Diverse picornaviruses are prevalent among free-living and laboratory rats (Rattus norvegicus) in Hungary and can cause disseminated infections

In this study, the full length genomes of three phylogenetically distant picornaviruses (family Picornaviridae) belonging to the genus Rosavirus (rat08/rRoB/HUN, MN116648), Kobuvirus (rat08/rAiA/HUN, MN116647), and Cardiovirus (rat08/rCaB/HUN, MN116646) were obtained from a single faecal sample of a free-living Norway rat (Rattus norvegicus) in Hungary using viral metagenomics and RT-PCR/Sanger sequencing. The acquired complete genomes were in silico analyzed in detail revealing the presence of a second minor open reading frame encoding an alternative Leader peptide (L*) in rat08/rCaB/HUN and a ca. 222 nt-long sequence repeat with compact secondary RNA structure in the 3′ UTR of rat08/rRoB/HUN. The studied rat picornaviruses were frequently detectable by RT-PCR with relatively high viral loads ranged between 8.99E+02 and 1.29E+06 copies/ml in rat faecal samples collected from five geographically distant locations throughout Hungary. The VP1 sequence-based phylogenetic analyses show the presence of multiple, mostly location-specific lineages for all three picornaviruses. Rat rosavirus and rat cardiovirus were identified in spleen while rat cardiovirus was also detected in liver, muscle and kidney samples with variable copy numbers (6.42E+01–1.90E+05 copies/μg total RNA) suggesting extra-intestinal dissemination. Both viruses were also prevalent (70.0% and 18.2%) among two populations of laboratory rats (“Wistar-type” and “hooded-type”) held in different, isolated laboratory animal units

Viruses in unexplained encephalitis cases in American black bears (Ursus americanus)

Viral infections were investigated in American black bears (Ursus americanus) from Nevada and northern California with and without idiopathic encephalitis. Metagenomics analyses of tissue pools revealed novel viruses in the genera Circoviridae, Parvoviridae, Anelloviridae, Polyomaviridae, and Papillomaviridae. The circovirus and parvovirus were of particular interest due to their potential importance as pathogens. We characterized the genomes of these viruses and subsequently screened bears by PCR to determine their prevalence. The circovirus (Ursus americanus circovirus, UaCV) was detected at a high prevalence (10/16, 67%), and the chaphamaparvovirus (Ursus americanus parvovirus, UaPV) was found in a single bear. We showed that UaCV is present in liver, spleen/lymph node, and brain tissue of selected cases by in situ hybridization (ISH) and PCR. Infections were detected in cases of idiopathic encephalitis and in cases without inflammatory brain lesions. Infection status was not clearly correlated with disease, and the significance of these infections remains unclear. Given the known pathogenicity of a closely related mammalian circovirus, and the complex manifestations of circovirus-associated diseases, we suggest that UaCV warrants further study as a possible cause or contributor to disease in American black bears

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Viruses in marine animals: Discovery, detection, and characterizarion

Diseases in marine animals are emerging at an increasing rate. Disease forecasting enabled by virus surveillance presents a proactive solution for managing emerging diseases. Broad viral surveys aid in disease forecasting by providing baseline data on viral diversity associated with various hosts, including many that are not associated with disease. However, these viruses can become pathogens due to expansion in host or geographic range, as well as when changing conditions shift the balance between commensal viruses and the host immune system. Therefore, it is extremely valuable to identify and characterize viruses present in many different hosts in a variety of environments, regardless of whether the hosts are symptomatic or not. The lack of a universal gene shared by all viruses makes virus surveillance difficult, because no single assay exists that can detect the enormous diversity of viruses. Viral metagenomics circumvents this issue by purifying viral particles directly from host tissues and sequencing the nucleic acids, allowing for virus identification. However, virus identification is only the first step, which should ideally be followed by complete sequencing of the viral genome to identify genes of interest and develop assays to reveal viral prevalence, tropism, ecology, and pathogenicity. This dissertation focuses on the discovery of novel viruses in marine animals, characterization of complete viral genomes, and the development of subsequent diagnostic assays for further analysis of virus ecology. First, viral metagenomics was used to explore the viruses present in the healthy Weddell seal (Leptonychotes weddellii) population in Antarctica, which led to the discovery of highly prevalent small, circular single-stranded DNA (ssDNA) viruses. The lack of knowledge regarding the viruses of Antarctic wildlife warrants this study to determine baseline viral communities in healthy animals that can be used to survey changes over time. From the healthy Weddell seals, viral metagenomics led to the discovery of 152 novel anellovirus genomes, encompassing two anellovirus species. Characterizing these viruses is important for understanding the prevalence and diversity of ssDNA viruses, which have only recently been described in marine animals. Furthermore, since emerging diseases can be caused by changing conditions affecting host susceptibility to a virus that was previously not related to disease (opportunistic pathogen), having baseline data allows for quick identification of the pathogen. In addition to determining baseline data, viral metagenomics can explore the role of viruses in disease. A novel virus, Asterias forbesi-associated circular virus (AfaCV), was discovered in the Atlantic sea star Asterias forbesi displaying symptoms of sea star wasting disease (SSWD). AfaCV was the first circular replicase-encoding ssDNA (CRESS-DNA) virus discovered in echinoderms, but it was only present in 10% of SSWD sea stars indicating it is not involved in the development of the disease. This dissertation also focuses on elucidating the role of two previously characterized viruses, chelonid fibropapillomatosis-associated herpesvirus (CHHV5; Chelonid herpesvirus 5, ChHV5) and Zalophus californianus anellovirus (ZcAV), in animal health. PCR amplicon sequencing was used to obtain large portions of the 132 kb genome of ChHV5, the putative etiological agent of the neoplastic sea turtle disease, fibropapillomatosis. Obtaining the genome of ChHV5 from Florida green, Kemp’s ridley, and loggerhead sea turtles provides data for phylogenetic analysis across geographic locations and sea turtle species, as well as a reference for designing downstream molecular assays to examine viral latency. ZcAV was first described from the lungs of captive sea lions involved in a mortality event. PCR could not detect ZcAV in the blood of infected animals, and since sea lions are a protected species, it is not possible to obtain lung biopsies from live sea lions to determine ZcAV prevalence or its role in sea lion health. To answer these important questions, an enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies to ZcAV in serum from wild sea lion populations. This newly developed ELISA showed that sea lions mount an immune response to ZcAV, and was used to determine the prevalence of ZcAV among wild sea lion populations. This dissertation makes an important contribution to marine science through discovery and characterization of viruses present in healthy and diseased marine animals. Several different methods were used for virus whole-genome sequencing including viral metagenomics, PCR amplicon sequencing, and target enrichment. These findings were expanded upon by developing and using PCR assays and a serological assay to screen for virus prevalence. These methods have implications for viral surveillance and understanding the role of novel viruses in animal health

Bacterial Profiling of the Scleractinian Coral Pocillopora damicornis

Fahsbender, E.1, Daniels, C.2, Cunning, R.3, Baker, A.3, and M. Breitbart2 1 University of South Florida, Tampa, Florida 2 University of South Florida, College of Marine Science, St. Petersburg, Florida 3 University of Miami, Rosenstiel School of Marine and Atmospheric Sciences, Miami, Florida Scleractinian corals are colonial animals capable of building reef structures, which serve as a habitat for many species, and are vital to maintaining diversity in marine ecosystems. Corals are dependent upon various clades of photosynthetic algae (zooxanthellae) for energy, as well as a diverse bacterial community that produces antibiotics to defend against pathogens. This study applies molecular methods to determine the bacterial community structure of the coral Pocillopora damicornis and examine whether zooxanthellae clade or sampling location influences bacterial community composition. Automated Ribosomal Intergenic Spacer Analysis (ARISA) was used to profile P. damicornis samples collected from five reef sites across the Pacific Ocean. ARISA targets the intergenic spacer, a region between 16S and 23S ribosomal genes, that varies in length amongst different bacteria, and can therefore produce a profile of the bacterial community. Preliminary results indicate 37 % average similarity among bacterial community profiles of different coral colonies, and show no clear relationship between bacterial community composition and zooxanthellae clade. Biogeographical comparisons are currently underway to determine if sampling location is a factor in structuring bacterial communities associated with P. damicornis

Development of a Serological Assay for the Sea Lion (Zalophus californianus) Anellovirus, ZcAV

New diseases in marine animals are emerging at an increasing rate, yet methodological limitations hinder characterization of viral infections. Viral metagenomics is an effective method for identifying novel viruses in diseased animals; however, determining virus pathogenesis remains a challenge. A novel anellovirus (Zalophus californianus anellovirus, ZcAV) was recently reported in the lungs of captive California sea lions involved in a mortality event. ZcAV was not detected by PCR in the blood of these animals, creating the inability to assess the prevalence of ZcAV in live sea lions. This study developed an enzyme-linked immunosorbent assay (ELISA) to detect antibodies to ZcAV in sea lion serum. To assess ZcAV prevalence, paired serum and lung samples (n = 96) from wild sea lions that stranded along the California coast were tested through ELISA and PCR, respectively. Over 50% of the samples tested positive for ZcAV by ELISA (34%), PCR (29%), or both (11%) assays. ZcAV is prevalent in stranded wild sea lion populations and results suggest that PCR assays alone may grossly underestimate ZcAV exposure. This ELISA provides a tool for testing live sea lions for ZcAV exposure and is valuable for subsequent studies evaluating the potential pathogenicity of this anellovirus

Diverse and Highly Recombinant Anelloviruses Associated with Weddell Seals in Antarctica

The viruses circulating among Antarctic wildlife remain largely unknown. In an effort to identify viruses associated withWeddell seals (Leptonychotes weddellii) inhabiting the Ross Sea, vaginal and nasal swabs, and faecal samples were collectedbetween November 2014 and February 2015. In addition, a Weddell seal kidney and South Polar skua (Stercorariusmaccormicki) faeces were opportunistically sampled. Using high throughput sequencing, we identified and recovered 152anellovirus genomes that share 63–70% genome-wide identities with other pinniped anelloviruses. Genome-wide pairwisecomparisons coupled with phylogenetic analysis revealed two novel anellovirus species, tentatively named torque tenoLeptonychotes weddellii virus (TTLwV) -1 and -2. TTLwV-1 (n¼133, genomes encompassing 40 genotypes) is highly recom-binant, whereas TTLwV-2 (n¼19, genomes encompassing three genotypes) is relatively less recombinant. This study documents ubiquitous TTLwVs among Weddell seals in Antarctica with frequent co-infection by multiple genotypes, how-ever, the role these anelloviruses play in seal health remains unknown

Chapparvovirus DNA Found in 4% of Dogs with Diarrhea

Feces from dogs in an unexplained outbreak of diarrhea were analyzed by viral metagenomics revealing the genome of a novel parvovirus. The parvovirus was named cachavirus and was classified within the proposed Chapparvovirus genus. Using PCR, cachavirus DNA was detected in two of nine tested dogs from that outbreak. In order to begin to elucidate the clinical impact of this virus, 2,053 canine fecal samples were screened using real-time PCR. Stool samples from 203 healthy dogs were positive for cachavirus DNA at a rate of 1.47%, while 802 diarrhea samples collected in 2017 and 964 samples collected in 2018 were positive at rates of 4.0% and 4.66% frequencies, respectively (healthy versus 2017-2018 combined diarrhea p-value of 0.05). None of 83 bloody diarrhea samples tested positive. Viral loads were generally low with average real-time PCR Ct values of 36 in all three positive groups. The species tropism and pathogenicity of cachavirus, the first chapparvovirus reported in feces of a placental carnivore, remains to be fully determined