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Viral glycoproteins drive membrane fusion in enveloped viruses and determine host range, tissue tropism and pathogenesis 1 . Despite their importance, there is a fragmentary understanding of glycoproteins within the Flaviviridae 2 , a large virus family that include pathogens such as hepatitis C, dengue and Zika viruses, and numerous other human, animal and emergent viruses. For many flaviviruses the glycoproteins have not yet been identified, for others, such as the hepaciviruses, the molecular mechanisms of membrane fusion remain uncharacterized 3 . Here we combine phylogenetic analyses with protein structure prediction to survey glycoproteins across the entire Flaviviridae. We find class II fusion systems, homologous to the Orthoflavivirus E glycoprotein in most species, including highly divergent jingmenviruses and large genome flaviviruses. However, the E1E2 glycoproteins of the hepaciviruses, pegiviruses and pestiviruses are structurally distinct, may represent a novel class of fusion mechanism, and are strictly associated with infection of vertebrate hosts. By mapping glycoprotein distribution onto the underlying phylogeny, we reveal a complex evolutionary history marked by the capture of bacterial genes and potentially inter-genus recombination. These insights, made possible through protein structure prediction, refine our understanding of viral fusion mechanisms and reveal the events that have shaped the diverse virology and ecology of the Flaviviridae., (© 2024. The Author(s).)

Background: Hainan Island and the Leizhou Peninsula, the southernmost part of mainland China, are areas where Aedes aegypti and Ae. albopictus are sympatric and are also high-incidence areas of dengue outbreaks in China. Many studies have suggested that Aedes endogenous viral components (EVEs) are enriched in piRNA clusters which can silence incoming viral genomes. Investigation the EVEs present in the piRNA clusters associated with viral infection of Aedes mosquitoes in these regions may provide a theoretical basis for novel transmission-blocking vector control strategies., Methods: In this study, specific primers for endogenous Flaviviridae elements (EFVEs) and endogenous Rhabdoviridae elements (ERVEs) were used to detect the distribution of Zika virus infection associated EVEs in the genomes of individuals of the two Aedes mosquitoes. Genetic diversity of EVEs with a high detection rate was also analyzed., Results: The results showed that many EVEs associated with Zika virus infection were detected in both Aedes species, with the detection rates were 47.68% to 100% in Ae. aegypti and 36.15% to 92.31% in sympatric Ae. albopictus populations. EVEs detection rates in another 17 Ae. albopictus populations ranged from 29.39% to 89.85%. Genetic diversity analyses of the four EVEs (AaFlavi53, AaRha61, AaRha91 and AaRha100) of Ae. aegypti showed that each had high haplotype diversity and low nucleotide diversity. The number of haplotypes in AaFlavi53 was 8, with the dominant haplotype being Hap_1 and the other 7 haplotypes being further mutated from Hap_1 in a lineage direction. In contrast, the haplotype diversity of the other three ERVEs (AaRha61, AaRha91 and AaRha100) was more diverse and richer, with the haplotype numbers were 9, 15 and 19 respectively. In addition, these EVEs all showed inconsistent patterns of both population differentiation and dispersal compared to neutral evolutionary genes such as the Mitochondrial COI gene., Conclusion: The EFVEs and ERVEs tested were present at high frequencies in the field Aedes mosquito populations. The haplotype diversity of the EFVE AaFlavi53 was relatively lower and the three ERVEs (AaRha61, AaRha91, AaRha100) were higher. None of the four EVEs could be indicative of the genetic diversity of the Ae. aegypti population. This study provided theoretical support for the use of EVEs to block arbovirus transmission, but further research is needed into the mechanisms by which these EVEs are antiviral to Aedes mosquitoes., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests including financial and nonfinancial interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The emergence and spread of novel viral pathogens is a major threat to human health, particularly in the context of climate and human-induced change in land use. Alongshan virus (ALSV) is a tick-borne virus associated with human disease, which was first identified in northeast China. More recently, several studies reported the emergence of ALSV in mammalian and arthropod hosts in multiple different countries outside of Asia, and the first viral genome sequencing data has become available. ALSV is a member of the Jingmenvirus group closely related to the Flaviviridae family. Unusually, the positive-sense, single-stranded RNA genome of ALSV is segmented and consists of four distinct segments, two of which show homology with the NS3 and NS5 protein encoding regions of non-segmented flaviviruses. Transmission of arthropod-borne pathogens will likely increase in the future due to environmental change mediated by a variety of environmental and ecological factors and increasing human encroachment into wild animal habitats. In this review, we present current knowledge of global ALSV distribution and emergence patterns, highlight genetic diversity, evolution and susceptible species. Finally, we discuss the role of this emerging tick-borne virus in the context of urbanization and global health.

In the last few years, there has been a dramatic increase in the number of discovered viruses that are transmitted by arthropods. Some of them are pathogenic for humans and mammals, and the pathogenic potential of others is unknown. The genus Orthoflavivirus belongs to the family Flaviviridae and includes arboviruses that cause severe human diseases with damage to the central nervous system and hemorrhagic fevers, as well as viruses with unknown vectors and viruses specific only to insects. The latter group includes Lammi virus, first isolated from a mosquito pool in Finland. It is known that Lammi virus successfully replicates in mosquito cell lines but not in mammalian cell cultures or mice. Lammi virus reduces the reproduction of West Nile virus during superinfection and thus has the potential to reduce the spread of West Nile virus in areas where Lammi virus is already circulating. In this work, we isolated Lammi virus from a pool of adult Aedes cinereus mosquitoes that hatched from larvae/pupae collected in Saint Petersburg, Russia. This fact may indicate transovarial transmission and trans-stadial survival of the virus.

After infection by flaviviruses like Zika and West Nile virus, eukaryotic hosts employ the well-conserved endoribonuclease Xrn1 to degrade the viral genomic RNA. Within the 3' untranslated regions, this enzyme encounters intricate Xrn1-resistant structures. This results in the accumulation of subgenomic flaviviral RNAs, an event that improves viral growth and aggravates viral pathogenicity. Xrn1-resistant RNAs have been established throughout the flaviviral genus, but not yet throughout the entire Flaviviridae family. In this work, we use previously determined characteristics of these structures to identify homologous sequences in many members of the genera pegivirus, hepacivirus and pestivirus. We used structural alignment and mutational analyses to establish that these sequences indeed represent Xrn1-resistant RNA and that they employ the general features of the flaviviral xrRNAs, consisting of a double pseudoknot formed by five base-paired regions stitched together by a crucial triple base interaction. Furthermore, we demonstrate that the pestivirus Bungowannah virus produces subgenomic RNA in vivo . Altogether, these results indicate that viruses make use of a universal Xrn1-resistant RNA throughout the Flaviviridae family.

RNA interference (RNAi)-mediated antiviral immunity is believed to be the primary defense against viral infection in mosquitoes. The production of virus-specific small RNA has been demonstrated in mosquitoes and mosquito-derived cell lines for viruses in all of the major arbovirus families. However, many if not all mosquitoes are infected with a group of viruses known as insect-specific viruses (ISVs), and little is known about the mosquito immune response to this group of viruses. Therefore, in this study, we sequenced small RNA from an Aedes albopictus -derived cell line infected with either Lammi virus (LamV) or Hanko virus (HakV). These viruses belong to two distinct phylogenetic groups of insect-specific flaviviruses (ISFVs). The results revealed that both viruses elicited a strong virus-derived small interfering RNA (vsiRNA) response that increased over time and that targeted the whole viral genome, with a few predominant hotspots observed. Furthermore, only the LamV-infected cells produced virus-derived Piwi-like RNAs (vpiRNAs); however, they were mainly derived from the antisense genome and did not show the typical ping-pong signatures. HakV, which is more distantly related to the dual-host flaviviruses than LamV, may lack certain unknown sequence elements or structures required for vpiRNA production. Our findings increase the understanding of mosquito innate immunity and ISFVs' effects on their host.

Pestiviruses are plus-stranded RNA viruses belonging to the family Flaviviridae. They comprise several important pathogens like classical swine fever virus and bovine viral diarrhea virus that induce economically important animal diseases. In 2017, the last update of pestivirus taxonomy resulted in demarcation of 11 species designated Pestivirus   A through Pestivirus K . Since then, multiple new pestiviruses have been reported including pathogens associated with disease in pigs or small ruminants. In addition, pestivirus sequences have been found during metagenomics analysis of different non-ungulate hosts (bats, rodents, whale, and pangolin), but the consequences of this pestivirus diversity for animal health still need to be established. To provide a systematic classification of the newly discovered viruses, we analyzed the genetic relationship based on complete coding sequences (cds) and deduced polyprotein sequences and calculated pairwise distances that allow species demarcation. In addition, phylogenetic analysis was performed based on a highly conserved region within the non-structural protein NS5B. Taking into account the genetic relationships observed together with available information about antigenic properties, host origin, and characteristics of disease, we propose to expand the number of pestivirus species to 19 by adding eight additional species designated Pestivirus   L through Pestivirus   S .

Jingmen tick virus (JMTV) is a recently identified virus which provides an unexpected connection between segmented and unsegmented RNA viruses. Recent investigations reveal that JMTV including JMTV-like virus (Alongshan virus) could be associated with human disease, suggesting the significance of JMTV in public health. To better understand the genetic diversity and host range of JMTV, a total of 164 rodents representing 8 species were collected in Qapqal Xibe county of Xinjiang Uygur Autonomous Region, China, and were screened for JMTVs using RT- PCR. Consequently, JMTV was identified in 42 rodents including 23 Microtus arvalis voles (24.5%), 9 Apodemus uralensis mice (29.0%), 5 Mus musculus mice, 1 Rhombomys opimus gerbil, 1 Meriones tamariscinus gerbil, 1 Meriones libycus gerbil, 1 Cricetulus migratorius hamster and 1 Microtus gregalis vole. Interestingly, nearly complete genome sequences were successfully recovered from 7 JMTV positive samples. Although the newly identified rodent JMTVs were closely related to those previously identified in ticks from China, based on the phylogenetic analysis of the S1, S2 and S3 segments, the newly identified rodent viruses clustered into two genetic groups. One group comprised of viruses only found in M. arvalis, while another group included viruses from A. uralensis, C. migratorius and M. gregalis. However, all rodent viruses clustered together in the S4 tree. Considering rodents live in close proximity to humans, more efforts are needed to investigate the role of rodents in the evolution and transmission of JMTV in nature., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

Viruses have developed innovative strategies to exploit the cellular machinery and overcome the antiviral defenses of the host, often using specifically structured RNA elements. Examples are found in the Flavivirus genus (in the family Flaviviridae ), where during flaviviral infection, pathogenic subgenomic flaviviral RNAs (sfRNAs) accumulate in the cell. These sfRNAs are formed when a host cell 5' to 3' exoribonuclease degrades the viral genomic RNA but is blocked by an exoribonuclease-resistant RNA structure (xrRNA) located in the viral genome's 3' untranslated region (UTR). Although known to exist in several Flaviviridae genera, the full distribution and diversity of xrRNAs in this family were unknown. Using the recently solved high-resolution structure of an xrRNA from the divergent flavivirus Tamana bat virus (TABV) as a reference, we used bioinformatic searches to identify xrRNAs in the remaining three genera of Flaviviridae : Pegivirus , Pestivirus , and Hepacivirus We biochemically and structurally characterized several examples, determining that they are genuine xrRNAs with a conserved fold. These new xrRNAs look superficially similar to the previously described xrRNAs but possess structural differences making them distinct from previous classes of xrRNAs. Overall, we have identified the presence of xrRNA in all four genera of Flaviviridae , but not in all species. Our findings thus require adjustments of previous xrRNA classification schemes and expand the previously known distribution of xrRNA in Flaviviridae. IMPORTANCE The members of the Flaviviridae comprise one of the largest families of positive-sense single-stranded RNA (+ssRNA) and are divided into the Flavivirus , Pestivirus , Pegivirus , and Hepacivirus genera. The genus Flavivirus contains many medically relevant viruses such as Zika virus, dengue virus, and Powassan virus. In these, a part of the RNA of the virus twists up into a distinct three-dimensional shape called an exoribonuclease-resistant RNA (xrRNA) that blocks the ability of the cell to "chew up" the viral RNA. Hence, part of the RNA of the virus remains intact, and this protected part is important for viral infection. These xrRNAs were known to occur in flaviviruses, but whether they existed in the other members of the family was not known. In this study, we identified a new subclass of xrRNA found not only in flaviviruses but also in the remaining three genera. The fact that these structured viral RNAs exist throughout the Flaviviridae family suggests they are important parts of the infection strategy of diverse pathogens, which could lead to new avenues of research., (Copyright © 2020 Szucs et al.)

Background: In 2014, a novel tick-borne virus of the Flaviviridae family was first reported in the Mogiana region of Brazil and named the Mogiana tick virus (MGTV). Thereafter, the Jingmen tick virus (JMTV), Kindia tick virus (KITV), and Guangxi tick virus (GXTV)-evolutionarily related to MGTV-were reported., Results: In the present study, we used small RNA sequencing (sRNA-seq) to detect viruses in ticks and discovered a new MGTV strain in Amblyomma testudinarium ticks collected in China's Yunnan Province in 2016. We obtained the full-length genome sequence of this MGTV strain Yunnan2016 (GenBank: MT080097, MT080098, MT080099 and MT080100) and recommended it for its inclusion in the NCBI RefSeq database for future studies on MGTV, JMTV, KITV and GXTV. Phylogenetic analysis showed that MGTV, JMTV, KITV and GXTV are monophyletic and belong to a MGTV group. Furthermore, this MGTV group of viruses may be phylogenetically related to geographical regions that were formerly part of the supercontinents Gondwana and Laurasia., Conclusions: To the best of our knowledge, this is the first study in which 5' and 3' sRNAs were used to generate full-length genome sequences of, but not limited to, RNA viruses. We also demonstrated the feasibility of using the sRNA-seq based method for the detection of viruses in pooled two and even possible one small ticks. MGTV may preserve the characteristic of ancient RNA viruses, which can be used to study the origin and evolution of RNA viruses. In addition, MGTV can be used as novel species for studies in phylogeography.

In recent decades, many new flavi-like viruses have been discovered predominantly in different invertebrates and, as was recently shown, some of them may cause disease in humans. The Jingmenvirus (JMV) group holds a special place among flaviviruses and flavi-like viruses because they have a segmented ssRNA(+) genome. We detected Alongshan virus (ALSV), which is a representative of the JMV group, in ten pools of adult Ixodes persulcatus ticks collected in two geographically-separated Russian regions. Three of the ten strains were isolated in the tick cell line IRE/CTVM19. One of the strains persisted in the IRE/CTVM19 cells without cytopathic effect for three years. Most ALSV virions purified from tick cells were spherical with a diameter of approximately 40.5 nm. In addition, we found smaller particles of approximately 13.1 nm in diameter. We obtained full genome sequences of all four segments of two of the isolated ALSV strains, and partial sequences of one segment from the third strain. Phylogenetic analysis on genome segment 2 of the JMV group clustered our novel strains with other ALSV strains. We found evidence for the existence of a novel upstream open reading frame in the glycoprotein-coding segment of ALSV and other members of the JMV group.

Human pegivirus 2 (HPgV-2) is a recently recognized pegivirus of the family Flaviviridae. To investigate the epidemic features of HPgV-2 circulating in the human immunodeficiency virus (HIV)-infected population, we tested for antibodies and viral RNA of HPgV-2 and hepatitis C virus (HCV) with retrospective plasma samples collected from 771 HIV infections with multiple risk behaviors in Honghe Prefecture of Yunnan Province. A total of 195 subjects (25.29%) were seroreactive to HPgV-2, and 41 (5.32%) were RNA positive. Although the positive rate of HPgV-2 antibodies in HIV/HCV-coinfected individuals (27.69%) was significantly higher than that of HIV monoinfections (20.82%) (p = 0.036), this is the first report of HPgV-2 viremia in HIV-infected individuals without HCV infection and the presence of two HPgV-2 lineages in China. Our data indicate that HPgV-2 can also be transmitted sexually, which might be facilitated when combined with HCV infection, injecting drug use, and risky sexual behavior, which appear to have a synergistic effect on HPgV-2 infection. Phylogenetic analysis of 26 near-full-length genome sequences showed that the HPgV-2 strains in China are divided into two clusters.

We describe a case of meningoencephalitis in which meta-transcriptomic (RNA) sequencing detected human pegivirus (HPgV) in brain tissue, cerebrospinal fluid, and serum in the absence of other pathogens. This is the first detection of HPgV in antemortem brain tissue, although it is uncertain whether HPgV is responsible for the observed encephalitis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

We applied an NGS based target capture approach to amplify HPgV-2 sequences from metagenomic libraries and enable full genome characterization. Despite expanded geographical sampling, sequence variability remains low, with diversity concentrated in approximately 3.3% of all amino acids. Serial samples from one HPgV-2 positive individual co-infected with comparable titers of HIV, HCV, and GBV-C showed that HPgV-2 remains highly stable over several weeks compared to other RNA viruses, despite a similarly error-prone polymerase. The consistent epidemiological association with and structural similarities to HCV, and the weak positive correlation of HCV and HPgV-2 titers shown here, suggests it may benefit from co-infection. While minimal selective pressure on HPgV-2 to evolve could suggest fitness, the rarity of HPgV-2 and the tight phylogenetic clustering of global strains likely indicates origination from a common source and a virus that is ill-suited to its host. Sporadic infections may explain the limited genetic diversity observed worldwide., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Approximately 2% of healthy persons are infected with human pegivirus (HPgV). HPgV is transmitted via vertical, sexual, and blood-borne routes. Recently, the association of HPgV infection with the risk of lymphoma was reported. Here, we examined the prevalence of chronic HPgV infection in liver transplantation (LT) recipients and patients with hepatectomy and the influence of HPgV infection after LT on clinical and perioperative factors. We enrolled 313 LT recipients and 187 patients with hepatectomy who received care at the Kyusyu University Hospital between May 1997 and September 2017. Of the 313 recipients and 187 patients enrolled in this study, 44 recipients (14.1%) and 2 patients (1.1%) had HPgV viremia, respectively. There was no significant association between HPgV infection and LT outcomes. Interestingly, one recipient was infected with HPgV during the peritransplant period, which was likely transmitted via blood transfusion because HPgV RNA was detected from the blood bag transfused to the recipient during LT. We reviewed the available literature on the prevalence HPgV infections in other organ-transplanted patients and whether they impacted clinical outcomes. They also had the higher prevalence of HPgV infection, while it appears to be of low or no consequences. In addition, HPgV infection induced the upregulation of interferon-stimulated gene (ISG) expression in peripheral blood mononuclear cells. LT recipients had higher HPgV viremia compared to patients with hepatectomy. Although HPgV infection was not associated with LT-related outcomes, it induced ISG expression in recipients., (© 2019 Wiley Periodicals, Inc.)

We obtained a Jingmen tick virus (JMTV) isolate, following inoculation of a tick pool with detectable Crimean-Congo hemorrhagic fever virus (CCHFV) RNA. We subsequently screened 7223 ticks, representing 15 species in five genera, collected from various regions in Anatolia and eastern Thrace, Turkey. Moreover, we tested specimens from various patient cohorts ( n = 103), and canine ( n = 60), bovine ( n = 20) and avian specimens ( n = 65). JMTV nucleic acids were detected in 3.9% of the tick pools, including those from several tick species from the genera Rhipicephalus and Haemaphysalis , and Hyalomma marginatum , the main vector of CCHFV in Turkey. Phylogenetic analysis supported two separate clades, independent of host or location, suggesting ubiquitous distribution in ticks. JMTV was not recovered from any human, animal or bird specimens tested. Near-complete viral genomes were sequenced from the prototype isolate and from three infected tick pools. Genome topology and functional organization were identical to the members of Jingmen group viruses. Phylogenetic reconstruction of individual viral genome segments and functional elements further supported the close relationship of the strains from Kosovo. We further identified probable recombination events in the JMTV genome, involving closely-related strains from Anatolia or China.

Jingmenvirus is a recently identified group of segmented RNA viruses phylogenetically linked with unsegmented Flaviviridae viruses. Primarily identified in various tick genera originating in China, Jingmenvirus geographical distribution has rapidly expanded to cover Africa, South America, Caribbean, and Europe. The identification of Jingmen-related viruses in various mammals, including febrile humans, opens the possibility that Jingmenviruses may be novel tick-borne arboviruses. In this study, we aimed at increasing knowledge of the host range, genetic diversity, and geographical distribution of Jingmenviruses by reporting for the first time the identification of Jingmenviruses associated with Rhipicephalus microplus ticks originating in the French Antilles (Guadeloupe and Martinique islands), with Amblyomma testudinarium ticks in Lao PDR, and with Ixodes ricinus ticks in metropolitan France, and from urine of Pteropus lylei bats in Cambodia. Analyses of the relationships between the different Jingmenvirus genomes resulted in the identification of three main phylogenic subclades, each of them containing both tick-borne and mammal-borne strains, reinforcing the idea that Jingmenviruses may be considered as tick-borne arboviruses. Finally, we estimated the prevalence of Jingmenvirus-like infection using luciferase immunoprecipitation assay screening (LIPS) of asymptomatic humans and cattle highly exposed to tick bites. Among 70 French human, 153 Laotian human, and 200 Caribbean cattle sera tested, only one French human serum was found (slightly) positive, suggesting that the prevalence of Jingmenvirus human and cattle infections in these areas is probably low. IMPORTANCE Several arboviruses emerging as new pathogens for humans and domestic animals have recently raised public health concern and increased interest in the study of their host range and in detection of spillover events. Recently, a new group of segmented Flaviviridae -related viruses, the Jingmenviruses, has been identified worldwide in many invertebrate and vertebrate hosts, pointing out the issue of whether they belong to the arbovirus group. The study presented here combined whole-genome sequencing of three tick-borne Jingmenviruses and one bat-borne Jingmenvirus with comprehensive phylogenetic analyses and high-throughput serological screening of human and cattle populations exposed to these viruses to contribute to the knowledge of Jingmenvirus host range, geographical distribution, and mammalian exposure., (Copyright © 2019 Temmam et al.)

Background: In racehorses, serum gamma-glutamyltransferase (GGT) activity is positively correlated with cumulative days in training and, when ≥100 IU/L, has been associated with poor performance. The prevalence of increased GGT activity in North American Thoroughbreds and its aetiopathogenesis are unknown. Four emerging viruses, pegivirus E (PgV E; equine pegivirus), hepacivirus A (HcV A; equine hepacivirus), pegivirus D (PgV D; Theiler's disease virus), and equine parvovirus-hepatitis (EqPV-H) have been identified in horses with clinical and subclinical hepatopathy. Available prevalence data indicate these viruses may commonly infect racehorses and contribute to increased liver enzyme activity in this population., Objectives: To investigate the association between viral infection and increased liver enzyme activity in racing Thoroughbreds., Study Design: Cross-sectional study., Methods: Prerace blood samples were collected from 802 Thoroughbreds and tested for GGT and sorbitol dehydrogenase (SDH) activity, and the presence of PgV E, HcV A, PgV D and EqPV-H nucleic acid., Results: Increased SDH and/or GGT were detected in 56.2% of the 802 serum samples. The infection prevalence and relative risk (RR) of having concurrently increased liver enzyme activity were: PgV E = 18.2% (RR = 0.820, 95% CI = 0.662-0.978, P = 0.03), HcV A = 2.5% (RR = 1.132, 95% CI = 0.719-1.466, P = 0.6), PgV D = 0.5% (RR = 0.875, 95% CI = 0.165-1.598, P>0.9), EqPV-H = 2.9% (RR = 0.916, 95% CI = 0.564-1.266, P = 0.7)., Main Limitations: Longitudinal samples were not tested., Conclusions: While viral infection was common among Thoroughbreds in this study, infection did not explain the high prevalence of increased liver enzyme activity. In fact, PgV E infection was associated with a reduced risk of having increased liver enzyme activity, indicating PgV E is unlikely to be a cause of hepatitis in horses. Importantly, like GGT, increased SDH activity was highly prevalent in this study, and provides additional evidence that hepatocellular injury was occurring in these horses., (© 2019 EVJ Ltd.)

Horses are often used as blood donors for commercial horse serum (HS) production and to manufacture biologicals. HS is an alternative for fetal bovine serum (FBS) used as a supplement for cell culture and vaccine production. Furthermore, HS is also frequently obtained in order to produce antisera toxins and pathogens. The advent of high-throughput sequencing (HTS) has promoted changes in virus detection, since previous knowledge of targets is not required. Thus, the present study aimed to describe the virome of five different batches of commercial HS from New Zealand (three batches) and Brazil and the United States (one batch each) using HTS. Each HS pool were processed and sequenced using an Illumina MiSeq platform. Sequences-related to viruses belonging to the Flaviviridae, Herpesviridae, and Parvoviridae families were detected. Particularly, equine hepacivirus (EqHV), equine pegivirus (EPgV), and Theiler's disease-associated virus (TDAV) were more frequent found in the batches analyzed. The presence of viral genomes in cell culture sera illustrates that these commercial sera can contain a mixture of different viruses and, therefore, can be regarded as potentially infectious for susceptible hosts. Moreover, the innocuity of commercial HS is important for the efficiency and security of diagnostics and the production of biological products., (Copyright © 2019 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Background: Flaviviridae viruses are single-stranded, positive-sense RNA viruses, which threat human constantly mediated by mosquitoes, ticks, and sandflies. Considering the recent increase in the prevalence of the family virus and its risk potential, we investigated the codon usage pattern to understand its evolutionary processes and provide some useful data to develop the medications for most of Flaviviridae viruses., Results: The overall extent of codon usage bias in 65 Flaviviridae viruses is low with the average value of GC contents being 50.5% and the highest value being 55.9%; the lowest value is 40.2%. ENC values of Flaviviridae virus genes vary from 48.75 to 57.83 with a mean value of 55.56. U- and A-ended codons are preferred in the Flaviviridae virus. Correlation analysis shows that the positive correlation between ENC value and GC content at the third nucleotide positions was significant in this family virus. The result of analysis of ENC, neutrality plot analysis, and correlation analysis revealed that codon usage bias of all the viruses was affected mainly by natural selection. Meanwhile, according to correspondence analysis (CoA) based on RSCU and phylogenetic analysis, the Flaviviridae viruses mainly are made up of two groups, Group I (Yellow fever virus, Apoi virus, Tembusu virus, Dengue virus 1, and others) and Group II (West Nile virus lineage 2, Japanese encephalitis virus, Usutu virus, Kedougou virus, and others)., Conclusions: All in, the bias of codon usage pattern is affected not only by compositional constraints but also by natural selection. Phylogenetic analysis also illustrates that codon usage bias of virus can serve as an effective means of evolutionary classification in Flaviviridae virus.

Background: In 2017, surveillance for tickborne diseases in China led to the identification of a patient who presented to a hospital in Inner Mongolia with a febrile illness that had an unknown cause. The clinical manifestation of the illness was similar to that of tickborne encephalitis virus (TBEV) infection, but neither TBEV RNA nor antibodies against the virus were detected., Methods: We obtained a blood specimen from the index patient and attempted to isolate and identify a causative pathogen, using genome sequence analysis and electron microscopy. We also initiated a heightened surveillance program in the same hospital to screen for other patients who presented with fever, headache, and a history of tick bites. We used reverse-transcriptase-polymerase-chain-reaction (RT-PCR) and cell-culture assays to detect the pathogen and immunofluorescence and neutralization assays to determine the levels of virus-specific antibodies in serum specimens from the patients., Results: We found that the index patient was infected with a previously unknown segmented RNA virus, which we designated Alongshan virus (ALSV) and which belongs to the jingmenvirus group of the family Flaviviridae. ALSV infection was confirmed by RT-PCR assay in 86 patients from Inner Mongolia and Heilongjiang who presented with fever, headache, and a history of tick bites. Serologic assays showed that seroconversion had occurred in all 19 patients for whom specimens were available from the acute phase and the convalescent phase of the illness., Conclusions: A newly discovered segmented virus was found to be associated with a febrile illness in northeastern China. (Funded by the National Key Research and Development Program of China and the National Natural Science Foundation of China.)., (Copyright © 2019 Massachusetts Medical Society.)

An equine parvovirus-hepatitis (EqPV-H) has been recently identified in association with equine serum hepatitis, also known as Theiler's disease. This disease was first described by Arnold Theiler in 1918 and is often observed after applications with blood products in equines. So far, the virus has only been described in the USA and China. In this study, we evaluated the presence of EqPV-H in several commercial serum samples to assess the potential risk of virus transmission by equine serum-based products for medical and research applications. In 11 out of 18 commercial serum samples, EqPV-H DNA was detectable with a viral load up to 10 5 copies/mL. The same serum batches as well as three additional samples were also positive for antibodies against the EqPV-H VP1 protein. The countries of origin with detectable viral genomes included the USA, Canada, New Zealand, Italy, and Germany, suggesting a worldwide distribution of EqPV-H. Phylogenetic analysis of the EqPV-H NS1 sequence in commercial serum samples revealed high similarities in viral sequences from different geographical areas. As horse sera are commonly used for the production of anti-sera, which are included in human and veterinary medical products, these results implicate the requirement for diagnostic tests to prevent EqPV-H transmission.

We report the discovery of the first cetacean pegivirus (family Flaviviridae) using a next-generation sequencing approach. One of two infected bottlenose dolphins had elevated activities of liver enzymes, which may suggest hepatocellular injury. Further research is needed to determine the epidemiology and pathogenicity of dolphin pegivirus., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Viral haemorrhagic fever can be caused by one of a diverse group of viruses that come from four different families of RNA viruses. Disease severity can vary from mild self-limiting febrile illness to severe disease characterized by high fever, high-level viraemia, increased vascular permeability that can progress to shock, multi-organ failure and death. Despite the urgent need, effective treatments and preventative vaccines are currently lacking for the majority of these viruses. A number of factors preclude the effective study of these diseases in humans including the high virulence of the agents involved, the sporadic nature of outbreaks of these viruses, which are typically in geographically isolated areas with underserviced diagnostic capabilities, and the requirements for high level bio-containment. As a result, animal models that accurately mimic human disease are essential for advancing our understanding of the pathogenesis of viral haemorrhagic fevers. Moreover, animal models for viral haemorrhagic fevers are necessary to test vaccines and therapeutic intervention strategies. Here, we present an overview of the animal models that have been established for each of the haemorrhagic fever viruses and identify which aspects of human disease are modelled. Furthermore, we discuss how experimental design considerations, such as choice of species and virus strain as well as route and dose of inoculation, have an influence on animal model development. We also bring attention to some of the pitfalls that need to be avoided when extrapolating results from animal models., (Copyright © 2016. Published by Elsevier Ltd.)

Lemurs are highly endangered mammals inhabiting the forests of Madagascar. In this study, we performed virus discovery on serum samples collected from 84 wild lemurs and identified viral sequence fragments from 4 novel viruses within the family Flaviviridae, including members of the genera Hepacivirus and Pegivirus. The sifaka hepacivirus (SifHV, two genotypes) and pegivirus (SifPgV, two genotypes) were discovered in the diademed sifaka (Propithecus diadema), while other pegiviral fragments were detected in samples from the indri (Indri indri, IndPgV) and the weasel sportive lemur (Lepilemur mustelinus, LepPgV). Although data are preliminary, each viral species appeared host species-specific and frequent infection was detected (18 of 84 individuals were positive for at least one virus). The complete coding sequence and partial 5' and 3' untranslated regions (UTRs) were obtained for SifHV and its genomic organization was consistent with that of other hepaciviruses, with one unique polyprotein and highly structured UTRs. Phylogenetic analyses showed the SifHV belonged to a clade that includes several viral species identified in rodents from Asia and North America, while SifPgV and IndPgV were more closely related to pegiviral species A and C, that include viruses found in humans as well as New- and Old-World monkeys. Our results support the current proposed model of virus-host co-divergence with frequent occurrence of cross-species transmission for these genera and highlight how the discovery of more members of the Flaviviridae can help clarify the ecology and evolutionary history of these viruses. Furthermore, this knowledge is important for conservation and captive management of lemurs.

Identifying novel viruses or assessing viral variation by NGS requires high sequencing coverage. More than 90% of total RNA is ribosomal (rRNA), making variant calling, virus discovery or transcriptomic profiling difficult. Current methods to increase informative reads suffer from drawbacks, either they cannot be used for some viruses, are optimized for a single species, or introduce bias. We describe a two-part approach combining reverse-transcription to create RNA/DNA hybrids which are then degraded with RNaseH/DNase sequentially that works for three medically relevant mosquito genera; Aedes, Anopheles, and Culex. We demonstrate depletion of rRNA from different samples, including whole mosquitoes and midgut contents from FTA cards. We describe novel insect-specific virus genomes from field collected mosquitoes. The protocol requires only common laboratory reagents and small oligonucleotides specific to rRNA. This approach can be adapted for other organisms, aiding virus diversity analyses, virus discovery and transcriptomics in both laboratory and field samples., (Copyright © 2018. Published by Elsevier Inc.)

Recent studies have suggested that human pegivirus 1 (HPgV-1) may have some pathogenic potential. In the southernmost region of Brazil, studies on HPgV-1 are scarce, and circulating genotypes have not yet been identified. The current study aimed to evaluate the prevalence of HPgV-1 among blood donors from the southernmost region of Brazil and identify the genotypes involved with associated factors. A cross-sectional study was conducted with 281 blood donors, who had their plasma subjected to RNA extraction, complementary DNA synthesis, HPgV-1 detection by nested polymerase chain reaction, and subsequent genotyping. The observed prevalence of HPgV-1-RNA was 21.7%. The only variable that was significantly associated with virus infection was the relationship status of the donor. Single or no fixed partner blood donors were twice as likely to have HPgV-1 (95% CI, 1.12 to 4.56; P = 0.02). Genotype 2-subtypes 2b (69%) and 2a (29%)-was the most prevalent. In the absence of risk factors for parenteral transmission, it is likely that sexual transmission was the route of infection in the individuals studied. Further work will be needed to determine whether this virus is inert in the population, or if there are potential deleterious effects in infected individuals., (© 2018 Wiley Periodicals, Inc.)

Metagenomic analysis of whole mosquitoes allows the genetic characterization of all associated viruses, including arboviruses and insect-specific viruses, plus those in their diet or infecting their parasites. We describe here the virome in mosquitoes, primarily Culex pipiens complex, Cx. tarsalis and Cx. erythrothorax, collected in 2016 from 31 counties in California, USA. The nearly complete genomes of 56 viruses, including 32 novel genomes, some from potentially novel RNA and DNA viral families or genera, were assembled and phylogenetically analyzed, significantly expanding the known Culex-associated virome. The majority of detected viral sequences originated from single-stranded RNA viral families with members known to infect insects, plants, or from unknown hosts. These reference viral genomes will facilitate the identification of related viruses in other insect species and to monitor changes in the virome of Culex mosquito populations to define factors influencing their transmission and possible impact on their insect hosts., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Human pegivirus (HPgV), previously called hepatitis G virus or GB virus C, is a lymphotropic virus with undefined pathology. Because many viruses from the family Flaviviridae, to which HPgV belongs, are neurotropic, we studied whether HPgV could infect the central nervous system. We tested serum and cerebrospinal fluid samples from 96 patients with a diagnosis of encephalitis for a variety of pathogens by molecular methods and serology; we also tested for autoantibodies against neuronal antigens. We found HPgV in serum and cerebrospinal fluid from 3 patients who had encephalitis of unclear origin; that is, all the markers that had been tested were negative. Single-strand confirmation polymorphism and next-generation sequencing analysis revealed differences between the serum and cerebrospinal fluid-derived viral sequences, which is compatible with the presence of a separate HPgV compartment in the central nervous system. It is unclear whether HPgV was directly responsible for encephalitis in these patients.

In 2013, two new viruses, equine pegivirus (EPgV) and Theiler's disease-associated virus (TDAV), both belonging to the genus Pegivirus within the family Flaviviridae, were identified. To investigate the geographical distribution and genetic diversity of these two viruses in China, we screened EPgV and TDAV infection in imported race horses and Chinese work horses by using reverse-transcription polymerase chain reaction (RT-PCR). EPgV was detected in 10.8 % (8/74) of the total horses tested, with a prevalence of 5.8 and 22.7 % in the race horses and work horses, respectively. No TDAV infection was found. A near full-length genome sequence of EPgV was obtained that showed an identity of 89.5-90.6 % at the nucleotide level and 98.1-98.3 % at the amino acid level with an American strain, C0035, and another Chinese strain, LW/216, respectively. Phylogenetic analysis showed two different clusters of the sequences from the race horses and work horses, indicating a difference in virus origin. Our results demonstrated a higher positive rate of EPgV in the Chinese work horses than in the imported race horses, a moderate genetic diversity of EPgV strains worldwide and possibly no liver pathogenesis for EPgV infection.

We report the presence of the second human pegivirus (HPgV-2) in Guangdong and Sichuan Provinces in China. The prevalence of HPgV-2 in hepatitis C virus/HIV-1-co-infected persons who inject drugs was 12.9% in Guangdong and 15.9% in Sichuan. This population is at high risk for HPgV-2 infection.

The human pegivirus (HPgV), classified in the Flaviviridae family - Pegivirus genus, is an RNA virus identified in 1995. HPgV is a lymphotrophic virus, with replication sites in bone marrow and lymphoid tissue, as well as in peripheral blood mononuclear cells (PBMCs). Transmission is through sexual and parenteral routes, and recent estimations suggest nearly 750 million people are infected with HPgV worldwide. Almost 25% of infected individuals can develop persistent infection. Until now, HPgV has been considered a non-pathogenic virus; however, epidemiological studies suggest a potential role in lymphoproliferative diseases, particularly in the development of non-Hodgkin lymphoma (NHL). The evidence of this is controversial and the role of HPgV in lymphomagenesis has not yet been demonstrated. Several studies report a high prevalence of HPgV infection in patients with NHL compared to controls and patients with other hematological diseases. Therefore, analytic studies show that HPgV could be related to an increased risk of NHL development. Conversely, other studies indicate no association between HPgV and NHL, so the role of HPgV in lymphomagenesis is not clear. This review summarizes the main findings related to HPgV's pathogenic potential and association with NHL.

In this study, we determined the complete coding sequence of a putative new member of the family Flaviviridae, named "Cyclopterus lumpus virus" (CLuV), which is associated with a serious disease in lumpfish (Cyclopterus lumpus). The virus was present in all tissues tested, but pathology was primarily observed in the liver and kidneys. CLuV shows low but distinct similarity to the unassigned Tamana bat virus (TABV). Unlike other known members of the family Flaviviridae, translation of the entire CLuV polyprotein is dependent on a - 1 ribosomal frameshift in the NS2A region.

Background: Human pegivirus type 2 (HPgV-2) is a novel blood-borne human pegivirus that mainly infects hepatitis C virus (HCV)-infected subjects. We have investigated the prevalence of HPgV-2 in China, its association with HCV and human immunodeficiency virus type 1 (HIV-1), and the impact on HCV viral load and liver damage., Methods: A cross-sectional study was conducted with both blood donors and HCV- and HIV-1-infected patients in Guangzhou, China. All subjects were screened for anti-HPgV-2 and HPgV-2 RNA. Demographic and clinical information were obtained from electronic medical records., Results: We tested 8198 serum or plasma samples. Only 0.15% (6/4017) of healthy blood donors were positive for anti-HPgV-2 and negative for HPgV-2 RNA. No HPgV-2 viremia was detected in hepatitis B virus- or HIV-1-monoinfected individuals. The relatively high frequency of HPgV-2 infection was observed in 1.23% (30/2440) and 0.29% (7/2440) of HCV-infected persons by serological assay and reverse-transcription polymerase chain reaction, respectively. Furthermore, anti-HPgV-2 and HPgV-2 RNA were detected in 8.91% (18/202) and 3.47% (7/202), respectively, of HCV/HIV-1-coinfected subjects. HPgV-2 persistent infection was documented in about 30% of anti-HPgV-2-positive individuals. In addition, HPgV-2 infection may not affect HCV-related liver injury and HCV viral load., Conclusions: Our results indicate the rarity of HPgV-2 infection in the general population and tight association with HCV, in particular with HCV/HIV-1 coinfection. HPgV-2 appears not to worsen HCV-related liver damage. Our study provides new findings about the association of HPgV-2 and HCV/HIV-1 and the impact of HPgV-2 infection on HCV replication and pathogenesis., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Eleven viral isolates derived mostly in albopictus C6/36 cells from mosquito pools collected in Southeast Asia and the Americas between 1966 and 2014 contained particles with electron microscopy morphology typical of reoviruses. Metagenomics analysis yielded the near complete genomes of three novel reoviruses, Big Cypress orbivirus, Ninarumi virus, and High Island virus and a new tetravirus, Sarawak virus. Strains of previously characterized Sathuvarachi, Yunnan, Banna and Parry's Lagoon viruses (Reoviridae), Bontang virus (Mesoniviridae), and Culex theileri flavivirus (Flaviviridae) were also characterized. The availability of these mosquito virus genomes will facilitate their detection by metagenomics or PCR to better determine their geographic range, extent of host tropism, and possible association with arthropod or vertebrate disease., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Human pegivirus-1 (HPgV-1) is a member of the Flaviviridae family whose genomic organization and mode of cellular entry is similar to that of hepatitis C virus (HCV). The E2 glycoprotein of HPgV-1 is the principle mediator in the virus-cell interaction and as such harbors most of HPgV-1's antigenic determinants. HPgV-1 persists in blood cell precursors which are increasingly used for cell therapy., Methods: We studied HPgV-1 prevalence in a large cohort of females donating fetal tissues for clinical use. PCR was used for screening and estimation of viral load in viremic plasma and fetal samples. Sequence analysis was performed for portions of the 5'-untranslated and E2 regions of HPgV-1 purified from donor plasmas. Sequencing was followed by phylogenetic analysis., Results: HPgV-1 was revealed in 13.7% of plasmas, 5.0% of fetal tissues, 5.4% of chorions, exceeding the prevalence of HCV in these types of samples. Transmission of HPgV-1 occurred in 25.8% of traceable mother-chorion-fetal tissues triads. For HPgV-1-positive donors, a high viral load in plasma appears to be a prerequisite for transmission. However, about one third of fetal samples acquired infection from non-viremic individuals. Sequencing of 5'-untranslated region placed most HPgV-1 samples to genotype 2a. At the same time, a portion of E2 sequence provided a much weaker support for this grouping apparently due to a higher variability. Polymorphisms were detected in important structural and antigenic motifs of E2., Conclusion: HPgV-1 is efficiently transmitted to fetus at early embryonic stages. A high variability in E2 may pose a risk of generation of pathogenic subtypes. Although HPgV-1 is considered benign and no longer tested mandatorily in blood banks, the virus may have adversary effects at target niches if delivered with infected graft upon cell transplantation. This argues for the necessity of HPgV-1 testing of cell samples aimed for clinical use.

Human Pegivirus-1 (HPgV-1) may have a beneficial impact on disease progression in human immunodeficiency virus-1 (HIV-1) infection. However, analysis of the genotypic diversity of HPgV-1 and its relevance to the progression of HIV-1 disease remains limited. A total of 1062 HIV-1-infected individuals were recruited in all sixteen prefectures of Yunnan province, China. The reverse transcription nested polymerase chain reaction (RT-nPCR), phylogenetic analyses, and clinical data analyses were used to detect HPgV-1 infection, determine genotype, and analyze HPgV-1 genotype impact on HIV-1 disease progression. The overall positive rate of HPgV-1 RNA was 23.4% (248/1062), and the frequency of HPgV-1 infection in injecting drug users (IDUs) (28.5%, 131/460) was significantly higher than in heterosexuals (19.4%, 117/602). Multiple genotypes were identified in 212 subjects with successful sequencing for the E2 gene, including genotype 7 (55.7%), genotype 3 (34.9%), genotype 4 (4.7%), genotype 2 (3.3%), and an unclassified group (1.4%). Moreover, genotype 7 predominated in IDUs, whereas genotype 3 was the most common in heterosexuals. Our results revealed that HPgV-1 genotype 7 groups exhibited significantly lower HIV-1 viral load and higher CD4⁺ cell counts. This finding suggests that HPgV-1 genotype 7 may be associated with a better progression of HIV-1 disease.

Arthropod-borne viruses (arboviruses) have in recent years become a tremendous global health concern resulting in substantial human morbidity and mortality. With the widespread utilization of molecular technologies such as next-generation sequencing and the advancement of bioinformatics tools, a new age of viral discovery has commenced. Many of the novel agents being discovered in recent years have been isolated from mosquitoes and exhibit a highly restricted host range. Strikingly, these insect-specific viruses have been found to be members of viral families traditionally associated with human arboviral pathogens, including but not limited to the families Flaviviridae, Togaviridae, Reoviridae, and Bunyaviridae. These agents therefore present novel opportunities in the fields of viral evolution and viral/vector interaction and have tremendous potential as agents for biocontrol of vectors and or viruses of medical importance., (© 2017 Elsevier Inc. All rights reserved.)

The Flaviviridae is a family of small enveloped viruses with RNA genomes of 9000-13 000 bases. Most infect mammals and birds. Many flaviviruses are host-specific and pathogenic, such as hepatitis C virus in the genus Hepacivirus. The majority of known members in the genus Flavivirus are arthropod borne, and many are important human and veterinary pathogens (e.g. yellow fever virus, dengue virus). This is a summary of the current International Committee on Taxonomy of Viruses (ICTV) report on the taxonomy of the Flaviviridae, which is available at www.ictv.global/report/flaviviridae.

To assess the potential role of ticks as carriers of West Nile virus (WNV) and Usutu virus (USUV), we tested 1721 ticks from 379 wild birds in Northwestern Italy between 2012 and 2014. Ticks were analyzed in pools using a pan-flavivirus real-time RT-PCR and positive pools were subjected to RT-PCR for USUV and WNV genome detection. All the tested samples resulted negative, suggesting that Ixodes spp. ticks, at least in our study area, are not competent vectors and not even exploitable sentinels for USUV and WNV.

Proposals are described for the assignment of recently reported viruses, infecting rodents, bats and other mammalian species, to new species within the Hepacivirus and Pegivirus genera (family Flaviviridae). Assignments into 14 Hepacivirus species (Hepacivirus A-N) and 11 Pegivirus species (Pegivirus A-K) are based on phylogenetic relationships and sequence distances between conserved regions extracted from complete coding sequences for members of each proposed taxon. We propose that the species Hepatitis C virus is renamed Hepacivirus C in order to acknowledge its unique historical position and so as to minimize confusion. Despite the newly documented genetic diversity of hepaciviruses and pegiviruses, members of these genera remain phylogenetically distinct, and differ in hepatotropism and the possession of a basic core protein; pegiviruses in general lack these features. However, other characteristics that were originally used to support their division into separate genera are no longer definitive; there is overlap between the two genera in the type of internal ribosomal entry site and the presence of miR-122 sites in the 5' UTR, the predicted number of N-linked glycosylation sites in the envelope E1 and E2 proteins, the presence of poly U tracts in the 3' UTR and the propensity of viruses to establish a persistent infection. While all classified hepaciviruses and pegiviruses have mammalian hosts, the recent description of a hepaci-/pegi-like virus from a shark and the likely existence of further homologues in other non-mammalian species indicate that further species or genera remain to be defined in the future.

Background: The identification of viruses in human blood is required for epidemiologic surveillance and to detect potentially emerging threats to blood transfusion safety., Study Design and Methods: Viral nucleic acids in plasma fractionation pools assembled from blood donors in the United States and Europe were analyzed by viral metagenomics., Results: Anelloviruses were detected in each of the 10 plasma pools. Human pegivirus A (HPgV; GB virus type C) sequences were identified in eight of the 10 pools, more than 90% of which belong to Genotype 2. The recently described human HPgV2 in Flaviviridae was not detected. A small number of sequence reads of human papillomavirus were also detected in three pools. In one pool, two different gemycircularvirus genomes were identified and fully sequenced. The capsid protein of one gemycircularvirus shared 83% to 84% identity to those of genomes from human serum and sewage. The presence of the gemycircularvirus genomes in the plasma pool was independently confirmed and the viral concentration estimated by digital PCR at more than 10(6) copies/mL assuming their origin from single donors., Conclusion: Further research is required to elucidate whether gemycircularviruses can infect humans or are indicative of contamination occurring during phlebotomy, plasma pool processing, or ongoing donor fungal infections., (© 2016 AABB.)

Since 2012, three viruses, known as equine hepacivirus (EqHV), equine pegivirus (EPgV) and Theiler's disease-associated virus (TDAV), have been discovered in equines. Given that these viruses are the newest members of the Flaviviridae family, genomic information concerning circulating EqHV, EPgV and TDAV strains around the world is limited. To date, no genetic surveillance studies have been performed on these three viruses in the equine population of China. Here, a total of 177 serum samples were collected from equines across China between 2014 and 2015. Using PCR, we detected viral RNA in the serum samples, six of which were EqHV positive and two of which were EPgV positive. Co-infection with the two viruses was not observed among the Chinese equines studied, and TDAV RNA was not detected in the equine serum samples collected for this study. Phylogenetic analysis of partial NS5B open reading frame (ORF), NS3 ORF, and 5' untranslated region nucleotide sequences from EqHV as well as partial NS3 ORF sequence from EPgV indicated that EqHV and EPgV have evolved into two main clades by themselves, both of which are circulating in China. Based on the partial NS5B and NS3 ORF sequences of EqHV, the sequences of one clade were also split into two subclades. This study enriches our knowledge of the geographic distribution of these three equine viruses.

We have used a metagenomic microarray to detect genomic RNA from human pegivirus in serum and cerebrospinal fluid from a patient suffering from severe encephalitis. No other pathogen was detected. HPgV in cerebrospinal fluid during encephalitis has never been reported before and its prevalence in cerebrospinal fluid needs further investigation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Next-generation sequencing has critical applications in virus discovery, diagnostics, and environmental surveillance. We used metagenomic sequence libraries for retrospective screening of plasma samples for the recently discovered human hepegivirus 1 (HHpgV-1). From a cohort of 150 hepatitis C virus (HCV)-positive case-patients, we identified 2 persons with HHpgV-1 viremia and a high frequency of human pegivirus (HPgV) viremia (14%). Detection of HHpgV-1 and HPgV was concordant with parallel PCR-based screening using conserved primers matching groups 1 (HPgV) and 2 (HHPgV-1) nonstructural 3 region sequences. PCR identified 1 HHPgV-1-positive person with viremia from a group of 195 persons with hemophilia who had been exposed to nonvirally inactivated factor VII/IX; 18 (9%) were HPgV-positive. Relative to HCV and HPgV, active infections with HHpgV-1 were infrequently detected in blood, even in groups that had substantial parenteral exposure. Our findings are consistent with lower transmissibility or higher rates of virus clearance for HHpgV-1 than for other bloodborne human flaviviruses.

Unlabelled: Viruses of the family Flaviviridae are important pathogens of humans and other animals and are currently classified into four genera. To better understand their diversity, evolutionary history, and genomic flexibility, we used transcriptome sequencing (RNA-seq) to search for the viruses related to the Flaviviridae in a range of potential invertebrate and vertebrate hosts. Accordingly, we recovered the full genomes of five segmented jingmenviruses and 12 distant relatives of the known Flaviviridae ("flavi-like" viruses) from a range of arthropod species. Although these viruses are highly divergent, they share a similar genomic plan and common ancestry with the Flaviviridae in the NS3 and NS5 regions. Remarkably, although these viruses fill in major gaps in the phylogenetic diversity of the Flaviviridae, genomic comparisons reveal important changes in genome structure, genome size, and replication/gene regulation strategy during evolutionary history. In addition, the wide diversity of flavi-like viruses found in invertebrates, as well as their deep phylogenetic positions, suggests that they may represent the ancestral forms from which the vertebrate-infecting viruses evolved. For the vertebrate viruses, we expanded the previously mammal-only pegivirus-hepacivirus group to include a virus from the graceful catshark (Proscyllium habereri), which in turn implies that these viruses possess a larger host range than is currently known. In sum, our data show that the Flaviviridae infect a far wider range of hosts and exhibit greater diversity in genome structure than previously anticipated., Importance: The family Flaviviridae of RNA viruses contains several notorious human pathogens, including dengue virus, West Nile virus, and hepatitis C virus. To date, however, our understanding of the biodiversity and evolution of the Flaviviridae has largely been directed toward vertebrate hosts and their blood-feeding arthropod vectors. Therefore, we investigated an expanded group of potential arthropod and vertebrate host species that have generally been ignored by surveillance programs. Remarkably, these species contained diverse flaviviruses and related viruses that are characterized by major changes in genome size and genome structure, such that these traits are more flexible than previously thought. More generally, these data suggest that arthropods may be the ultimate reservoir of the Flaviviridae and related viruses, harboring considerable genetic and phenotypic diversity. In sum, this study revises the traditional view on the evolutionary history, host range, and genomic structures of a major group of RNA viruses., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The known genetic diversity of the hepaciviruses and pegiviruses has increased greatly in recent years through the discovery of viruses related to hepatitis C virus and human pegivirus in bats, bovines, equines, primates, and rodents. Analysis of these new species is important for research into animal models of hepatitis C virus infection and into the zoonotic origins of human viruses. Here, we provide the first systematic phylogenetic and evolutionary analysis of these two genera at the whole-genome level. Phylogenies confirmed that hepatitis C virus is most closely related to viruses from horses whereas human pegiviruses clustered with viruses from African primates. Within each genus, several well-supported lineages were identified and viral diversity was structured by both host species and location of sampling. Recombination analyses provided evidence of interspecific recombination in hepaciviruses, but none in the pegiviruses. Putative mosaic genome structures were identified in NS5B gene region and were supported by multiple tests. The identification of interspecific recombination in the hepaciviruses represents an important evolutionary event that could be clarified by future sampling of novel viruses. We also identified parallel amino acid changes shared by distantly related lineages that infect similar types of host. Notable parallel changes were clustered in the NS3 and NS4B genes and provide a useful starting point for experimental studies of the evolution of Hepacivirus host-virus interactions., (© The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Hepatitis C virus (HCV), a major causative agent of acute and chronic liver disease, belongs to the Flaviviridæ family and contains a single-strand positive-sense RNA genome, which upon virus entry and uncoating, functions as mRNAs and thus can be directly translated into proteins by host cell machinery. To date the HCV origin remains unclear and HCV life cycle and pathogenesis are not enlightened processes due to the absence of HCV efficient cell cultures systems or animals models. Here we show that rabbit and hare HCV-like viruses, RHCV and HHCV respectively, are formed after the inoculation of genomic DNA in Madin-Darby bovine kidney cell line cultures. RHCV is closely related to the HCV-1a/HCV-1b genotypes and HHCV is more closely related to the HCV-1b genotype. These findings could contribute to the understanding of HCV origin as well as clarify the virus life cycle, pathogenesis, evolution and diversity.