens epithelium-derived growth factor (LEDGF)/p75 in contrast to CPSF6 predominantly functions to direct HIV-1 integration to interior regions of transcription units. Though CPSF6 and LEDGF/p75 can reportedly interact with the capsid and integrase proteins of both primate and nonprimate lentiviruses, the extents to which these different viruses target SPADs versus LADs, as well as their dependencies on CPSF6 and LEDGF/p75 for integration targeting, are largely unknown. Here, we mapped 5,489,157 primate and nonprimate lentiviral integration sites in HEK293T and Jurkat T cells as well as derivative cells that were knocked out or knocked down for host factor expression. Despite marked preferences of all lentiviruses to target genes for integration, nonprimate lentiviruses only marginally favored SPADs, with corresponding upticks in LAD-proximal integration. While LEDGF/p75 knockout disrupted the intragenic integration profiles of all lentiviruses similarly, CPSF6 depletion specifically counteracted SPAD integration targeting by primate lentiviruses. CPSF6 correspondingly failed to appreciably interact with nonprimate lentiviral capsids. We conclude that primate lentiviral capsid proteins evolved to interact with CPSF6 to optimize PIC localization for integration into transcriptionally active SPADs. IMPORTANCE Integration is the defining step of the retroviral life cycle and underlies the inability to cure HIV/AIDS through the use of intensified antiviral therapy. The reservoir of latent, replication-competent proviruses that forms early during HIV infection reseeds viremia when patients discontinue medication. HIV cure research is accordingly focused on the factors that guide provirus formation and associated chromatin environments that regulate transcriptional reactivation, and studies of orthologous infectious agents such as nonprimate lentiviruses can inform basic principles of HIV biology. HIV-1 utilizes the integrase-binding protein LEDGF/p75 and the capsid interactor CPSF6 to target speckle-associated domains (SPADs) for integration. However, the extent to which these two host proteins regulate integration of other lentiviruses is largely unknown. Here, we mapped millions of retroviral integration sites in cell lines that were depleted for LEDGF/p75 and/or CPSF6. Our results reveal that primate lentiviruses uniquely target SPADs for integration in a CPSF6-dependent manner., (Copyright © 2020 Li et al.)

Coronaviruses are enveloped RNA viruses capable of causing respiratory, enteric, or systemic diseases in a variety of mammalian hosts that vary in clinical severity from subclinical to fatal. The host range and tissue tropism are largely determined by the coronaviral spike protein, which initiates cellular infection by promoting fusion of the viral and host cell membranes. Companion animal coronaviruses responsible for causing enteric infection include feline enteric coronavirus, ferret enteric coronavirus, canine enteric coronavirus, equine coronavirus, and alpaca enteric coronavirus, while canine respiratory coronavirus and alpaca respiratory coronavirus result in respiratory infection. Ferret systemic coronavirus and feline infectious peritonitis virus, a mutated feline enteric coronavirus, can lead to lethal immuno-inflammatory systemic disease. Recent human viral pandemics, including severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and most recently, COVID-19, all thought to originate from bat coronaviruses, demonstrate the zoonotic potential of coronaviruses and their potential to have devastating impacts. A better understanding of the coronaviruses of companion animals, their capacity for cross-species transmission, and the sharing of genetic information may facilitate improved prevention and control strategies for future emerging zoonotic coronaviruses. This article reviews the clinical, epidemiologic, virologic, and pathologic characteristics of nine important coronaviruses of companion animals.

A rare genotype G13P[18] group A rotavirus (RVA/Horse-tc/JPN/MK9/2019/G13P[18]) was isolated from a diarrhoeic foal for the first time in 28 years. The genotype constellation of the virus was assigned to G13-P[18]-I6-R9-C9-M6-A6-N9-T12-E14-H11 and was the same as that of the first isolated strain, RVA/Horse-tc/GBR/L338/1991/G13P[18]. Phylogenetic analysis suggests that the virus is related to RVA/Horse-tc/GBR/L338/1991/G13P[18] and is distant from typical equine rotaviruses of the G3P[12] and G14P[12] genotypes.

Background: Ross River virus (RRV) is a zoonotic alphavirus transmitted by several mosquito species. Until recently, endemic transmission was only considered possible in the presence of marsupial reservoirs., Methods: RRV seroprevalence was investigated in placental mammals (including horses, cows, goats, pigs, dogs, rats, and mice) in Fiji, where there are no marsupials. A total of 302 vertebrate serum samples were collected from 86 households from 10 communities in Western Fiji., Results: Neutralizing antibodies against RRV were detected in 28% to 100% of sera depending on the species, and neutralization was strong even at high dilutions., Conclusions: These results are unlikely to be due to cross-reactions. Chikungunya is the only other alphavirus known to be present in the Pacific Islands, but it rarely spills over into non-humans, even during epidemics. The study findings, together with a recent report of high RRV seroprevalence in humans, strongly suggest that RRV is circulating in Fiji in the absence of marsupial reservoirs. Considering that all non-human vertebrates present in Fiji are pan-global in distribution, RRV has the potential to further expand its geographic range. Further surveillance of RRV and access to RRV diagnostics will be critical for the early detection of emergence and outbreaks., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Equine infectious anemia (EIA), a disease caused by equine infectious anemia virus (EIAV), is considered an obstacle to the development of the horse industry. There is no treatment or vaccine available for EIA, and its pathogenesis, as well as the immune response against the virus, is not fully understood. Therefore, an immunohistochemistry assay was developed for the detection of viral antigens in tissues of equids naturally infected with EIAV. Sections of organs of six equids from Apodi-RN, Brazil, that tested positive for EIA by serological tests (ELISA and AGID) were fixed in 10% formalin solution and embedded in paraffin. Immunohistochemistry was performed using a polyclonal anti-EIAV antibody. EIAV antigens were observed in red spleen pulp cells and hepatic sinusoids, as well as bronchiolar and alveolar epithelial cells of the lungs and proximal and distal tubules of the kidneys. The presence of EIAV in the spleen and liver was expected due to viral tropism by macrophages, which are abundantly present in these organs. However, EIAV was also found in lung and kidney epithelial cells, indicating that the virus infects cell types other than macrophages. In conclusion, the immunohistochemical assay standardized in this study was able to detect EIAV antigens in spleen, liver, kidney and lung cells from naturally infected EIAV equids. Immunostaining observed in the spleen confirms viral tropism by mononuclear phagocytes; however, the presence of EIAV in lung and kidney epithelial cells indicates that virus may be eliminated in urine and/or oronasal secretions, suggesting new routes for viral excretion.

Equine hepacivirus (EHV) belongs to the hepacivirus A and is related to hepatitis C virus (HCV). This virus shows hepatic tropism and is known to chronically infect horses. EHV has been reported from various countries, but the prevalence in Mongolia, where large horse populations are pastured, remains unknown. This study collected serum samples from horses in six areas across Mongolia, in order to investigate the status of infection. The possibility of human infection was also examined. The results showed an infection rate among horses of about 40 % in all regions. However, no evidence of EHV viremia was found in human serum. A mutation characteristic of Mongolian EHV was found in the 5'-untranslated region of the viral sequence. Molecular phylogenetic trees for core, NS3, and NS5B sequences showed the formation of two clusters depending on the area from which samples were taken. The same results were obtained from molecular phylogenetic analyses using the full genome. From detailed calculations of genetic diversity calculated using the full genome, EHV appears divisible into two subgenotypes. Blood samples were collected again after a 7-month interval to examine infection persistence. Seventeen of 19 horses retested showed positive results for EHV after 7 months, suggesting a high rate of persistent infection. These results indicate a relatively higher frequency of EHV infection in Mongolia than in Europe or North America, with virus strains divided into at least two subgenotypes., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to declare., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The equine encephalitis viruses, Venezuelan (VEEV), East (EEEV) and West (WEEV), belong to the genus alphavirus, family Togaviridae and still represent a threat for human and animal public health in the Americas. In both, these infections are characterized by high viremia, rash, fever, encephalitis and death. VEEV encephalitis is similar, clinically, to other arboviral diseases, such as dengue, Zika or chikungunya. Most of the alphaviruses are transmitted between vertebrates and mosquitoes. They are able to replicate in a wide number of hosts, including mammals, birds, reptiles, amphibian and arthropods. The VEEV has enzootic and epizootic transmission cycles. At the enzootic one, enzootic strains (subtype I, serotypes D-F and serotypes II-VI) are continuously circulating between mosquitoes and wild rodents in tropical forests and mangroves of the Americas. The main reseroivrs are wild rodent species of the subfamily Sigmodontinae. However, bats can be also accidental reservoirs of VEEV. In this article, we reviewed the main features, epidemiology, clinical aspects and the current perspectives of the VEEV.

West Nile virus (WNV) is one of the most widely distributed mosquito-borne viruses in the world. In North Africa, it causes human cases of meningoencephalitis with fatalities in Algeria and in Tunisia, whereas only horses were affected in Morocco. The aims of this study were to detect WNV in mosquitoes and to determine seroprevalence of WNV in Moroccan horses by the detection of IgG antibodies. A total of 1455 mosquitoes belonging to four different species were grouped by collection site, date, and sex with 10 specimens per pool and tested for 38 arboviruses using a high-throughput chip based on the BioMark Dynamic array system. Out of 146 mosquito pools tested, one pool was positive for WNV. This positive pool was confirmed by real time RT-PCR. The serosurvey showed that 33.7% (31/92) of horses were positive for competitive enzyme-linked immunosorbent assay (cELISA) test. The flavivirus-sphere microsphere immnoassay (MIA) test, targeting three flaviviruses (WNV, Usutu virus (USUV) and Tick borne encephalitic virus (TBEV)) showed that 23 sera out of 31 were positive for WNV, two for USUV, two for USUV or WNV, and four for an undetermined flavivirus. Virus neutralization tests with USUV and WNV showed that 28 of 31 sera were positive for WNV and all sera were negative for USUV. This study reports, for the first time, the detection of WNV from Culex pipiens mosquitoes in Morocco and its circulation among horses. This highlights that the detection of arboviruses in mosquitoes could serve as an early warning signal of a viral activity to prevent future outbreaks in animals and humans., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)