Genomic Analysis of Infectious Bursal Disease Virus in Nigeria: Identification of Unique Mutations of Yet Unknown Biological Functions in Both Segments A and B.

Infectious bursal disease (IBD) is a viral poultry disease known worldwide for impacting the economy and food security. The disease is endemic in Nigeria, with reported outbreaks in vaccinated poultry flocks. To gain insight into the dynamics of infectious bursal disease virus (IBDV) evolution in Nigeria, near-complete genomes of four IBDVs were evaluated. Amino acid sequences in the hypervariable region of the VP2 revealed conserved markers (222A, 242I, 256I, 294I and 299S) associated with very virulent (vv) IBDV, including the serine-rich heptapeptide motif (SWSASGS). Based on the newly proposed classification for segments A and B, the IBDVs clustered in the A3B5 group (where A3 are IBDVs with vvIBDV-like segment A, and where B5 are from non-vvIBDV-like segment B) form a monophyletic subcluster. Unique amino acid mutations with yet-to-be-determined biological functions have been observed in both segments. Amino acid sequences of the Nigerian IBDVs showed that they are reassortant viruses. Circulation of reassortant IBDVs may be responsible for the vaccination failures observed in the Nigerian poultry population. Close monitoring of changes in the IBDV genome is recommended to nip deleterious changes in the bud through the identification and introduction of the most appropriate vaccine candidates and advocacy/extension programs for properly implementing disease control.

Host Response of Syrian Hamster to SARS-CoV-2 Infection including Differences with Humans and between Sexes.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the importance of having proper tools and models to study the pathophysiology of emerging infectious diseases to test therapeutic protocols, assess changes in viral phenotypes, and evaluate the effects of viral evolution. This study provided a comprehensive characterization of the Syrian hamster (Mesocricetus auratus) as an animal model for SARS-CoV-2 infection using different approaches (description of clinical signs, viral load, receptor profiling, and host immune response) and targeting four different organs (lungs, intestine, brain, and PBMCs). Our data showed that both male and female hamsters were susceptible to the infection and developed a disease similar to the one observed in patients with COVID-19 that included moderate to severe pulmonary lesions, inflammation, and recruitment of the immune system in the lungs and at the systemic level. However, all animals recovered within 14 days without developing the severe pathology seen in humans, and none of them died. We found faint evidence for intestinal and neurological tropism associated with the absence of lesions and a minimal host response in intestines and brains, which highlighted another crucial difference with the multiorgan impairment of severe COVID-19. When comparing male and female hamsters, we observed that males sustained higher viral RNA shedding and replication in the lungs, suffered from more severe symptoms and histopathological lesions, and triggered higher pulmonary inflammation. Overall, these data confirmed the Syrian hamster as a suitable model for mild to moderate COVID-19 and reflected sex-related differences in the response against the virus observed in humans.

Discovery of a coronavirus in the Eurasian badger (Meles meles) belonging to a putative new genus.

In the aftermath of COVID-19, coronaviruses gained renewed attention by the scientific community. The study reports the identification and genetic characterization of a novel coronavirus in the European badger (Meles meles) obtained in the framework of passive surveillance implemented in Italian wildlife in response to the pandemic. Positive samples were characterized using next generation sequencing as well as genetic and phylogenetic analyses, aiming for taxonomic placement under ICTV guidelines of the viruses contained in each sample. Results obtained for six conserved domains within the polyprotein showed that the virus clustered as outgroup and shared <46% amino acid identity with other coronaviruses, supporting the assumption that it belongs to a new putative genus Epsiloncoronavirus. This finding highlights that mammals still hide diverse coronaviruses whose zoonotic and epizootic potential remains unknown.

Transcriptome Profiling of Oncorhynchus mykiss Infected with Low or Highly Pathogenic Viral Hemorrhagic Septicemia Virus (VHSV).

The rainbow trout (Oncorhynchus mykiss) is the most important produced species in freshwater within the European Union, usually reared in intensive farming systems. This species is highly susceptible to viral hemorrhagic septicemia (VHS), a severe systemic disease widespread globally throughout the world. Viral hemorrhagic septicemia virus (VHSV) is the etiological agent and, recently, three classes of VHSV virulence (high, moderate, and low) have been proposed based on the mortality rates, which are strictly dependent on the viral strain. The molecular mechanisms that regulate VHSV virulence and the stimulated gene responses in the host during infection are not completely unveiled. While some preliminary transcriptomic studies have been reported in other fish species, to date there are no publications on rainbow trout. Herein, we report the first time-course RNA sequencing analysis on rainbow trout juveniles experimentally infected with high and low VHSV pathogenic Italian strains. Transcriptome analysis was performed on head kidney samples collected at different time points (1, 2, and 5 days post infection). A large set of notable genes were found to be differentially expressed (DEGs) in all the challenged groups (e.s. trim63a, acod1, cox-2, skia, hipk1, cx35.4, ins, mtnr1a, tlr3, tlr7, mda5, lgp2). Moreover, the number of DEGs progressively increased especially during time with a greater amount found in the group infected with the high VHSV virulent strain. The gene ontology (GO) enrichment analysis highlighted that functions related to inflammation were modulated in rainbow trout during the first days of VHSV infection, regardless of the pathogenicity of the strain. While some functions showed slight differences in enrichments between the two infected groups, others appeared more exclusively modulated in the group challenged with the highly pathogenic strain.

Identification of Dobrava-Belgrade Virus in Apodemus flavicollis from North-Eastern Italy during Enhanced Mortality.

Hantaviruses include several zoonotic pathogens that cause different syndromes in humans, with mortality rates ranging from 12 to 40%. Most commonly, humans get infected through the inhalation of aerosols or dust particles contaminated with virus-containing rodent excreta. Hantaviruses are specifically associated with the host species, and human cases depend on the presence and the dynamics of reservoir hosts. In this letter, we report the identification of Dobrava-Belgrade virus (DOBV) in the yellow-necked mouse (Apodemus flavicollis) from Italy. The virus was detected in the mountainous area of the province of Udine, bordering Austria and Slovenia, during an event of enhanced mortality in wild mice and voles. Despite serological evidence in rodents and humans that suggested the circulation of hantaviruses in Italy since 2000, this is the first virological confirmation of the infection. Phylogenetic analyses across the whole genome of the two detected viruses confirmed the host-specificity of DOBV sub-species and showed the highest identity with viruses identified in Slovenia and Croatia from both A. flavicollis and humans, with no signs of reassortment. These findings highlight the need for ecologists, veterinarians and medical doctors to come together in a coordinated approach in full compliance with the One Health concept.

Clinical Tick-Borne Encephalitis in a Roe Deer (Capreolus capreolus L.).

Tick-borne encephalitis virus (TBEV) is the causative agent of tick-borne encephalitis (TBE), a severe zoonosis occurring in the Palearctic region mainly transmitted through Ixodes ticks. In Italy, TBEV is restricted to the north-eastern part of the country. This report describes for the first time a case of clinical TBE in a roe deer (Capreolus capreolus L.). The case occurred in the Belluno province, Veneto region, an area endemic for TBEV. The affected roe deer showed ataxia, staggering movements, muscle tremors, wide-base stance of the front limbs, repetitive movements of the head, persistent teeth grinding, hypersalivation and prolonged recumbency. An autopsy revealed no significant lesions to explain the neurological signs. TBEV RNA was detected in the brain by real-time RT-PCR, and the nearly complete viral genome (10,897 nucleotides) was sequenced. Phylogenetic analysis of the gene encoding the envelope protein revealed a close relationship to TBEV of the European subtype, and 100% similarity with a partial sequence (520 nucleotides) of a TBEV found in ticks in the bordering Trento province. The histological examination of the midbrain revealed lymphohistiocytic encephalitis, satellitosis and microgliosis, consistent with a viral etiology. Other viral etiologies were ruled out by metagenomic analysis of the brain. This report underlines, for the first time, the occurrence of clinical encephalitic manifestations due to TBEV in a roe deer, suggesting that this pathogen should be included in the frame of differential diagnoses in roe deer with neurologic disease.

Increased virulence of Italian infectious hematopoietic necrosis virus (IHNV) associated with the emergence of new strains.

Infectious hematopoietic necrosis virus (IHNV) is the causative agent of IHN triggering a systemic syndrome in salmonid fish. Although IHNV has always been associated with low levels of mortality in Italian trout farming industries, in the last years trout farmers have experienced severe disease outbreaks. However, the observed increasing virulence of IHNV is still based on empirical evidence due to the poor and often confounding information from the field. Virulence characterization of a selection of sixteen Italian isolates was performed through in vivo challenge of juvenile rainbow trout to confirm field evidence. The virulence of each strain was firstly described in terms of cumulative mortality and survival probability estimated by Kaplan-Meier curves. Furthermore, parametric survival models were applied to analyze the mortality rate profiles. Hence, it was possible to characterize the strain-specific mortality peaks and to relate their topology to virulence and mortality. Indeed, a positive correlation between maximum mortality probability and virulence was observed for all the strains. Results also indicate that more virulent is the strain, the earliest and narrowest is the mortality peak. Additionally, intra-host viral quantification determined in dead animals showed a significant correlation between viral replication and virulence. Whole-genome phylogeny conducted to determine whether there was a relation between virulence phenotype and IHNV genetics evidenced no clear clustering according to phenotype. Moreover, a root-to-tip analysis based on genetic distances and sampling date of Italian IHNV isolates highlighted a relevant temporal signal indicating an evolving nature of the virus, over time, with the more virulent strains being the more recent ones. This study provides the first systematic characterization of Italian IHNV's virulence. Overall results confirm field data and point out an abrupt increase in IHNV virulence, with strains from 2015-2019 showing moderate to high virulence in rainbow trout. Further investigations are needed in order to extensively clarify the relation between evolution and virulence of IHNV and investigate the genetic determinants of virulence of this viral species in rainbow trout.

Heterogeneity of Early Host Response to Infection with Four Low-Pathogenic H7 Viruses with a Different Evolutionary History in the Field.

Once low-pathogenic avian influenza viruses (LPAIVs) of the H5 and H7 subtypes from wild birds enter into poultry species, there is the possibility of them mutating into highly pathogenic avian influenza viruses (HPAIVs), resulting in severe epizootics with up to 100% mortality. This mutation from a LPAIV to HPAIV strain is the main cause of an AIV's major economic impact on poultry production. Although AIVs are inextricably linked to their hosts in their evolutionary history, the contribution of host-related factors in the emergence of HPAI viruses has only been marginally explored so far. In this study, transcriptomic sequencing of tracheal tissue from chickens infected with four distinct LP H7 viruses, characterized by a different history of pathogenicity evolution in the field, was implemented. Despite the inoculation of a normalized infectious dose of viruses belonging to the same subtype (H7) and pathotype (LPAI), the use of animals of the same age, sex and species as well as the identification of a comparable viral load in the target samples, the analyses revealed a heterogeneity in the gene expression profile in response to infection with each of the H7 viruses administered.

Characterization of changes in the hemagglutinin that accompanied the emergence of H3N2/1968 pandemic influenza viruses.

The hemagglutinin (HA) of A/H3N2 pandemic influenza viruses (IAVs) of 1968 differed from its inferred avian precursor by eight amino acid substitutions. To determine their phenotypic effects, we studied recombinant variants of A/Hong Kong/1/1968 virus containing either human-type or avian-type amino acids in the corresponding positions of HA. The precursor HA displayed receptor binding profile and high conformational stability typical for duck IAVs. Substitutions Q226L and G228S, in addition to their known effects on receptor specificity and replication, marginally decreased HA stability. Substitutions R62I, D63N, D81N and N193S reduced HA binding avidity. Substitutions R62I, D81N and A144G promoted viral replication in human airway epithelial cultures. Analysis of HA sequences revealed that substitutions D63N and D81N accompanied by the addition of N-glycans represent common markers of avian H3 HA adaptation to mammals. Our results advance understanding of genotypic and phenotypic changes in IAV HA required for avian-to-human adaptation and pandemic emergence.

Interface between Bats and Pigs in Heavy Pig Production.

Bats are often claimed to be a major source for future viral epidemics, as they are associated with several viruses with zoonotic potential. Here we describe the presence and biodiversity of bats associated with intensive pig farms devoted to the production of heavy pigs in northern Italy. Since chiropters or signs of their presence were not found within animal shelters in our study area, we suggest that fecal viruses with high environmental resistance have the highest likelihood for spillover through indirect transmission. In turn, we investigated the circulation of mammalian orthoreoviruses (MRVs), coronaviruses (CoVs) and astroviruses (AstVs) in pigs and bats sharing the same environment. Results of our preliminary study did not show any bat virus in pigs suggesting that spillover from these animals is rare. However, several AstVs, CoVs and MRVs circulated undetected in pigs. Among those, one MRV was a reassortant strain carrying viral genes likely acquired from bats. On the other hand, we found a swine AstV and a MRV strain carrying swine genes in bat guano, indicating that viral exchange at the bat-pig interface might occur more frequently from pigs to bats rather than the other way around. Considering the indoor farming system as the most common system in the European Union (EU), preventive measures should focus on biosecurity rather than displacement of bats, which are protected throughout the EU and provide critical ecosystem services for rural settings.

Multifactorial Causes of Chronic Mortality in Juvenile Sturgeon (Huso huso).

This investigation focused on an episode of chronic mortality observed in juvenile Huso huso sturgeons. The examined subjects underwent pathological, microbiological, molecular, and chemical investigations. Grossly severe body shape deformities, epaxial muscle softening, and multifocal ulcerative dermatitis were the main observed findings. The more constant histopathologic findings were moderate to severe rarefaction and disorganization of the lymphohematopoietic lymphoid tissues, myofiber degeneration, atrophy and interstitial edema of skeletal epaxial muscles, and degeneration and atrophy of the gangliar neurons close to the myofibers. Chemical investigations showed a lower selenium concentration in affected animals, suggesting nutritional myopathy. Other manifestations were nephrocalcinosis and splenic vessel wall hyalinosis. Septicemia due to bacteria such as Aeromonas veronii, Shewanella putrefaciens, Citrobacter freundii, Chryseobacterium sp., and pigmented hyphae were found. No major sturgeon viral pathogens were detected by classical methods. Next-generation sequencing (NGS) analysis confirmed the absence of viral pathogens, with the exception of herpesvirus, at the order level; also, the presence of Aeromonas veronii and Shewanella putrefaciens was confirmed at the family level by the metagenomic classification of NGS data. In the absence of a primary yet undetected biological cause, it is supposed that environmental stressors, including nutritional imbalances, may have led to immune system impairment, facilitating the entry of opportunistic bacteria and mycotic hyphae.

Unexpected rabies variant identified in kinkajou (Potos flavus), Mato Grosso, Brazil.

A second case of a novel rabies variant described once in a capuchin monkey from Mato Grosso, Brazil, was discovered in a rabid wild kinkajou from the same region, indicating a public health risk following exposure to either of the two animals.

Unexpected Genetic Diversity of Two Novel Swine MRVs in Italy.

Mammalian Orthoreoviruses (MRV) are segmented dsRNA viruses in the family Reoviridae. MRVs infect mammals and cause asymptomatic respiratory, gastro-enteric and, rarely, encephalic infections. MRVs are divided into at least three serotypes: MRV1, MRV2 and MRV3. In Europe, swine MRV (swMRV) was first isolated in Austria in 1998 and subsequently reported more than fifteen years later in Italy. In the present study, we characterized two novel reassortant swMRVs identified in one same Italian farm over two years. The two viruses shared the same genetic backbone but showed evidence of reassortment in the S1, S4, M2 segments and were therefore classified into two serotypes: MRV3 in 2016 and MRV2 in 2018. A genetic relation to pig, bat and human MRVs and other unknown sources was identified. A considerable genetic diversity was observed in the Italian MRV3 and MRV2 compared to other available swMRVs. The S1 protein presented unique amino acid signatures in both swMRVs, with unexpected frequencies for MRV2. The remaining genes formed distinct and novel genetic groups that revealed a geographically related evolution of swMRVs in Italy. This is the first report of the complete molecular characterization of novel reassortant swMRVs in Italy and Europe, which suggests a greater genetic diversity of swMRVs never identified before.

Characterization of novel, pathogenic field strains of infectious bronchitis virus (IBV) in poultry in Trinidad and Tobago.

Avian coronaviruses, including infectious bronchitis virus (IBV) and turkey coronavirus (TCoV), are economically important viruses affecting poultry worldwide. IBV is responsible for causing severe losses to the commercial poultry sector globally. The objectives of this study were to identify the viruses that were causing outbreaks of severe respiratory disease in chickens in Trinidad and Tobago (T&T) and to characterize the strains. Swab samples were collected from birds showing severe respiratory signs in five farms on the island of Trinidad. Samples were tested for the presence of IBV, as well as avian influenza virus (AIV), Newcastle disease virus (NDV) and avian metapneumovirus (aMPV) by real-time reverse transcription polymerase chain reaction (qRT-PCR). All samples from the five farms tested negative for AIV, NDV and aMPV; however, samples from clinically affected birds in all five of the farms tested positive for IBV. Genetic data revealed the presence of TCoV in chickens on two of the farms. Interestingly, these two farms had never reared turkeys. Phylogenetic analysis showed that IBV S1 sequences formed two distinct clusters. Two sequences grouped with vaccine strains within the GI-1 lineage, whereas three sequences grouped together, but separately from other defined lineages, forming a likely new lineage of IBV. Pairwise comparison revealed that the three unique variant strains within the distinct lineage of IBV were significantly different in their S1 nucleotide coding regions from viruses in the closest lineage (16% difference) and locally used vaccine strains (>20% difference). Results also suggested that one of the samples was a recombinant virus, generated from a recombination event between a Trinidad virus of the GI-1 lineage and a Trinidad virus of the newly defined lineage. Many amino acid differences were also observed between the S1 coding regions of the circulating field and vaccine strains, indicating that the IBV vaccines may not be protective. Vaccine-challenge studies are however needed to prove this.

Spatial spread and emergence of reassortant H5 highly pathogenic avian influenza viruses in Iran.

Since 2005, H5Nx highly pathogenic avian influenza (HPAI) viruses of the Goose/Guangdong (Gs/GD) lineage have spread worldwide, affecting poultry and wild birds in Asia, Europe, Africa and North America. So far, the role of Western Asia and the Middle East in the diffusion dynamics of this virus has been poorly explored. In order to investigate the genetic diversity and the role of Iran in the transmission dynamics of the Gs/GD lineage, we sequenced the complete genome of twenty-eight H5Nx viruses which were circulating in the country between 2016 and 2018. We reported the first characterization of the HPAI H5N6 subtype of clade 2.3.4.4B in Iran and gave evidence of the high propensity of the Gs/GD H5 AIVs to reassort, describing six novel H5N8 genotypes of clade 2.3.4.4B, some of them likely generated in this area, and one H5N1 reassortant virus of clade 2.3.2.1c. Our spatial analyses demonstrated that the viruses resulted from different viral introductions from Asia and Europe and provided evidence of virus spread from Iran to the Middle East. Therefore, Iran may represent a hot-spot for virus introduction, dissemination and for the generation of new genetic variability. Increasing surveillance efforts in this high-risk area is of utmost importance for the early detection of novel emerging strains with zoonotic potential.

Genomic Sequence of a New Alphavirus Detected in Comber (Serranus cabrilla).

The comber alphavirus was isolated from a fish cell line from the brain of an apparently healthy Serranus cabrilla specimen collected during wild fish surveillance in southern Italy. The comber alphavirus is a new member of the genus Alphavirus, family Togaviridae.

Sequencing of animal viruses: quality data assurance for NGS bioinformatics.

BACKGROUND: Next generation sequencing (NGS) is becoming widely used among diagnostics and research laboratories, and nowadays it is applied to a variety of disciplines, including veterinary virology. The NGS workflow comprises several steps, namely sample processing, library preparation, sequencing and primary/secondary/tertiary bioinformatics (BI) analyses. The latter is constituted by a complex process extremely difficult to standardize, due to the variety of tools and metrics available. Thus, it is of the utmost importance to assess the comparability of results obtained through different methods and in different laboratories. To achieve this goal, we have organized a proficiency test focused on the bioinformatics components for the generation of complete genome sequences of salmonid rhabdoviruses. METHODS: Three partners, that performed virus sequencing using different commercial library preparation kits and NGS platforms, gathered together and shared with each other 75 raw datasets which were analyzed separately by the participants to produce a consensus sequence according to their own bioinformatics pipeline. Results were then compared to highlight discrepancies, and a subset of inconsistencies were investigated more in detail. RESULTS: In total, we observed 526 discrepancies, of which 39.5% were located at genome termini, 14.1% at intergenic regions and 46.4% at coding regions. Among these, 10 SNPs and 99 indels caused changes in the protein products. Overall reproducibility was 99.94%. Based on the analysis of a subset of inconsistencies investigated more in-depth, manual curation appeared the most critical step affecting sequence comparability, suggesting that the harmonization of this phase is crucial to obtain comparable results. The analysis of a calibrator sample allowed assessing BI accuracy, being 99.983%. CONCLUSIONS: We demonstrated the applicability and the usefulness of BI proficiency testing to assure the quality of NGS data, and recommend a wider implementation of such exercises to guarantee sequence data uniformity among different virology laboratories.

Disentangling the role of Africa in the global spread of H5 highly pathogenic avian influenza.

The role of Africa in the dynamics of the global spread of a zoonotic and economically-important virus, such as the highly pathogenic avian influenza (HPAI) H5Nx of the Gs/GD lineage, remains unexplored. Here we characterise the spatiotemporal patterns of virus diffusion during three HPAI H5Nx intercontinental epidemic waves and demonstrate that Africa mainly acted as an ecological sink of the HPAI H5Nx viruses. A joint analysis of host dynamics and continuous spatial diffusion indicates that poultry trade as well as wild bird migrations have contributed to the virus spreading into Africa, with West Africa acting as a crucial hotspot for virus introduction and dissemination into the continent. We demonstrate varying paths of avian influenza incursions into Africa as well as virus spread within Africa over time, which reveal that virus expansion is a complex phenomenon, shaped by an intricate interplay between avian host ecology, virus characteristics and environmental variables.

Complete Genome Sequences of Three Rabbit Endogenous Lentivirus Type K Viruses Obtained from Commercial Meat Rabbits in Italy.

Rabbit endogenous lentivirus type K (RELIK) was discovered in the genome of the European rabbit (Oryctolagus cuniculus). In our study, we present three complete genome sequences of RELIK viruses generated using a target amplification approach performed on the RNA of commercial rabbits from Italy.

First whole genome characterization of porcine astrovirus detected in swine faeces in Italy.

Porcine astroviruses (PoAstV) are found in the gastrointestinal tract of healthy and diseased pigs worldwide. However, their role in causing enteric disease in pigs and other animals has not been elucidated. In the present report, we describe for the first time in Italy, the identification and genetic characterization, through whole genome sequencing, of a PoAstV2 in pigs in Northeast Italy in 2015. This instance is the first detection of PoAstV2 in pigs in Italy. The phylogenetic analysis of the complete ORF2 segment highlights the high similarity of this virus to those circulating that same year in Japan. There are very few full astrovirus genomes available, and the present data represent an important contribution towards a better understanding of the characteristics and evolution of these viruses.