The Possible Role of Prion-Like Viral Protein Domains on the Emergence of Novel Viruses as SARS-CoV-2.

Self-replicating proteins or prions deviate from the central dogma of replication. The discovery of prion-like domains in coronavirus SARS-CoV-2 suggests their possible role in viral evolution. Here, we have outlined the possible role of self-replicating protein-like domains in the emergence of novel viruses. Further studies are needed to understand the function of these viral self-replicating protein-like domains and whether they could be antiviral target(s) for the development of effective antiviral agents in the future.

Novel putative pathogenic viruses identified in pangolins by mining metagenomic data.

The pangolin is the only scaly mammal in the world and also an important reservoir of pathogenic viruses. Habitat loss and poaching have been shrinking the survival range of pangolins. More information on pangolin virus populations is needed to better understand and assess potential disease risks. In this study, viral metagenomic data were used to reinvestigate the virome in pangolin lung tissue. Complete genome sequences of two novel anelloviruses were acquired and clustered with the referenced feline strains belonging to genus Tettorquevirus and genus Etatorquevirus, respectively. Two genomes belonging to the genus Gemykibivirus, and species Bat-associated cyclovirus 9 were detected, respectively. One genome with a large contig belonging to the genus Senecavirus were also characterized, according to phylogenetic analysis, which can be presumed to be a novel species. In addition, a full genome of endogenous retroviruse (ERV) was assembled from the lungs of pangolin, and this virus may have the possibility of cross-species transmission during the evolution. This virological investigation has increased our understanding of the virome carried by pangolins and provided a reference baseline for possible zoonotic infectious diseases in the future.

The Virome of Piper nigrum: Identification, Genomic Characterization, Prevalence, and Transmission of Three New Viruses of Black Pepper in China.

Viral diseases are one of the main categories of diseases that cause substantial yield losses in black pepper. Disease symptoms in black pepper are generally complex and are often caused by both known and undescribed viruses. To identify and clarify the etiology of viral diseases in black pepper in Hainan, China, we conducted high-throughput sequencing (HTS) by targeting purified double-stranded RNA (dsRNA) and ribosomal RNA depleted total RNA (rRNA-depleted totRNA). Analysis of the data revealed the presence of one known virus, piper yellow mottle virus (PYMoV), and three newly identified viruses: black pepper virus F (BPVF) in the genus Fabavirus, black pepper virus E (BPVE) in the genus Enamovirus, and black pepper virus B (BPVB) in the genus Badnavirus. The dominant viruses in P. nigrum sampled in Hainan are PYMoV, with an incidence of 100%, followed by BPVF (84%, 133 of 158) and BPVB (66%, 105 of 158). Mechanical inoculation of sap extracts from source plants containing PYMoV, BPVF, and BPVB gave negative results on both herbaceous and woody host plants 60 days postinoculation (dpi). BPVF and PYMoV were successfully transmitted to virus-free seedlings of black pepper through bark grafting, while BPVB was experimentally undetectable up to 150 dpi. Seed transmission experiments showed that no target viruses were present in all 59 germinated seedlings. This study provides information on diagnosis, prevalence, and transmission of black-pepper-associated viruses.

The discovery and global distribution of novel mosquito-associated viruses in the last decade (2007-2017).

In the last decade, virus hunting and discovery has gained pace. This achievement has been driven by three major factors: (a) advancements in sequencing technologies, (b) scaled-up routine arbovirus surveillance strategies, and (c) the "hunt" for emerging pathogens and novel viruses. Many novel viruses have been discovered from a myriad of hosts, vectors, and environmental samples. To help promote understanding of the global diversity and distribution of mosquito-associated viruses and facilitate future studies, we review mosquito-associated viruses discovered between years 2007 and 2017, across the world. In the analyzed period, novel mosquito-associated viruses belonging to 25 families and a general group of unclassified viruses were categorized. The top three discovered novel mosquito-associated viruses belonged to families Flaviviridae (n=32), Rhabdoviridae (n=16), and Peribunyaviridae (n=14). Also, 67 unclassified viruses were reported. Majority of these novel viruses were identified from Culex spp, Anopheles spp, Aedes spp, and Mansonia spp mosquitoes, respectively. Notably, the number of these discovered novels is not representative of intercontinental virus diversity but rather is influenced by the number of studies done in the study period. Some of these newly discovered mosquito-associated viruses have medical significance, either directly or indirectly. For instance, in the study period, 14 novel mosquito-borne viruses that infect mammalian cells in vitro were reported. These viruses pose a danger to the global health security on emerging viral diseases. On the other hand, some of the newly discovered insect specific viruses described herein have potential application as future biocontrol and vaccine agents against known pathogenic arboviruses. Overall, this review outlines the crucial role played by mosquitoes as viral vectors in the global virosphere.

Evolution and Emergence of Pathogenic Viruses: Past, Present, and Future.

Incidences of emerging/re-emerging deadly viral infections have significantly affected human health despite extraordinary progress in the area of biomedical knowledge. The best examples are the recurring outbreaks of dengue and chikungunya fever in tropical and sub-tropical regions, the recent epidemic of Zika in the Americas and the Caribbean, and the SARS, MERS, and influenza A outbreaks across the globe. The established natural reservoirs of human viruses are mainly farm animals, and, to a lesser extent, wild animals and arthropods. The intricate "host-pathogen-environment" relationship remains the key to understanding the emergence/re-emergence of pathogenic viruses. High population density, rampant constructions, poor sanitation, changing climate, and the introduction of anthropophilic vectors create selective pressure on host-pathogen reservoirs. Nevertheless, the knowledge and understanding of such zoonoses and pathogen diversity in their known non-human reservoirs are very limited. Prevention of arboviral infections using vector control methods has not been very successful. Currently, new approaches to protect against food-borne infections, such as consuming only properly cooked meats and animal products, are the most effective control measures. Though significant progress in controlling human immunodeficiency virus and hepatitis viruses has been achieved, the unpredictable nature of evolving viruses and the rare occasions of outbreaks severely hamper control and preventive modalities.

Pathogen dynamics and discovery of novel viruses and enzymes by deep nucleic acid sequencing of wastewater.

Wastewater contains an extensive reservoir of genetic information, yet largely unexplored. Here, we analyzed by high-throughput sequencing total nucleic acids extracted from wastewater samples collected during a 17 month-period in Berlin, Germany. By integrating global wastewater datasets and applying a novel computational approach to accurately identify viral strains within sewage RNA-sequencing data, we demonstrated the emergence and global dissemination of a specific astrovirus strain. Astrovirus abundance and sequence variation mirrored temporal and spatial patterns of infection, potentially serving as footprints of specific timeframes and geographical locations. Additionally, we revealed more than 100,000 sequence contigs likely originating from novel viral species, exhibiting distinct profiles in total RNA and DNA datasets and including undescribed bunyaviruses and parvoviruses. Finally, we identified thousands of new CRISPR-associated protein sequences, including Transposase B (TnpB), a class of compact, RNA-guided DNA editing enzymes. Collectively, our findings underscore the potential of high-throughput sequencing of total nucleic acids derived from wastewater for a broad range of applications.

Engineered Resistance to Tobamoviruses.

Tobacco mosaic virus (TMV) was the first virus to be studied in detail and, for many years, TMV and other tobamoviruses, particularly tomato mosaic virus (ToMV) and tobamoviruses infecting pepper (Capsicum spp.), were serious crop pathogens. By the end of the twentieth and for the first decade of the twenty-first century, tobamoviruses were under some degree of control due to introgression of resistance genes into commercial tomato and pepper lines. However, tobamoviruses remained important models for molecular biology, biotechnology and bio-nanotechnology. Recently, tobamoviruses have again become serious crop pathogens due to the advent of tomato brown rugose fruit virus, which overcomes tomato resistance against TMV and ToMV, and the slow but apparently inexorable worldwide spread of cucumber green mottle mosaic virus, which threatens all cucurbit crops. This review discusses a range of mainly molecular biology-based approaches for protecting crops against tobamoviruses. These include cross-protection (using mild tobamovirus strains to 'immunize' plants against severe strains), expressing viral gene products in transgenic plants to inhibit the viral infection cycle, inducing RNA silencing against tobamoviruses by expressing virus-derived RNA sequences in planta or by direct application of double-stranded RNA molecules to non-engineered plants, gene editing of host susceptibility factors, and the transfer and optimization of natural resistance genes.

Diversity and independent evolutionary profiling of rodent-borne viruses in Hainan, a tropical island of China.

The risk of emerging infectious diseases (EID) is increasing globally. More than 60% of EIDs worldwide are caused by animal-borne pathogens. This study aimed to characterize the virome, analyze the phylogenetic evolution, and determine the diversity of rodent-borne viruses in Hainan Province, China. We collected 682 anal and throat samples from rodents, combined them into 28 pools according to their species and location, and processed them for next-generation sequencing and bioinformatics analysis. The diverse viral contigs closely related to mammals were assigned to 22 viral families. Molecular clues of the important rodent-borne viruses were further identified by polymerase chain reaction for phylogenetic analysis and annotation of genetic characteristics such as arenavirus, coronavirus, astrovirus, pestivirus, parvovirus, and papillomavirus. We identified pestivirus and bocavirus in Leopoldoms edwardsi from Huangjinjiaoling, and bocavirus in Rattus andamanensis from the national nature reserves of Bangxi with low amino acid identity to known pathogens are proposed as the novel species, and their rodent hosts have not been previously reported to carry these viruses. These results expand our knowledge of viral classification and host range and suggest that there are highly diverse, undiscovered viruses that have evolved independently in their unique wildlife hosts in inaccessible areas.

Virome Profiling, New Virus Identification and the Prevalence and Distribution of Viruses Infecting Chieh-Qua (Benincasa hispida Cogn. var. chieh-qua How) in China.

The cucurbit vegetable chieh-qua (Benincasa hispida var. chieh-qua How) is an important crop in South China and southeast Asian countries. Viral diseases cause substantial loss of chieh-qua yield. To identify the viruses that affect chieh-qua in China, ribosomal RNA-depleted total RNA sequencing was performed using chieh-qua leaf samples with typical viral symptoms. The virome of chieh-qua comprises four known viruses (melon yellow spot virus (MYSV), cucurbit chlorotic yellows virus (CCYV), papaya ringspot virus (PRSV) and watermelon silver mottle virus (WSMoV) and two novel viruses: cucurbit chlorotic virus (CuCV) in the genus Crinivirus and chieh-qua endornavirus (CqEV) in the genus Alphaendornavirus. The complete genomes of the two novel viruses in chieh-qua and three other isolates of CuCV in pumpkin, watermelon and cucumber were determined and the recombination signals of pumpkin and watermelon isolates of CuCV were detected. A reverse transcriptase PCR indicated that the dominant viruses of chieh-qua in Hainan are MYSV (66.67%) and CCYV (55.56%), followed by CuCV (27.41%), WSMoV (7.41%), cucumber mosaic virus (8.15%), zucchini yellow mosaic virus (6.67%), PRSV (6.67%) and CqEV (35.56%). Our findings support diagnostic and prevalence studies of viruses infecting chieh-qua in China, enabling sustainable control strategies for cucurbit viruses worldwide.

Virome of Pseudostellaria heterophylla: Identification and characterization of three novel carlaviruses and one novel amalgavirus associated with viral diseases of Pseudostellaria heterophylla.

Pseudostellaria heterophylla is a traditional Chinese herbal medicine, which has been cultivated for hundreds of years. Viral diseases of P. heterophylla occur widely and limit the yield and quality of this medicinal plant. In this study, five leaf samples of P. heterophylla with typical viral symptoms were collected from four main producing regions that are distributed in Fujian, Guizhou, and Anhui Provinces in China and analyzed by next-generation sequencing. Comprehensive bioinformatics analyses revealed that nine viruses in five genera Carlavirus, Potyvirus, Fabavirus, Cucumovirus, and Amalgavirus infected P. heterophylla. Among these viruses, three novel and two known carlaviruses, tentatively designated Pseudostellaria heterophylla carlavirus 1, 2, and 3 (PhCV1, PhCV2, and PhCV3), Jasmine virus C isolate Ph (Ph-JVC) and Stevia carlavirus 1 isolate Ph (Ph-StCV1), respectively, were first identified in P. heterophylla. PhCV1-3 share a similar genomic organization and clear sequence homology with members in the genus Carlavirus and could potentially be classified as new species of this genus. One novel amalgavirus, tentatively designated P. heterophylla amalgavirus 1 (PhAV1), was first identified in P. heterophylla. It had a typical genomic organization of the genus Amalgavirus. In PhAV1, the + 1 programmed ribosomal frameshifting, which is prevalent in most amalgaviruses, was identified and used in the expression of RNA-dependent RNA polymerase (RdRp). Combined with a phylogenetic analysis, PhAV1 could potentially be classified as new species of the genus Amalgavirus. In addition, multiple Broad bean wilt virus 2 (BBWV2) variants, Turnip mosaic virus (TuMV), and Cucumber mosaic virus (CMV), which have been reported in P. heterophylla, were also detected in this study. The distribution of PhCV1-3, Ph-JVC, Ph-StCV1, TuMV, BBWV2, and CMV in four production regions in Fujian, Guizhou, and Anhui Provinces was determined. This study increased our understanding of P. heterophylla virome and provided valuable information for the development of a molecular diagnostic technique and control of viral diseases in P. heterophylla.

Co-circulation of distinct shrew-borne hantaviruses in the far east of Russia.

Insectivores are the new emerging reservoir of hantaviruses. Here, we describe Lena virus (LENV), a novel hantavirus harbored by the Laxmann`s shrew (Sorex caecutiens), which is also the host of Artybash virus (ARTV). Genetic analysis of the complete genomic sequence shows that LENV is in distant relation to ARTV and other Sorex-borne hantaviruses, suggesting that LENV has emerged from cross-species transmission. Additionally, new genetic variant of ARTV, designated as ARTV-St, was identified in tundra shrews (Sorex tundrensis). Finally, distinct insectivore-borne hantaviruses are co-circulating in the same localities of far eastern Russia: LENV, ARTV and Yakeshi in the forest site, while ARTV, ARTV-St, and Kenkeme virus in the meadow field site.

Discovery and Characterization of Novel RNA Viruses in Aquatic North American Wild Birds.

Wild birds are recognized viral reservoirs but our understanding about avian viral diversity is limited. We describe here three novel RNA viruses that we identified in oropharyngeal/cloacal swabs collected from wild birds. The complete genome of a novel gull metapneumovirus (GuMPV B29) was determined. Phylogenetic analyses indicated that this virus could represent a novel avian metapneumovirus (AMPV) sub-group, intermediate between AMPV-C and the subgroup of the other AMPVs. This virus was detected in an American herring (1/24, 4.2%) and great black-backed (4/26, 15.4%) gulls. A novel gull coronavirus (GuCoV B29) was detected in great black-backed (3/26, 11.5%) and American herring (2/24, 8.3%) gulls. Phylogenetic analyses of GuCoV B29 suggested that this virus could represent a novel species within the genus Gammacoronavirus, close to other recently identified potential novel avian coronaviral species. One GuMPV-GuCoV co-infection was detected. A novel duck calicivirus (DuCV-2 B6) was identified in mallards (2/5, 40%) and American black ducks (7/26, 26.9%). This virus, of which we identified two different types, was fully sequenced and was genetically closest to other caliciviruses identified in Anatidae, but more distant to other caliciviruses from birds in the genus Anas. These discoveries increase our knowledge about avian virus diversity and host distributions.

Detection of Diverse Novel Bat Astrovirus Sequences in the Czech Republic.

Astroviruses are a major cause of gastroenteritis in humans and animals. Recently, novel groups of astroviruses were identified in apparently healthy insectivorous bats. We report the detection of diverse novel astrovirus sequences in nine different European bat species: Eptesicus serotinus, Hypsugo savii, Myotis emarginatus, M. mystacinus, Nyctalus noctula, Pipistrellus nathusii or P. pygmaeus, P. pipistrellus, Vespertilio murinus, and Rhinolophus hipposideros. In six bat species, astrovirus sequences were detected for the first time. One astrovirus strain detected in R. hipposideros clustered phylogenetically with Chinese astrovirus strains originating from bats of the families Rhinolophidae and Hipposideridae. All other Czech astrovirus sequences from vesper bats formed, together with one Hungarian sequence, a separate monophyletic lineage within the bat astrovirus group. These findings provide new insights into the molecular epidemiology, ecology, and prevalence of astroviruses in European bat populations.

A novel, highly divergent ssDNA virus identified in Brazil infecting apple, pear and grapevine.

Fruit trees of temperate and tropical climates are of great economical importance worldwide and several viruses have been reported affecting their productivity and longevity. Fruit trees of different Brazilian regions displaying virus-like symptoms were evaluated for infection by circular DNA viruses. Seventy-four fruit trees were sampled and a novel, highly divergent, monopartite circular ssDNA virus was cloned from apple, pear and grapevine trees. Forty-five complete viral genomes were sequenced, with a size of approx. 3.4 kb and organized into five ORFs. Deduced amino acid sequences showed identities in the range of 38% with unclassified circular ssDNA viruses, nanoviruses and alphasatellites (putative Replication-associated protein, Rep), and begomo-, curto- and mastreviruses (putative coat protein, CP, and movement protein, MP). A large intergenic region contains a short palindromic sequence capable of forming a hairpin-like structure with the loop sequence TAGTATTAC, identical to the conserved nonanucleotide of circoviruses, nanoviruses and alphasatellites. Recombination events were not detected and phylogenetic analysis showed a relationship with circo-, nano- and geminiviruses. PCR confirmed the presence of this novel ssDNA virus in field plants. Infectivity tests using the cloned viral genome confirmed its ability to infect apple and pear tree seedlings, but not Nicotiana benthamiana. The name "Temperate fruit decay-associated virus" (TFDaV) is proposed for this novel virus.

New technologies and challenges of novel virus detection.

The use of new cell substrates for the development of biologicals, particularly tumorigenic and tumor-derived cell lines, can pose a major regulatory challenge due to safety concerns related to the presence of novel viruses, latent and occult viruses including oncogenic viruses, and endogenous retroviruses, since these may not be detected by the currently recommended conventional assays. This report is a summary of our laboratory's experiences using advanced nucleic acid-based technologies to evaluate a Madin-Darby canine kidney (MDCK) cell line and the insect Sf9 cell line derived from Spodoptera frugiperda, and presents some ongoing efforts to address the challenges of novel virus detection.