Bioinformatics resources for SARS-CoV-2 discovery and surveillance.

In early January 2020, the novel coronavirus (SARS-CoV-2) responsible for a pneumonia outbreak in Wuhan, China, was identified using next-generation sequencing (NGS) and readily available bioinformatics pipelines. In addition to virus discovery, these NGS technologies and bioinformatics resources are currently being employed for ongoing genomic surveillance of SARS-CoV-2 worldwide, tracking its spread, evolution and patterns of variation on a global scale. In this review, we summarize the bioinformatics resources used for the discovery and surveillance of SARS-CoV-2. We also discuss the advantages and disadvantages of these bioinformatics resources and highlight areas where additional technical developments are urgently needed. Solutions to these problems will be beneficial not only to the prevention and control of the current COVID-19 pandemic but also to infectious disease outbreaks of the future.

Diagnosis and analysis of unexplained cases of childhood encephalitis in Australia using metatranscriptomic sequencing.

Encephalitis is most often caused by a variety of infectious agents identified through diagnostic tests utilizing cerebrospinal fluid. We investigated the clinical characteristics and potential aetiological agents of unexplained encephalitis through metagenomic sequencing of residual clinical samples from multiple tissue types and independent clinical review. Forty-three specimens were collected from 18 encephalitis cases with no cause identified by the Australian Childhood Encephalitis study. Samples were subjected to total RNA sequencing ('metatranscriptomics') to determine the presence and abundance of potential pathogens, and to describe the possible aetiologies of unexplained encephalitis. Using this protocol, we identified five RNA and two DNA viruses associated with human infection from both non-sterile and sterile sites, which were confirmed by PCR. These comprised two human rhinoviruses, two human seasonal coronaviruses, two polyomaviruses and one picobirnavirus. Human rhinovirus and seasonal coronaviruses may be responsible for five of the encephalitis cases. Immune-mediated encephalitis was considered likely in six cases and metatranscriptomics did not identify a possible pathogen in these cases. The aetiology remained unknown in nine cases. Our study emphasizes the importance of respiratory viruses in the aetiology of unexplained child encephalitis and suggests that non-central-nervous-system sampling in encephalitis clinical guidelines and protocols could improve the diagnostic yield.

Redefining the invertebrate RNA virosphere.

Current knowledge of RNA virus biodiversity is both biased and fragmentary, reflecting a focus on culturable or disease-causing agents. Here we profile the transcriptomes of over 220 invertebrate species sampled across nine animal phyla and report the discovery of 1,445 RNA viruses, including some that are sufficiently divergent to comprise new families. The identified viruses fill major gaps in the RNA virus phylogeny and reveal an evolutionary history that is characterized by both host switching and co-divergence. The invertebrate virome also reveals remarkable genomic flexibility that includes frequent recombination, lateral gene transfer among viruses and hosts, gene gain and loss, and complex genomic rearrangements. Together, these data present a view of the RNA virosphere that is more phylogenetically and genomically diverse than that depicted in current classification schemes and provide a more solid foundation for studies in virus ecology and evolution.

Molecular epidemiology of enterovirus from children with herpangina or hand, foot, and mouth disease in Hangzhou, 2016.

Enteroviruses (EVs) are the major cause of hand, foot, and mouth disease (HFMD) and herpangina in children. In this study, we conducted a molecular investigation of EVs in throat swab samples from children in Hangzhou, China with a diagnosis of HFMD or herpangina. EVs were detected using one-step real-time RT-PCR, and their serotypes were determined based on partial VP1 gene sequences. The molecular typing results revealed the presence of six different EV serotypes in HFMD cases, including coxsackievirus (CV) A16 (20/30, 66.7%), CVA4 (3/30, 10.0%), CVA6 (3/30, 10.0%), EVA71 (2/30, 6.7%), CVB4 (1/30, 3.3%), and CVB5 (1/30, 3.3%). Eleven different EV serotypes were detected in herpangina cases, among which CVA4 was the most frequently detected serotype (105/170, 61.8%), followed by CVA16 (30/170, 17.6%), CVB4 (9/170, 5.3%), CVA6 (6/170, 3.5%), CVB3 (5/170, 2.9%), CVA10 (3/170, 1.8%), EVA71 (4/170, 2.4%), Echo9 (3/170, 1.8%), CVA9 (2/170, 1.2%), CVB1 (3/170, 1.8%) and CVA5 (1/170, 0.6%). The nucleotide sequence identity of EV strains from the same subtype ranged from 80.7% to 100%, and most of the EVs were closely related to virus strains found in Australia and mainland China. In conclusion, CVA 16 and CVA 4 were the main serotypes causing HFMD and herpangina, respectively, in children in Hangzhou in 2016. Most of these EVs were closely related to virus strains from Australia and mainland China.

Divergent Viruses Discovered in Arthropods and Vertebrates Revise the Evolutionary History of the Flaviviridae and Related Viruses.

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.

A tick-borne segmented RNA virus contains genome segments derived from unsegmented viral ancestors.

Although segmented and unsegmented RNA viruses are commonplace, the evolutionary links between these two very different forms of genome organization are unclear. We report the discovery and characterization of a tick-borne virus--Jingmen tick virus (JMTV)--that reveals an unexpected connection between segmented and unsegmented RNA viruses. The JMTV genome comprises four segments, two of which are related to the nonstructural protein genes of the genus Flavivirus (family Flaviviridae), whereas the remaining segments are unique to this virus, have no known homologs, and contain a number of features indicative of structural protein genes. Remarkably, homology searching revealed that sequences related to JMTV were present in the cDNA library from Toxocara canis (dog roundworm; Nematoda), and that shared strong sequence and structural resemblances. Epidemiological studies showed that JMTV is distributed in tick populations across China, especially Rhipicephalus and Haemaphysalis spp., and experiences frequent host-switching and genomic reassortment. To our knowledge, JMTV is the first example of a segmented RNA virus with a genome derived in part from unsegmented viral ancestors.

Taxonomy of the order Mononegavirales: update 2016.

In 2016, the order Mononegavirales was emended through the addition of two new families (Mymonaviridae and Sunviridae), the elevation of the paramyxoviral subfamily Pneumovirinae to family status (Pneumoviridae), the addition of five free-floating genera (Anphevirus, Arlivirus, Chengtivirus, Crustavirus, and Wastrivirus), and several other changes at the genus and species levels. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Diverse RNA viruses in the venom-related microenvironment of different animal phyla.

Venom is known as the source of natural antimicrobial products. Previous studies have largely focused on the expression of venom-related genes and the biochemical components of venom. With the advent of metagenomic sequencing, many more microorganisms, especially viruses, have been identified in highly diverse environments. Herein, we investigated the RNA virome in the venom-related microenvironment through analysis of a large volume of venom-related RNA-sequencing data mined from public databases. From this, we identified viral sequences belonging to thirty-six different viruses, of which twenty-two were classified as 'novel' as they exhibited less than 90 per cent amino acid identity to known viruses in the RNA-dependent RNA polymerase. Most of these novel viruses possessed genome structures similar to their closest relatives, with specific alterations in some cases. Phylogenetic analyses revealed that these viruses belonged to at least twenty-two viral families or unclassified groups, some of which were highly divergent from known taxa. Although further analysis failed to find venom-specific viruses, some viruses seemingly had much higher abundance in the venom-related microenvironment than in other tissues. In sum, our study provides insights into the RNA virome of the venom-related microenvironment from diverse animal phyla.

The evolutionary and molecular history of a chikungunya virus outbreak lineage.

In 2018-2019, Thailand experienced a nationwide spread of chikungunya virus (CHIKV), with approximately 15,000 confirmed cases of disease reported. Here, we investigated the evolutionary and molecular history of the East/Central/South African (ECSA) genotype to determine the origins of the 2018-2019 CHIKV outbreak in Thailand. This was done using newly sequenced clinical samples from travellers returning to Sweden from Thailand in late 2018 and early 2019 and previously published genome sequences. Our phylogeographic analysis showed that before the outbreak in Thailand, the Indian Ocean lineage (IOL) found within the ESCA, had evolved and circulated in East Africa, South Asia, and Southeast Asia for about 15 years. In the first half of 2017, an introduction occurred into Thailand from another South Asian country, most likely Bangladesh, which subsequently developed into a large outbreak in Thailand with export to neighbouring countries. Based on comparative phylogenetic analyses of the complete CHIKV genome and protein modelling, we identified several mutations in the E1/E2 spike complex, such as E1 K211E and E2 V264A, which are highly relevant as they may lead to changes in vector competence, transmission efficiency and pathogenicity of the virus. A number of mutations (E2 G205S, Nsp3 D372E, Nsp2 V793A), that emerged shortly before the outbreak of the virus in Thailand in 2018 may have altered antibody binding and recognition due to their position. This study not only improves our understanding of the factors contributing to the epidemic in Southeast Asia, but also has implications for the development of effective response strategies and the potential development of new vaccines.

Diversity and connectedness of brine shrimp viruses in global hypersaline ecosystems.

Brine shrimp (Artemia) has existed on Earth for 400 million years and has major ecological importance in hypersaline ecosystems. As a crucial live food in aquaculture, brine shrimp cysts have become one of the most important aquatic products traded worldwide. However, our understanding of the biodiversity, prevalence and global connectedness of viruses in brine shrimp is still very limited. A total of 143 batches of brine shrimp (belonging to seven species) cysts were collected from six continents including 21 countries and more than 100 geographic locations worldwide during 1977-2019. In total, 55 novel RNA viruses were identified, which could be assigned to 18 different viral families and related clades. Eleven viruses were dsRNA viruses, 16 were +ssRNA viruses, and 28 were-ssRNA viruses. Phylogenetic analyses of the RNA-directed RNA polymerase (RdRp) showed that brine shrimp viruses were often grouped with viruses isolated from other invertebrates and fungi. Remarkably, most brine shrimp viruses were related to those from different hosts that might feed on brine shrimp or share the same ecological niche. A notable case was the novel brine shrimp noda-like virus 3, which shared 79.25% (RdRp) and 63.88% (capsid proteins) amino acid identity with covert mortality nodavirus (CMNV) that may cause losses in aquaculture. In addition, both virome composition and phylogenetic analyses revealed global connectedness in certain brine shrimp viruses, particularly among Asia and Northern America. This highlights the incredible species diversity of viruses in these ancient species and provides essential data for the prevalence of RNA viruses in the global aquaculture industry. More broadly, these findings provide novel insights into the previously unrecognized RNA virosphere in hypersaline ecosystems worldwide and demonstrate that human activity might have driven the global connectedness of brine shrimp viruses.