Vertebrate Viruses Research Articles

Characterization of oral virome and microbiome revealed distinctive microbiome disruptions in paediatric patients with hand, foot and mouth disease

While the underlying determinants are unclear, hand, foot and mouth disease (HFMD) presents a wide spectrum of clinical manifestations with varying severity in different individuals. Recently, many studies identified the human microbiome as a critical factor in the pathogenesis of various diseases. Therefore, we here investigated the ecological dynamics of the oral microbiome changes during the HFMD infection. After targeted enrichment of all known vertebrate viruses, the virome profiles of symptomatic and asymptomatic HFMD patients were examined and revealed to be significantly altered from those of healthy individuals, with nine discriminative viruses detected. Further characterization of the prokaryotic microbiome revealed an elevated level of Streptococcus sp. as the most important signature of the symptomatic HFMD cohort, positively correlating to the level of enterovirus A RNA. In addition, we found that while coxsackievirus A5 is detected in saliva RNA of all asymptomatic cases, coxsackievirus A6 dominates the majority of the symptomatic cohort.

Divergent Influenza-Like Viruses of Amphibians and Fish Support an Ancient Evolutionary Association.

Influenza viruses (family Orthomyxoviridae) infect a variety of vertebrates, including birds, humans, and other mammals. Recent metatranscriptomic studies have uncovered divergent influenza viruses in amphibians, fish and jawless vertebrates, suggesting that these viruses may be widely distributed. We sought to identify additional vertebrate influenza-like viruses through the analysis of publicly available RNA sequencing data. Accordingly, by data mining, we identified the complete coding segments of five divergent vertebrate influenza-like viruses. Three fell as sister lineages to influenza B virus: salamander influenza-like virus in Mexican walking fish (Ambystoma mexicanum) and plateau tiger salamander (Ambystoma velasci), Siamese algae-eater influenza-like virus in Siamese algae-eater fish (Gyrinocheilus aymonieri) and chum salmon influenza-like virus in chum salmon (Oncorhynchus keta). Similarly, we identified two influenza-like viruses of amphibians that fell as sister lineages to influenza D virus: cane toad influenza-like virus and the ornate chorus frog influenza-like virus, in the cane toad (Rhinella marina) and ornate chorus frog (Microhyla fissipes), respectively. Despite their divergent phylogenetic positions, these viruses retained segment conservation and splicing consistent with transcriptional regulation in influenza B and influenza D viruses, and were detected in respiratory tissues. These data suggest that influenza viruses have been associated with vertebrates for their entire evolutionary history.

Human cytomegalovirus evades ZAP detection by suppressing CpG dinucleotides in the major immediate early 1 gene.

The genomes of RNA and small DNA viruses of vertebrates display significant suppression of CpG dinucleotide frequencies. Artificially increasing dinucleotide frequencies results in substantial attenuation of virus replication, suggesting that these compositional changes may facilitate recognition of non-self RNA sequences. Recently, the interferon inducible protein ZAP, was identified as the host factor responsible for sensing CpG in viral RNA, through direct binding and possibly downstream targeting for degradation. Using an arrayed interferon stimulated gene expression library screen, we identified ZAPS, and its associated factor TRIM25, as inhibitors of human cytomegalovirus (HCMV) replication. Exogenous expression of ZAPS and TRIM25 significantly reduced virus replication while knockdown resulted in increased virus replication. HCMV displays a strikingly heterogeneous pattern of CpG representation with specific suppression of CpG motifs within the IE1 major immediate early transcript which is absent in subsequently expressed genes. We demonstrated that suppression of CpG dinucleotides in the IE1 gene allows evasion of inhibitory effects of ZAP. We show that acute virus replication is mutually exclusive with high levels of cellular ZAP, potentially explaining the higher levels of CpG in viral genes expressed subsequent to IE1 due to the loss of pressure from ZAP in infected cells. Finally, we show that TRIM25 regulates alternative splicing between the ZAP short and long isoforms during HCMV infection and interferon induction, with knockdown of TRIM25 resulting in decreased ZAPS and corresponding increased ZAPL expression. These results demonstrate for the first time that ZAP is a potent host restriction factor against large DNA viruses and that HCMV evades ZAP detection through suppression of CpG dinucleotides within the major immediate early 1 transcript. Furthermore, TRIM25 is required for efficient upregulation of the interferon inducible short isoform of ZAP through regulation of alternative splicing.

Modern taxonomy of viruses of vertebrates

The modern taxonomy of viruses of vertebrates is presented according to the information of ICTV issue 07.2019, ratification 03.2020. The leading criteria of taxonomy of viruses are named: type and structure of viral genome, mechanism of replication and morphology of virion. The periods of formation of taxonomic ranks of viruses are characterized: in 1966–1970 genera of viruses were formed, in 1971–1975 – families and subfamilies, since 1990 – orders, in 2018–2019 – realms, kingdoms, phylums, subphylums, classes, suborders, subgenеres. The nomenclature of viruses is described. Viruses belong to the Viruses domain. Viruses of vertebrates (1878 species) belong to 4 realms, 5 kingdoms, 10 phylums, 2 subphylums, 20 classes, 26 orders, 3 suborders, 45 families (of which 15 – DNA-genomic and 30 – RNA-genomic), 33 subfamilies, 345 genera and 49 subgenera. Taxonomic ranks of DNA- and RNA-genomic viruses of vertebrates are described. The DNA-genome family Anelloviridae and the unclassified RNA-genomic genus Deltavirus are not included in any realm. The family Birnaviridae is not classified within the kingdom Orthornavirae. The family of DNA-genomic Hepadnaviridae is included in the realm of RNA-containing viruses Riboviria on the grounds that the replication of hepadnaviruses occurs through the stage of RNA on the principle of reverse transcription, as in the family Retroviridae. The main taxonomic features of DNA- and RNA-genomic viruses of vertebrates are described: type and structure of viral genome (DNA or RNA, number of strands, conformation, fragmentation, polarity), shape and size of virions, presence of outer lipoprotein shell, type of capsid symmetry (spiral, iсosahedral). Some families, in addition to viruses of vertebrates, contain viruses of invertebrates and plants, in particular: families Poxviridae, Iridoviridae, Parvoviridae, Circoviridae, Smacoviridae, Genomoviridae, Rhabdoviridae, Nyamiviridae, Peribunyaviridae, Phenuiviridae, Nairoviviridae, Nodaviridae, Reoviridae and Birnaviridae – viruses of insects; families Genomoviridae, Rhabdoviridae, Phenuiviridae and Reoviridae – viruses of plants; family Nyamiviridae – viruses of nematodes, cestodes, sipunculidеs and echinoderms; family Rhabdoviridae – viruses of nematodes; family Reoviridae – Eriocheir sinensis reovirus; family Birnaviridae – viruses of tellines and rotifers.

Coronavirus discovery by metagenomic sequencing: a tool for pandemic preparedness.

IntroductionThe SARS-CoV-2 pandemic of 2020 is a prime example of the omnipresent threat of emerging viruses that can infect humans. A protocol for the identification of novel coronaviruses by viral metagenomic sequencing in diagnostic laboratories may contribute to pandemic preparedness.AimThe aim of this study is to validate a metagenomic virus discovery protocol as a tool for coronavirus pandemic preparedness.MethodsThe performance of a viral metagenomic protocol in a clinical setting for the identification of novel coronaviruses was tested using clinical samples containing SARS-CoV-2, SARS-CoV, and MERS-CoV, in combination with databases generated to contain only viruses of before the discovery dates of these coronaviruses, to mimic virus discovery.ResultsClassification of NGS reads using Centrifuge and Genome Detective resulted in assignment of the reads to the closest relatives of the emerging coronaviruses. Low nucleotide and amino acid identity (81% and 84%, respectively, for SARS-CoV-2) in combination with up to 98% genome coverage were indicative for a related, novel coronavirus. Capture probes targeting vertebrate viruses, designed in 2015, enhanced both sequencing depth and coverage of the SARS-CoV-2 genome, the latter increasing from 71% to 98%.ConclusionThe model used for simulation of virus discovery enabled validation of the metagenomic sequencing protocol. The metagenomic protocol with virus probes designed before the pandemic, can assist the detection and identification of novel coronaviruses directly in clinical samples.

Virome Sequencing in Patients With Myocarditis.

Polymerase chain reaction analyses of cardiac tissues have detected viral sequences in up to 67% of cases of myocarditis. However, viruses have not been implicated in giant cell myocarditis (GCM). Furthermore, efforts to detect viruses implicated in myocarditis have been unsuccessful in more accessible samples such as peripheral blood. We used Virome Capture Sequencing for Vertbrate Viruses (VirCapSeq-VERT), a method that simultaneously screens for all known vertebrate viruses, to investigate viruses in 33 patients with myocarditis. We investigated peripheral blood mononuclear cells (n=24), plasma (n=27), endomyocardial biopsies (n=2), and cardiac tissue samples from explanted hearts (n=13). Nine patients (27%) had GCM and 4 patients (13%) had fulminant myocarditis. We found the following viruses in the blood of patients with myocarditis: Epstein Barr virus (n=11, 41%), human pegivirus (n=1, 4%), human endogenous retrovirus K (n=27, 100%), and anellovirus (n=15, 56%). All tissue samples from fulminant myocarditis (n=2) and GCM (n=13) contained human endogenous retrovirus K. No nucleic acids from viruses previously implicated in myocarditis or other human illnesses were detected in relevant amounts in cardiac tissue samples from GCM or in blood samples from other types of myocarditis. These findings do not exclude a role for viral infection in GCM but do suggest that if viruses are implicated, the mechanism is likely to be indirect rather than due to cytotoxic infection of myocardium.

Transmission of plant and vertebrate viruses by arthropods

Many plant and vertebrate viruses use mobile vectors to be transmitted between hosts. These vectors, mainly arthropods, acquire or inoculate the virus by feeding on plant extract or vertebrate blood. Several virus transmission modes have been characterized based on the tight interactions between the virus and the vector. Some viruses are internalized into cells and migrate through different tissues and organs before being released. In the vector, the virus can replicate in some cases. Other viruses are retained, specifically or non-specifically, on the vector mouthparts. Acquiring knowledge on the molecular mechanisms of virus transmission by arthropods consists in studying (i) virus receptors in the vectors, (ii) the mode of virus uptake into vector cells, (iii) virus localization and transport in the vector, and (iv) viral determinants required for transmission. This review, although non exhaustive, presents a state-of-the-art of plant and vertebrate virus transmission by arthropods, notably by pointing to their similarities and differences.

Investigation of viral etiology in potentially malignant disorders and oral squamous cell carcinomas in non-smoking, non-drinking patients.

Head and neck squamous cell carcinomas (HNSCC) are the seventh most frequent cancers. Among HNSCCs, oral squamous cell carcinomas (OSCCs) include several anatomical locations of the oral cavity, but exclude the oropharynx. The known risk factors for OSCCs are mainly alcohol consumption and tobacco use for at least 75–80% of cases. In addition to these risk factors, Human papillomavirus (HPV) types 16 and 18, classified as high-risk (HR) HPV genotypes, are considered as risk factors for oropharyngeal cancers, but their role in the development of OSCC remains unclear. We tested the hypothesis of viral etiology in a series of 68 well-characterized OSCCs and 14 potentially malignant disorders (PMD) in non-smoking, non-drinking (NSND) patients using broad-range, sensitive molecular methodologies. Deep-sequencing of the transcriptome did not reveal any vertebrate virus sequences other than HPV transcripts, detected in only one case. In contrast, HPV DNA was detected in 41.2% (28/68) and 35.7% (5/14) of OSCC and PMD cases, respectively. Importantly, 90.9% (30/33) of these belonged to the Betapapillomavirus genus, but no viral transcripts were detected. Finally, high-throughput sequencing revealed reads corresponding to transcripts of the Trichomonas vaginalis virus (TVV), which were confirmed by RT-PCR in two OSCCs. Our results strongly suggest that Alphapapillomavirus genotypes classified as HR are not involved in the development of OSCCs in NSND patients and that known oncogenic infectious agents are absent in these specific OSCCs. Any possible direct or indirect role of Betapapillomavirus genus members and TVV in OSCCs remains speculative and requires further investigation.

Synonymous Dinucleotide Usage: A Codon-Aware Metric for Quantifying Dinucleotide Representation in Viruses.

Distinct patterns of dinucleotide representation, such as CpG and UpA suppression, are characteristic of certain viral genomes. Recent research has uncovered vertebrate immune mechanisms that select against specific dinucleotides in targeted viruses. This evidence highlights the importance of systematically examining the dinucleotide composition of viral genomes. We have developed a novel metric, called synonymous dinucleotide usage (SDU), for quantifying dinucleotide representation in coding sequences. Our method compares the abundance of a given dinucleotide to the null hypothesis of equal synonymous codon usage in the sequence. We present a Python3 package, DinuQ, for calculating SDU and other relevant metrics. We have applied this method on two sets of invertebrate- and vertebrate-specific flaviviruses and rhabdoviruses. The SDU shows that the vertebrate viruses exhibit consistently greater under-representation of CpG dinucleotides in all three codon positions in both datasets. In comparison to existing metrics for dinucleotide quantification, the SDU allows for a statistical interpretation of its values by comparing it to a null expectation based on the codon table. Here we apply the method to viruses, but coding sequences of other living organisms can be analysed in the same way.

Capsid Structure of a Marine Algal Virus of the Order Picornavirales.

The order Picornavirales includes viruses that infect different kinds of eukaryotes and that share similar properties. The capsid proteins (CPs) of viruses in the order that infect unicellular organisms, such as algae, presumably possess certain characteristics that have changed little over the course of evolution, and thus these viruses may resemble the Picornavirales ancestor in some respects. Herein, we present the capsid structure of Chaetoceros tenuissimus RNA virus type II (CtenRNAV-II) determined using cryo-electron microscopy at a resolution of 3.1 Å, the first alga virus belonging to the family Marnaviridae of the order Picornavirales A structural comparison to related invertebrate and vertebrate viruses revealed a unique surface loop of the major CP VP1 that had not been observed previously, and further, revealed that another VP1 loop obscures the so-called canyon, which is a host-receptor binding site for many of the mammalian Picornavirales viruses. VP2 has an N-terminal tail, which has previously been reported as a primordial feature of Picornavirales viruses. The above-mentioned and other critical structural features provide new insights on three long-standing theories about Picornavirales: (i) the canyon hypothesis, (ii) the primordial VP2 domain swap, and (iii) the hypothesis that alga Picornavirales viruses could share characteristics with the Picornavirales ancestor.IMPORTANCE Identifying the acquired structural traits in virus capsids is important for elucidating what functions are essential among viruses that infect different hosts. The Picornavirales viruses infect a broad spectrum of hosts, ranging from unicellular algae to insects and mammals and include many human pathogens. Those viruses that infect unicellular protists, such as algae, are likely to have undergone fewer structural changes during the course of evolution compared to those viruses that infect multicellular eukaryotes and thus still share some characteristics with the Picornavirales ancestor. This article describes the first atomic capsid structure of an alga Marnavirus, CtenRNAV-II. A comparison to capsid structures of the related invertebrate and vertebrate viruses identified a number of structural traits that have been functionally acquired or lost during the course of evolution. These observations provide new insights on past theories on the viability and evolution of Picornavirales viruses.

Characterization of tick viromes collected from dogs in China

In this study, we explored the viral diversity and community structures of the ticks collected from dogs and detected possible tick-borne viruses (TBVs) using metagenomic analysis. Additionally, full-length sequences and the structural, phylogenetic and molecular features of the possible TBVs were characterized using bioinformatics tools. The sequence run produced 12,254,268 reads and 6,667,259 unique reads from the tick pools. Further analysis of the viral reads revealed that 92.73% were similar to ssRNA viruses, and 90.5% of the total viral sequences belonged to Non-classified viral families. Of the 29 classified viral families, most virome sequences were homologous with vertebrate viruses from Circoviridae, plant viruses from Virgaviridae, phages from Microviridae, insect viruses from Baculoviridae, and giant viruses from Mimiviridae. In the process of assembly of reads, large contigs representing four virus families were identified, including Phenuiviridae, Chuviridae, Parvoviridae and Non-classified families. Some sequences of Non-classified family shared a highly divergent amino acid sequence identities from existing virus sequences. The importance of these newly identified virus contigs to public and veterinary health needs additional researches. Because of the lack of the acquired viral reads from these families, we only demonstrated the complete sequences of Circoviridae in detail. The complete canine circovirus (CaCV)-NC21 genome detected from police dogs was 2,063 bp long with a GC content of 53.6%. Our results suggested that tick viromes collected from dogs contained diverse sequences with a broad range of animal, insect, plant, and phage viruses, which may reflect the ecological characteristics of the dogs and their ticks. Furthermore, our study revealed the existence of the partial contigs belonging to the four virus families in ticks. More researches are needed to verify their importance to public and veterinary health. As well, our detection of CaCv-NC21 demonstrated that ticks can harbor and potentially transmit canine circoviruses.

Mapping the virome in lab-reared and wild-caught Aedes albopictus mosquitoes

Mosquitoes are known vectors that can harbor a wide array of viruses that range from classical arboviruses that can infect vertebrates to insect-specific viruses that are restricted only in mosquito. Recent advances in the field of metagenomics and bioinformatics have strongly supported studies on mosquito-associated viruses. However, the study for mosquito virome profile is still inadequate. In the present study, we aimed to investigate and compare the virome structure and profile between laboratory reared and wild-caught Aedes albopictus mosquito. Four mosquito samples (egg, larvae, pupae, adult) of laboratory rearedand two (larvae and adult)of field collected from Guangzhou of China were prepared for next generation sequencing (NGS) and analyzed. The results indicated that the common viruses presented in all samples were High Island virus, Aedes flavivirus, and Guato virus, whereas the Wenzhou sobemo-like virus 4 and Hubei mosquito virus 2 were the most dominant virus and presented only in wild mosquito samples. Additionally, unclassified Papillomaviridae viruses was the most dominant vertebrate virus, which was significantly detected only in wild adult mosquitoes. Important to note, the viral composition of larvae obtained from the wild was the most diverse group with many viruses belonging to virus families including Flaviviridae, Totiviridae, Tymoviridae, Rhabdoviridae, Orthomyxoviridae, Iflaviridae and unclassified viruses. This observed diverse nature can be attributed to influence of environment substrates and water in which the mosquito larvae inhabit. In conclusion, our findings provide essential information necessary in understanding the structure and diversity of Aedes virome. Keywords: mosquito, virome, Aedes albopictus, next generation sequencing.

Deep Immunoprofiling By Flow Cytometry and NGS Reveals Distinct T Cell Profile in Pediatric Hepatitis-Associated Aplastic Anemia

Hepatitis-associated aplastic anemia (HAAA) is a rare variant of bone marrow (BM) failure that is typically diagnosed 2-3 months after an acute attack of hepatitis in patients that are seronegative for known hepatitis viruses. Although very little is known about its etiology, an autoimmune mechanism is presumed based on clinical response to immunosuppressive therapy (IST) and reports of oligoclonal T-lymphocyte expansion and skewing of CDR3 region length. However due to the rareness of this disease, the published evidence is still limited. We analyzed 18 pediatric patients consecutively diagnosed with HAAA in the Czech Republic between 2004-2019. Based on BM biopsy examination, 14 patients had aplastic anemia (AA) and interestingly 4 patients were histologically consistent with refractory cytopenia of childhood (RCC). Median age of these patients was 6.6 years (range 1-18) with a male to female ratio of 2:1. The prevalence of HAAA in our cohort of pediatric patients with BM failure (n=107; excluding patients with known genetic cause) was 17%. The presence of a paroxysmal nocturnal hemoglobinuria clone was excluded and cytogenetic evaluation showed no abnormalities. We examined both the diagnostic BM and peripheral blood by flow cytometry in all patients (with the exception of one peripheral blood sample) and performed deep immunophenotyping of T cells in 7 of them. Ten patients were selected for T cell receptor beta (TRB) repertoire and whole-exome sequencing (WES) based on the availability of DNA samples isolated from the BM before the start of the treatment. TRB sequencing was performed following the SOP developed by the EuroClonality-NGS working group with DNA input normalized to the equivalent of 20 000 CD3+ cells per sample based on flow cytometry data. Bioinformatic analysis was performed with ARResT/Interrogate. WES was performed on Illumina NextSeq 500 and libraries prepared using the Agilent SureSelectXT Human All Exon V6+UTRs kit. Vertebrate virus detection was performed using the VirCapSeq-VERT probe capture enrichment set and massive parallel sequencing from 8 BM DNA samples taken at the time of diagnosis. We found significant activation of CD8 positive T cells based on the expression of HLA-DR compared to non-HAAA RCC and AA patients (n=20 and n=21 respectively, Mann-Whitney p<0.0001). Three out of 7 patients with deep T cell immunophenotype available showed a decrease of naive CD27pos45RAposCD8pos T cells. From TRB repertoire analysis we were able to identify a total of 5 private immunodominant clones in 3 patients with relative abundance of up to 11% from all productive TRB sequences. These clones were not found in any previous publications or clonotype databases based on their CDR3 sequence. We detected 5 clones shared by 3 or more patients; these were previously published in other cohorts and are not specific for HAAA. WES analysis did not reveal any previously published variant in genes associated with immune dysregulation or immunodeficiency, or any candidate variant for functional validation. No relevant vertebrate virus signal was detected in the 8 samples. Out of all 18 patients, 3 patients underwent MSD-HSCT without preceding immunotherapy. Fifteen patients were treated according to standard European Working Group on MDS/SAA protocols with anti-thymocyte globulin (horse n=6; rabbit n=9), corticosteroids and cyclosporin A. In total, 7 patients reached complete remission on IST by day 120 and 7 patients with insufficient response underwent MUD-HSCT. One patient died shortly after MUD-HSCT because of toxicity. We did not observe any correlation of T cell populations at diagnosis or day 120 of IST (activated and exhausted T cells) and clinical response. Similarly, the presence of an immunodominant clone or oligoclonal TRB repertoire with lower diversity at diagnosis had no significant impact on the outcome of these patients in our cohort. In conclusion, we present the biggest cohort of pediatric patients with HAAA analyzed by next-generation sequencing and flow cytometry so far. Even though our results show significantly increased activation of CD8 positive T-cells and presence of expanded clones, we did not confirm that these factors would be useful and significant for the prediction of treatment outcome. Large number of our patients did not fully respond to IST, requiring HSCT. Supported by AZV 18-07-00430, 16-32568A and PRIMUS/17/MED/11. Disclosures No relevant conflicts of interest to declare.

Susceptibility of Indian major carp Labeo rohita to tilapia lake virus

Infection with tilapia lake virus (TiLV) has been reported to cause large-scale mortalities in tilapines, the second most cultured species in the world. Interestingly, in polyculture system, although tilapia is reported to suffer high mortalities due to infection with TiLV, the disease has not been reported in co-cultured species. Nevertheless, natural host switch is a frequent phenomenon among viruses of lower vertebrates, especially fish viruses. To analyse the risk of spread of virus to new host species, it is essential to study the susceptibility of other fish species to the virus. In India, tilapia is generally cultured along with Indian major carps (IMC) in polyculture systems. Keeping this in consideration, in the present study, the susceptibility of rohu Labeo rohita, an important major carp, to TiLV was evaluated following experimental infection and it was compared with a susceptible host, i.e. Nile tilapia Oreochromis niloticus. The results revealed that the experimentally-infected rohu did not exhibit any clinical signs and mortality. On the other hand, in tilapia, a cumulative mortality of 80% was observed during the experimental period of 12 days with clinical signs typical of TiLV disease. In histopathological examination of liver, one of the main target organ of TiLV, typical syncytial cells were observed in tilapia whereas, no alterations were observed in rohu. Importantly, in rohu, the virus copy numbers in liver and intestine were almost negligible (<3 virus copies) and did not increase with time course, and the virus was not detected in brain at any time point by qRT-PCR. However, in tilapia, there was significant increase in virus copy numbers in liver, brain as well as intestine up to 6 days post-infection (dpi), and thereafter, a marginal decrease was observed up to 12 dpi. Therefore, based on lack of clinical signs and mortality pattern, histopathological findings and results of qRT-PCR, it can be concluded that TiLV did not replicate in rohu, and it is not susceptible to infection with TiLV.

Hepatitis E virus genotype 3 strains and a plethora of other viruses detected in raw and still in tap water

In this study, next generation sequencing was used to explore the virome in 20L up to 10,000L water from different purification steps at two Swedish drinking water treatment plants (DWTPs), and in tap water. One DWTP used ultrafiltration (UF) with 20 nm pores, the other UV light treatment after conventional treatment of the water. Viruses belonging to 26 different families were detected in raw water, in which 6–9 times more sequence reads were found for phages than for known environmental, plant or vertebrate viruses. The total number of viral reads was reduced more than 4-log10 after UF and 3-log10 over UV treatment. However, for some viruses the reduction was 3.5-log10 after UF, as for hepatitis E virus (HEV), which was also detected in tap water, with sequences similar to those in raw water and after treatment. This indicates that HEV had passed through the treatment and entered into the supply network. However, the viability of the viruses is unknown. In tap water 10–130 International Units of HEV RNA/mL were identified, which is a comparable low amount of virus. The risk of getting infected through consumption of tap water is probably negligible, but needs to be investigated. The HEV strains in the waters belonged to subtypes HEV3a and HEV3c/i, which is associated with unknown source of infection in humans infected in Sweden. None of these subtypes are common among pigs or wild boar, the major reservoirs for HEV, indicating that water may play a role in transmitting this virus. The results indicate that monitoring small fecal/oral transmitted viruses in DWTPs may be considered, especially during community outbreaks, to prevent potential transmission by tap water.

Inherent Virus Characteristics and Host Range Drive the Zoonotic and Emerging Potential of Viruses

Background: Understanding the zoonotic and emerging potential of viruses is important to inform disease control and prevention programs. We demonstrate that inherent virus characteristics and virus host-range allow prediction of the zoonotic and disease emergence potential of virus species. Methods: We examined recent International Committee on Taxonomy of Viruses (ICTV) virus species (n=4958) data to identify 1479 vertebrate virus species and their host range. Species and host range were then used as key predictors of two host-associated outcomes - zoonotic potential (confirmed and potential) and disease emergence. Findings: We categorised 2010 viruses as Animalia viruses, including 1479 vertebrate infecting viruses. Vertebrate viruses were further categorised as human (n=404), bat (n=219), rodent (n=186), non-human primate (n=206) and livestock (n=267). In addition to 164 known zoonotic viruses (zoonoses confirmed), we report 231 viruses that infect humans and have also been reported from other vertebrate hosts (zoonotic potential). Our model predicted 27 viruses - not reported as zoonotic to date - to have zoonotic potential. Some of these viruses (n=11, 40·7%) have already been isolated from humans and other vertebrate host(s). Most of the predicted zoonotic (confirmed) risk viruses belonged to the virus families Peribunyaviridae (n=4) and Flaviviridae (n=9). Viruses with highest predicted probabilities included Thiafora orthonairovirus (p=0·92) and Uukuniemi phlebovirus (p=0·91). We identified 12 viruses - not reported as emerging human viruses to date - with an emergence risk. Most of these viruses belonged to the families Arenaviridae (n=3) and Flaviviridae (n=5). Viruses with the highest predicted probabilities included mucambo virus (p=0·88) and lymphocytic choriomeningitis mammarenavirus (p=0·67). We report significant interactions in the host range as well as host-viral traits, not previously reported, that influence the zoonotic and emergence potential of viruses. Interpretation: We conclude that inherent virus characteristics are important to identify their zoonotic and emerging potential. RNA viruses reported from non-human primates, livestock viruses not reported from invertebrates and bat viruses neither reported from invertebrates nor from birds were more likely to be zoonotic. Zoonotic pathogens also reported from non-human primates were more likely to emerge. We recommend the influential role of phylogenetic relatedness and social closeness amongst humans and other vertebrates to be interpreted given the significant interactions that occur in host-range as well as host-viral traits that influence the zoonotic potential of a virus. Funding: Department of Education and Training, Australian Government. Declaration of Interest: We declare no competing interests. Ethical Approval: Not required.

Chichen type III interferon produced by silkworm bioreactor induces ISG expression and restricts ALV-J infection in vitro

Type III interferon (IFN-λ) has recently been shown to exert a significant antiviral impact against viruses in vertebrates. Avian leukosis virus subgroup J (ALV-J), which causes tumor disease and immunosuppression in infected chicken, is a retrovirus that is difficult to prevent and control because of a lack of vaccines and drugs. Here, we obtained chicken IFN-λ (chIFN-λ) using a silkworm bioreactor and demonstrated that chIFN-λ has antiviral activity against ALV-J infection of both chicken embryo fibroblast cell line (DF1) and epithelial cell line (LMH). We found that chIFN-λ triggered higher levels of particular type III interferon-stimulated genes (type III ISGs) including myxovirus resistance protein (Mx), viperin (RSAD2), and interferon-inducible transmembrane protein 3 (IFITM3) in DF1 and LMH cells. Furthermore, over-expression of Mx, viperin, and IFITM3 could inhibit ALV-J infection in DF1 and LMH cells. Therefore, these results suggested that the anti-ALV-J function of chIFN-λ was specifically implemented by induction of expression of type III ISGs. Our data identified chIFN-λ as a critical antiviral agent of ALV-J infection and provides a potentially and attractive platform for the production of commercial chIFN-λ.

Proteomics Computational Analyses Suggest that the Antennavirus Glycoprotein Complex Includes a Class I Viral Fusion Protein (α-Penetrene) with an Internal Zinc-Binding Domain and a Stable Signal Peptide

A metatranscriptomic study of RNA viruses in cold-blooded vertebrates identified two related viruses from frogfish (Antennarius striatus) that represent a new genus Antennavirus in the family Arenaviridae (Order: Bunyavirales). Computational analyses were used to identify features common to class I viral fusion proteins (VFPs) in antennavirus glycoproteins, including an N-terminal fusion peptide, two extended alpha-helices, an intrahelical loop, and a carboxyl terminal transmembrane domain. Like mammarenavirus and hartmanivirus glycoproteins, the antennavirus glycoproteins have an intracellular zinc-binding domain and a long virion-associated stable signal peptide (SSP). The glycoproteins of reptarenaviruses are also class I VFPs, but do not contain zinc-binding domains nor do they encode SSPs. Divergent evolution from a common progenitor potentially explains similarities of antennavirus, mammarenavirus, and hartmanivirus glycoproteins, with an ancient recombination event resulting in a divergent reptarenavirus glycoprotein.

Effects of Arbovirus Multi-Host Life Cycles on Dinucleotide and Codon Usage Patterns.

Arthropod-borne viruses (arboviruses) of vertebrates including dengue, zika, chikungunya, Rift Valley fever, and blue tongue viruses cause extensive morbidity and mortality in humans, agricultural animals, and wildlife across the globe. As obligate intercellular pathogens, arboviruses must be well adapted to the cellular and molecular environment of both their arthropod (invertebrate) and vertebrate hosts, which are vastly different due to hundreds of millions of years of separate evolution. Here we discuss the comparative pressures on arbovirus RNA genomes as a result of a dual host life cycle, focusing on pressures that do not alter amino acids. We summarize what is currently known about arboviral genetic composition, such as dinucleotide and codon usage, and how cyclical infection of vertebrate and invertebrate hosts results in different genetic profiles compared with single-host viruses. To serve as a comparison, we compile what is known about arthropod tRNA, dinucleotide, and codon usages and compare this with vertebrates. Additionally, we discuss the potential roles of genetic robustness in arboviral evolution and how it may vary from other viruses. Overall, both arthropod and vertebrate hosts influence the resulting genetic composition of arboviruses, but a great deal remains to be investigated.

Virome capture sequencing does not identify active viral infection in unicentric and idiopathic multicentric Castleman disease.

Castleman disease (CD) describes a spectrum of heterogeneous disorders defined by characteristic lymph node histopathology. Enlarged lymph nodes demonstrating CD histopathology can occur in isolation (unicentric CD; UCD) sometimes accompanied by mild symptoms, or at multiple sites (multicentric CD, MCD) with systemic inflammation and cytokine-driven multi-organ dysfunction. The discovery that Kaposi sarcoma herpesvirus/human herpesvirus (HHV)-8 drives MCD in a subset of patients has led to the hypotheses that UCD and MCD patients with negative HHV-8 testing by conventional methods may represent false negatives, or that these cases are driven by another virus, known or unknown. To investigate these hypotheses, the virome capture sequencing for vertebrate viruses (VirCapSeq-VERT) platform was employed to detect RNA transcripts from known and novel viruses in fresh frozen lymph node tissue from CD patients (12 UCD, 11 HHV-8-negative MCD [idiopathic MCD; iMCD], and two HHV-8-positive MCD) and related diseases (three T cell lymphoma and three Hodgkin lymphoma). This assay detected HHV-8 in both HHV-8-positive cases; however, HHV-8 was not found in clinically HHV-8-negative iMCD or UCD cases. Additionally, no novel viruses were discovered, and no single known virus was detected with apparent association to HHV-8-negative CD cases. Herpesviridae family members, notably including Epstein-Barr virus (EBV), were detected in 7 out of 12 UCD and 5 of 11 iMCD cases with apparent correlations with markers of disease severity in iMCD. Analysis of a separate cohort of archival formalin-fixed, paraffin-embedded lymph node tissue by In situ hybridization revealed significantly fewer EBV-positive cells in UCD and iMCD compared to tissue from HHV-8-positive MCD and EBV-associated lymphoproliferative disorder. In an additional cohort, quantitative testing for EBV by PCR in peripheral blood during disease flare did not detect systemic EBV viremia, suggesting detection lymph node tissue is due to occult, local reactivation in UCD and iMCD. This study confirms that HHV-8 is not present in UCD and iMCD patients. Further, it fails to establish a clear association between any single virus, novel or known, and CD in HHV-8-negative cases. Given that distinct forms of CD exist with viral and non-viral etiological drivers, CD should be considered a group of distinct and separate diseases with heterogeneous causes worthy of further study.