Ocrelizumab B cell depletion has no effect on HERV RNA expression in PBMC in MS patients.

BACKGROUND: Epstein barr virus (EBV) infection of B cells is now understood to be one of the triggering events for the development of Multiple Sclerosis (MS), a progressive immune-mediated disease of the central nervous system. EBV infection is also linked to expression of human endogenous retroviruses (HERVs) of the HERV-W group, a further risk factor for the development of MS. Ocrelizumab is a high-potency disease-modifying treatment (DMT) for MS, which depletes B cells by targeting CD20. OBJECTIVES: We studied the effects of ocrelizumab on gene expression in peripheral blood mononuclear cells (PBMC) from paired samples from 20 patients taken prior to and 6 months after beginning ocrelizumab therapy. We hypothesised that EBV and HERV-W loads would be lower in post-treatment samples. METHODS: Samples were collected in Paxgene tubes, subject to RNA extraction and Illumina paired end short read mRNA sequencing with mapping of sequence reads to the human genome using Salmon and differential gene expression compared with DeSeq2. Mapping was also performed separately to the HERV-D database of HERV sequences and the EBV reference sequence. RESULTS: Patient samples were more strongly clustered by individual rather than disease type (relapsing/remitting or primary progressive), treatment (pre and post), age, or sex. Fourteen genes, all clearly linked to B cell function were significantly down regulated in the post treatment samples. Interestingly only one pre-treatment sample had detectable EBV RNA and there were no significant differences in HERV expression (of any group) between pre- and post-treatment samples. CONCLUSIONS: While EBV and HERV expression are clearly linked to triggering MS pathogenesis, it does not appear that high level expression of these viruses is a part of the ongoing disease process or that changes in virus load are associated with ocrelizumab treatment.

Lack of detection of SARS-CoV-2 in wildlife from Kerala, India in 2020-21.

Spillover of SARS-CoV-2 into a variety of wild and domestic animals has been an ongoing feature of the human pandemic. The establishment of a new reservoir in white-tailed deer in North America and increasing divergence of the viruses circulating in them from those circulating in the human population has highlighted the ongoing risk this poses for global health. Some parts of the world have seen more intensive monitoring of wildlife species for SARS-CoV-2 and related coronaviruses but there are still very large gaps in geographical and species-specific information. This paper reports negative results for SARS-CoV-2 PCR based testing using a pan coronavirus end point RDRP PCR and a Sarbecovirus specific E gene qPCR on lung and or gut tissue from wildlife from the Indian State of Kerala. These animals included: 121 Rhinolophus rouxii (Rufous Horsehoe Bat), six Rhinolophus bedommei (Lesser Woolly Horseshoe Bat), 15 Rossettus leschenaultii (Fulvous Fruit Bat), 47 Macaca radiata (Bonnet macaques), 35 Paradoxurus hermaphroditus (Common Palm Civet), five Viverricula indica (Small Indian Civet), four Herpestes edwardsii (Common Mongoose), ten Panthera tigris (Bengal Tiger), eight Panthera pardus fusca (Indian Leopard), four Prionailurus bengalensis (Leopard cats), two Felis chaus (Jungle cats), two Cuon alpinus (Wild dogs) and one Melursus ursinus (sloth bear).

Veterinarians' knowledge and experience of avian influenza and perspectives on control measures in the UK.

BACKGROUND: The scale of the outbreak of highly pathogenic avian influenza (HPAI) in 2021-23 due to the influenza A/H5N1 virus is unprecedented. METHODS: An online survey was designed to explore veterinarians' experiences of and confidence in treating avian species, experiences of dealing with suspected HPAI and perspectives on control measures in the UK. The survey ran between December 2021 and March 2022. RESULTS: Survey responses were received from 26 veterinarians. Although veterinarians are well placed to communicate HPAI-related information and guidance, a lack of confidence around treating birds and dealing with suspected cases of HPAI represent key barriers for non-specialist practices, and this limits opportunities to educate clients. LIMITATIONS: This study presents the views of a small group of self-selected respondents and may over-represent veterinarians with existing interests in avian species and/or avian influenza and who engage with online fora. CONCLUSIONS: Improved training and resources designed to increase confidence with avian species, along with guidance on diagnosing and reporting notifiable diseases, are needed for first opinion practices. Governing bodies should clarify regulations on treating birds in veterinary practices when HPAI outbreak numbers are high.

UK flockdown: A survey of smallscale poultry keepers and their understanding of governmental guidance on highly pathogenic avian influenza (HPAI).

The scale of the current outbreak of highly pathogenic avian influenza (HPAI) due to the A/H5N1 virus in the United Kingdom is unprecedented. In addition to its economic impact on the commercial poultry sector, the disease has devastated wild bird colonies and represents a potential public health concern on account of its zoonotic potential. Although the implementation of biosecurity measures is paramount to reducing the spread of HPAI in domestic and commercial settings, little is known about the attitudes and perspectives of backyard poultry keepers, who often keep their flocks in close proximity to the public. A large nationwide survey of backyard poultry keepers was undertaken in December 2021-March 2022, contemporaneous with the enforcement of an Avian Influenza Prevention Zone (AIPZ) and additional housing measures in England, Scotland and Wales. The survey explored keepers' understanding of the clinical manifestations of HPAI, compliance with housing and biosecurity measures, attitudes towards obligatory culling on confirmation of HPAI in their flocks, and the potential use of vaccination to control HPAI. Summary statistical analysis of the closed question responses was supplemented with qualitative data analysis and corpus linguistic approaches to draw out key themes and salient patterns in responses to open text questions. Survey responses were received from 1559 small-scale poultry keepers across the United Kingdom. Awareness of the HPAI outbreak was very high (99.0%). The majority of respondents learned of it via social media (53%), with Defra (49.7%), British Hen Welfare Trust (33.8%) and the APHA (22.0%) identified as the principal sources of information. Analysis revealed that backyard keepers lacked knowledge of the clinical signs of avian influenza and legal requirements relating to compliance with biosecurity measures. Some respondents dismissed the seriousness of HPAI and were unwilling to comply with the measures in force. The issue of obligatory culling proved highly emotive, and some expressed a lack of trust in authorities. Most respondents (93.1%) indicated a willingness to pay for vaccination if the option was available. Communications on biosecurity measures that are relevant to large-scale industrial setups are inappropriate for backyard contexts. Understanding the barriers that backyard keepers face is essential if official agencies are to communicate biosecurity information effectively to such groups. Lack of trust in authorities is likely to make elimination of the virus in the UK difficult. We make recommendations for tailoring HPAI-related information for backyard contexts, to aid future HPAI control measures in the UK.

Lack of detection of SARS-CoV-2 in British wildlife 2020-21 and first description of a stoat (Mustela erminea) Minacovirus.

Repeat spillover of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into new hosts has highlighted the critical role of cross-species transmission of coronaviruses and establishment of new reservoirs of virus in pandemic and epizootic spread of coronaviruses. Species particularly susceptible to SARS-CoV-2 spillover include Mustelidae (mink, ferrets and related animals), cricetid rodents (hamsters and related animals), felids (domestic cats and related animals) and white-tailed deer. These predispositions led us to screen British wildlife with sarbecovirus-specific quantitative PCR and pan coronavirus PCR assays for SARS-CoV-2 using samples collected during the human pandemic to establish if widespread spillover was occurring. Fourteen wildlife species (n=402) were tested, including: two red foxes (Vulpes vulpes), 101 badgers (Meles meles), two wild American mink (Neogale vison), 41 pine marten (Martes martes), two weasels (Mustela nivalis), seven stoats (Mustela erminea), 108 water voles (Arvicola amphibius), 39 bank voles (Myodes glareolous), 10 field voles (Microtus agrestis), 15 wood mice (Apodemus sylvaticus), one common shrew (Sorex aranaeus), two pygmy shrews (Sorex minutus), two hedgehogs (Erinaceus europaeus) and 75 Eurasian otters (Lutra lutra). No cases of SARS-CoV-2 were detected in any animals, but a novel minacovirus related to mink and ferret alphacoronaviruses was detected in stoats recently introduced to the Orkney Islands. This group of viruses is of interest due to pathogenicity in ferrets. The impact of this virus on the health of stoat populations remains to be established.

Antiviral activity of salt-coated materials against SARS-CoV-2.

The SARS-CoV-2 pandemic demonstrated the importance of human coronaviruses and the need to develop materials to prevent the spread of emergent respiratory viruses. Coating of surfaces with antiviral materials is a major interest in controlling spread of viruses, especially in high-risk or high-traffic areas. A number of different coatings for surfaces have been proposed, each with their own advantages and disadvantages. Here we show that simple salt coating on a range of surfaces, including a novel biomass aerogel can reduce the infectivity of SARS-CoV-2 placed onto the surface. This suggests that a simple to apply coating could be applied to a range of materials and have an antiviral effect against SARS-CoV-2, as well as other potential emerging viruses.

Exploring the responses of smallscale poultry keepers to avian influenza regulations and guidance in the United Kingdom, with recommendations for improved biosecurity messaging.

Understanding how smallscale ('backyard') poultry keepers interpret and respond to governmental directives designed to reduce the transmission of highly pathogenic avian influenza (HPAI) is of paramount importance in preparing for future HPAI outbreaks. Qualitative insights from open questions in an online survey conducted during the 2021-22 HPAI season (1,559 responses) shed light on smallscale poultry keepers' understanding of, and responses to, governmental directives to control HPAI exposure and onwards transmission. A follow-up participatory workshop (21 participants) explored the HPAI-related information sources used by smallscale poultry keepers, their trust in these sources, perceptions of HPAI-related risk, and interpretation of, opinions on and adherence to government regulations and communications regarding biosecurity and housing measures. This paper draws on a multi-scale behaviour change model to explore barriers to compliance with HPAI-related regulations. Insights from behaviour settings theory reveal how poultry-keeping settings and routines might be 'disrupted' and 're-configured' to improve long-term biosecurity and reduce the risk of HPAI exposure. The findings highlight the need for HPAI-related guidance that is tailored to smallscale poultry keepers. This guidance should include clear action points and simple, practical, affordable and sustainable suggestions for improving compliance with biosecurity measures.

Sarbecoviruses of British horseshoe bats; sequence variation and epidemiology.

Horseshoe bats are the natural hosts of the Sarbecovirus subgenus that includes SARS-CoV and SARS-CoV- 2. Despite the devastating impact of the COVID-19 pandemic, there is still little known about the underlying epidemiology and virology of sarbecoviruses in their natural hosts, leaving large gaps in our pandemic preparedness. Here we describe the results of PCR testing for sarbecoviruses in the two horseshoe bat species (Rhinolophus hipposideros and R. ferrumequinum) present in Great Britain, collected in 2021-22 during the peak of COVID-19 pandemic. One hundred and ninety seven R. hipposideros samples from 33 roost sites and 277 R. ferrumequinum samples from 20 roost sites were tested. No coronaviruses were detected in any samples from R. ferrumequinum whereas 44 and 56 % of individual and pooled (respectively) faecal samples from R. hipposideros across multiple roost sites tested positive in a sarbecovirus-specific qPCR. Full genome sequences were generated from three of the positive samples (and partial genomes from two more) using Illumina RNAseq on unenriched samples. Phylogenetic analyses showed that the obtained sequences belong to the same monophyletic clade, with >95 % similarity to previously-reported European isolates from R. hipposideros. The sequences differed in the presence or absence of accessory genes ORF 7b, 9b and 10. All lacked the furin cleavage site of SARS-CoV-2 spike gene and are therefore unlikely to be infective for humans. These results demonstrate a lack, or at least low incidence, of SARS-CoV-2 spill over from humans to susceptible GB bats, and confirm that sarbecovirus infection is widespread in R. hipposideros. Despite frequently sharing roost sites with R. ferrumequinum, no evidence of cross-species transmission was found.

Associations between small ruminant lentivirus infection and total milk yield and somatic cell count in a dairy sheep flock.

BACKGROUND: Small ruminant lentiviruses (SRLVs) are lentiviruses of sheep and goats, formerly known as maedi-visna (MV) in sheep and caprine encephalitis and arthritis in goats. In sheep, SRLVs commonly cause progressive pneumonia, wasting and indurative mastitis. SRLVs have a long latent period, and chronic production losses are often not recognised until very late. Few studies quantifying the production losses in ewes have been published, and none have been published under UK flock husbandry conditions. METHODS: Production records of milk yield and somatic cell count (SCC) from a dairy flock of 319 milking East Friesian × Lacaune ewes identified as MV infected via routine serological screening for SRLV antibodies were used in multivariable linear regression modelling to estimate the impact of SRLV status on total milk yield and SCC. RESULTS: Milk yield was reduced in seropositive ewes by 8.1%-9.2% over an entire lactation. SCC counts were not significantly different in SRLV-infected and unifected animals. LIMITATIONS: Further parameters, such as body condition score or clinical mastitis, that were not available may have clarified the underlying cause of milk yield drop. CONCLUSIONS: The study demonstrates substantial production losses in an SRLV-affected flock and highlights the impact of the virus on a farm's economic viability.

Genome Reference Assembly for Bottlenecked Southern Australian Koalas.

Koala populations show marked differences in inbreeding levels and in the presence or absence of the endogenous Koala retrovirus (KoRV). These genetic differences among populations may lead to severe disease impacts threatening koala population viability. In addition, the recent colonization of the koala genome by KoRV provides a unique opportunity to study the process of retroviral adaptation to vertebrate genomes and the impact this has on speciation, genome structure, and function. The genome build described here is from an animal from the bottlenecked Southern population free of endogenous and exogenous KoRV. It provides a more contiguous genome build than the previous koala reference derived from an animal from a more outbred Northern population and is the first koala genome from a KoRV polymerase-free animal.

Failure to detect Schmallenberg virus RNA in ram semen in the UK (2016-2018).

Background: Schmallenberg virus (SBV) is a midge-borne arbovirus that first emerged in the European ruminant population in 2011 and has since settled to an endemic pattern of disease outbreaks on an approximately 4-year cycle when herd immunity from the previous circulation drops to a point allowing renewed widescale virus circulation. The impacts of trade restrictions on genetic products (semen, embryos) from affected areas were severe, particularly after the discovery that the virus is intermittently shed in the semen of a small number of bulls. The trade in small ruminant (ram and goat) semen is less than that of bulls; nonetheless, there has been no study into the shedding rate of SBV in ram semen. Methods: Semen samples (n = 65) were collected as part of UK ram trials and artificial insemination studies around the period of the 2016-2018 SBV recirculation. Semen was preserved in RNAlater for shipping, and RNA extraction with RNeasy and S gene RT-quantitative PCR performed for SBV nucleic acid detection. Results: No SBV RNA was detected in any samples. Conclusions: While larger numbers of animals would be needed to completely exclude the possibility of SBV shedding in ram semen, this trial nonetheless highlights that this is likely a rare event if it occurs at all and is unlikely to play a role in disease transmission.

Clearance of Maedi-visna infection in a longitudinal study of naturally infected rams is associated with homozygosity for the TMEM154 resistance allele.

Maedi-visna (MV) is a lentiviral disease of sheep responsible for severe production losses in affected flocks. There are no vaccination or treatment options with control reliant on test and cull strategies. The most common diagnostic methods used at present are combination ELISAs for Gag and Env proteins with virus variability making PCR diagnostics still largely an experimental tool. To assess variability in viral loads and diagnostic tests results, serology, DNA and RNA viral loads were measured in the blood of 12 naturally infected rams repeatedly blood sampled over 16 months. Six animals tested negative in one or more tests at one or more time points and would have been missed on screening programmes reliant on one test method or a single time point. In addition the one animal homozygous for the 'K' allele of the TMEM154 E35K SNP maintained very low viral loads in all assays and apparently cleared infection to below detectable limits at the final time point it was sampled. This adds crucial data to the strong epidemiological evidence that this locus represents a genuine resistance marker for MV infection and is a strong candidate for selective breeding of sheep for resistance to disease.

Serum Cytokine Alterations Associated with Age of Patients with Nephropathia Epidemica.

Nephropathia epidemica (NE) is a zoonotic disease caused by hantaviruses transmitted from rodents, endemic in the Republic of Tatarstan, Russia. The disease presents clinically with mild, moderate, and severe forms, and time-dependent febrile, oliguric, and polyuric stages of the disease are also recognized. The patient's cytokine responses have been suggested to play a central role in disease pathogenesis; however, little is known about the different patterns of cytokine expression in NE in cohorts of different ages and sexes. Serum samples and clinical records were collected from 139 patients and 57 controls (healthy donors) and were used to analyze 48 analytes with the Bio-Plex multiplex magnetic bead-based antibody detection kits. Principal component analysis of 137 patient and 55 controls (for which there was full data) identified two components that individually accounted for >15% of the total variance in results and together for 38% of the total variance. PC1 represented a proinflammatory TH17/TH2 cell antiviral cytokine profile and PC2 a more antiviral cytokine profile with patients tending to display one or the other of these. Severity of disease and stage of illness did not show any correlation with PC1 profiles; however, significant differences were seen in patients with high PC1 profiles vs. lower for a number of individual clinical parameters: High PC1 patients showed a reduced number of febrile days, but higher maximum urine output, higher creatinine levels, and lower platelet levels. Overall, the results of this study point towards a stronger proinflammatory profile occurring in younger NE patients, this being associated with markers of acute kidney injury and low levels of high-density cholesterol. This is consistent with previous work indicating that the pathology of NE is immune driven, with an inflammatory immune response being associated with disease and that this immune response is more extreme in younger patients.

Retroviral integrations contribute to elevated host cancer rates during germline invasion.

Repeated retroviral infections of vertebrate germlines have made endogenous retroviruses ubiquitous features of mammalian genomes. However, millions of years of evolution obscure many of the immediate repercussions of retroviral endogenisation on host health. Here we examine retroviral endogenisation during its earliest stages in the koala (Phascolarctos cinereus), a species undergoing germline invasion by koala retrovirus (KoRV) and affected by high cancer prevalence. We characterise KoRV integration sites (IS) in tumour and healthy tissues from 10 koalas, detecting 1002 unique IS, with hotspots of integration occurring in the vicinity of known cancer genes. We find that tumours accumulate novel IS, with proximate genes over-represented for cancer associations. We detect dysregulation of genes containing IS and identify a highly-expressed transduced oncogene. Our data provide insights into the tremendous mutational load suffered by the host during active retroviral germline invasion, a process repeatedly experienced and overcome during the evolution of vertebrate lineages.

Role of Viruses in the Pathogenesis of Multiple Sclerosis.

Multiple sclerosis (MS) is an immune inflammatory disease, where the underlying etiological cause remains elusive. Multiple triggering factors have been suggested, including environmental, genetic and gender components. However, underlying infectious triggers to the disease are also suspected. There is an increasing abundance of evidence supporting a viral etiology to MS, including the efficacy of interferon therapy and over-detection of viral antibodies and nucleic acids when compared with healthy patients. Several viruses have been proposed as potential triggering agents, including Epstein-Barr virus, human herpesvirus 6, varicella-zoster virus, cytomegalovirus, John Cunningham virus and human endogenous retroviruses. These viruses are all near ubiquitous and have a high prevalence in adult populations (or in the case of the retroviruses are actually part of the genome). They can establish lifelong infections with periods of reactivation, which may be linked to the relapsing nature of MS. In this review, the evidence for a role for viral infection in MS will be discussed with an emphasis on immune system activation related to MS disease pathogenesis.

Retrieval of the Complete Coding Sequence of the UK-Endemic Tatenale Orthohantavirus Reveals Extensive Strain Variation and Supports Its Classification as a Novel Species.

Orthohantaviruses are globally distributed viruses, associated with rodents and other small mammals. However, data on the circulation of orthohantaviruses within the UK, particularly the UK-endemic Tatenale virus, is sparse. In this study, 531 animals from five rodent species were collected from two locations in northern and central England and screened using a degenerate, pan- orthohantavirus RT-PCR assay. Tatenale virus was detected in a single field vole (Microtus agrestis) from central England and twelve field voles from northern England. Unbiased high-throughput sequencing of the central English strain resulted in the recovery of the complete coding sequence of a novel strain of Tatenale virus, whilst PCR-primer walking of the northern English strain recovered almost complete coding sequence of a previously identified strain. These findings represented the detection of a third lineage of Tatenale virus in the United Kingdom and extended the known geographic distribution of these viruses from northern to central England. Furthermore, the recovery of the complete coding sequence revealed that Tatenale virus was sufficiently related to the recently identified Traemersee virus, to meet the accepted criteria for classification as a single species of orthohantavirus.

Novel insights into viral infection and oncogenesis from koala retrovirus (KoRV) infection of HEK293T cells.

Koala retrovirus is thought to be an underlying cause of high levels of neoplasia and immunosuppression in koalas. While epidemiology studies suggest a strong link between KoRV and disease it has been difficult to prove causality because of the complex nature of the virus, which exists in both endogenous and exogenous forms. It has been difficult to identify koalas completely free of KoRV, and infection studies in koalas or koala cells are fraught with ethical and technical difficulties, respectively. This study uses KoRV infection of the susceptible human cell line HEK293T and RNAseq to demonstrate gene networks differentially regulated upon KoRV infection. Many of the pathways identified are those associated with viral infection, such as cytokine receptor interactions and interferon signalling pathways, as well as viral oncogenesis pathways. This study provides strong evidence that KoRV does indeed behave similarly to infectious retroviruses in stimulating antiviral and oncogenic cellular responses. In addition, it provides novel insights into KoRV oncogenesis with the identification of a group of histone family genes that are part of several oncogenic pathways as upregulated in KoRV infection.

Koala retrovirus viral load and disease burden in distinct northern and southern koala populations.

Koala retrovirus (KoRV) displays features of both an endogenous and exogenous virus and is linked to neoplasia and immunosuppression in koalas. This study explores the apparent differences in the nature and impact of KoRV infection between geographically and genetically separated "northern" and "southern" koala populations, by investigating the disease status, completeness of the KoRV genome and the proviral (DNA) and viral (RNA) loads of 71 northern and 97 southern koalas. All northern animals were positive for all KoRV genes (gag, pro-pol and env) in both DNA and RNA forms, whereas many southern animals were missing one or more KoRV genes. There was a significant relationship between the completeness of the KoRV genome and clinical status in this population. The proviral and viral loads of the northern population were significantly higher than those of the southern population (P < 0.0001), and many provirus-positive southern animals failed to express any detectable KoRV RNA. Across both populations there was a positive association between proviral load and neoplasia (P = 0.009). Potential reasons for the differences in the nature of KoRV infection between the two populations are discussed.