Epidemiological study of Kabuto Mountain virus, a novel uukuvirus, in Japan.

Kabuto Mountain virus (KAMV), the new member of the genus Uukuvirus, was isolated from the tick Haemaphysalis flava in 2018 in Japan. To date, there is no information on KAMV infection in human and animals. Therefore, serological surveillance of the infection among humans and wild mammals was conducted by virus-neutralization (VN) test and indirect immunofluorescence assay (IFA). Sera of 24 humans, 59 monkeys, 171 wild boars, 233 Sika deer, 7 bears, and 27 nutria in Yamaguchi Prefecture were analyzed by VN test. The positive ratio of humans, monkeys, wild boars, and Sika deer were 20.8%, 3.4%, 33.9% and 4.7%, respectively. No positive samples were detected in bears and nutria. The correlation coefficients between VN test and IFA in human, monkey, wild boar, and Sika deer sera were 0.5745, 0.7198, 0.9967 and 0.9525, respectively. In addition, KAMV was detected in one pool of Haemaphysalis formosensis ticks in Wakayama Prefecture. These results indicated that KAMV or KAMV-like virus is circulating among many wildlife and ticks, and that this virus incidentally infects humans.

Genomic characterization of two novel viruses infecting Barleria cristata L. from the genera Orthotospovirus and Polerovirus.

Barleria cristata L. has become naturalized in South Africa, where it is commonly used as an ornamental. In 2019, plants of B. cristata showing putative viral symptoms were collected from two locations in Gauteng, South Africa. RNAtag-seq libraries were prepared and sequenced using an Illumina HiSeq 2500 platform. De novo assembly of the resulting data revealed the presence of a novel member of the family Tospoviridae associated with the plants from both locations, and this virus was given the tentative name "barleria chlorosis-associated virus". Segments L, M, and S have lengths of 8752, 4760, and 2906 nt, respectively. Additionally, one of the samples was associated with a novel polerovirus, provisionally named "barleria polerovirus 1", with a complete genome length of 6096 nt. This is the first study to show the association of viruses with a member of the genus Barleria.

Host Cell Restriction Factors of Bunyaviruses and Viral Countermeasures.

The Bunyavirales order comprises more than 500 viruses (generally defined as bunyaviruses) classified into 12 families. Some of these are highly pathogenic viruses infecting different hosts, including humans, mammals, reptiles, arthropods, birds, and/or plants. Host cell sensing of infection activates the innate immune system that aims at inhibiting viral replication and propagation. Upon recognition of pathogen-associated molecular patterns (PAMPs) by cellular pattern recognition receptors (PRRs), numerous signaling cascades are activated, leading to the production of interferons (IFNs). IFNs act in an autocrine and paracrine manner to establish an antiviral state by inducing the expression of hundreds of IFN-stimulated genes (ISGs). Some of these ISGs are known to restrict bunyavirus infection. Along with other constitutively expressed host cellular factors with antiviral activity, these proteins (hereafter referred to as "restriction factors") target different steps of the viral cycle, including viral entry, genome transcription and replication, and virion egress. In reaction to this, bunyaviruses have developed strategies to circumvent this antiviral response, by avoiding cellular recognition of PAMPs, inhibiting IFN production or interfering with the IFN-mediated response. Herein, we review the current knowledge on host cellular factors that were shown to restrict infections by bunyaviruses. Moreover, we focus on the strategies developed by bunyaviruses in order to escape the antiviral state developed by the infected cells.

A novel emaravirus comprising five RNA segments is associated with ringspot disease in oak.

We report the complete nucleotide sequence of the genome of a novel virus in ringspot-diseased common oak (Quercus robur L.). The newly identified pathogen is associated with leaf symptoms such as mottle, chlorotic spots and ringspots on diseased trees. High-throughput sequencing (HTS, Illumina RNASeq) was used to explore the virome of a ringspot-diseased oak that had chlorotic ringspots of suspected viral origin on leaves for several years. Bioinformatic analysis of the HTS dataset followed by RT-PCR enabled us to determine complete sequences of four RNA genome segments of a novel virus. These sequences showed high similarity to members of the genus Emaravirus, which includes segmented negative-stranded RNA viruses of economic importance. To verify the ends of each RNA, we conducted rapid amplification of cDNA ends (RACE). We identified an additional genome segment (RNA 5) by RT-PCR using a genus-specific primer (PDAP213) to the conserved 3´ and 5´termini in order to amplify full-length genome segments. RNA 5 encodes a 21-kDa protein that is homologous to the silencing suppressor P8 of High Plains wheat mosaic virus. The five viral RNAs were consistently detected by RT-PCR in ringspot-diseased oaks in Germany, Sweden, and Norway. We conclude that the virus represents a new member of the genus Emaravirus affecting oaks in Germany and in Scandinavia, and we propose the name "common oak ringspot-associated emaravirus" (CORaV).

Molecular Characteristics of Jujube Yellow Mottle-Associated Virus Infecting Jujube (Ziziphus jujuba Mill.) Grown at Aksu in Xinjiang of China.

Chinese jujube (Ziziphus jujuba Mill.) is a native fruit crop in China. Leaf mottle and dapple fruit disease is prevalent in cultivated jujube plants grown at Aksu in Xinjiang Uygur Autonomous Region of China. Jujube yellow mottle-associated virus (JYMaV), a tentative member in the genus Emaravirus, was recently identified from mottle-diseased jujube plants grown in Liaoning Province in China, but its incidence and genetic diversity in China is unknown. In this study, the genome sequences of three JYMaV isolates from two jujube cultivars and one jujube variant were determined by high-throughput sequencing (HTS) for small RNA and rRNA-depleted RNA coupled with RT-PCR assays. Comparison of these sequences together with sequences of the viral RNA segments derived by primer set 3C/5H-based RT-PCR revealed that genetic diversity was present in the virus populations and high sequence variation occurred at the non-translational regions of each of the viral genomic segments. Field investigation confirmed the close association of the virus with leaf mottle symptoms of jujube plants. Furthermore, this study revealed that P5 encoded in the viral RNA5 displayed a nuclear localization feature differing from the plasmodesma (PD) subcellular localization of the virus movement protein (P4), and the two proteins could interact with each other in the BiFC assays. Our study provides a snapshot of JYMaV genetic diversity in its natural hosts.

A survey of RNA viruses in mosquitoes from Mozambique reveals novel genetic lineages of flaviviruses and phenuiviruses, as well as frequent flavivirus-like viral DNA forms in Mansonia.

BACKGROUND: Mosquito-borne diseases involving arboviruses represent expanding threats to sub-Saharan Africa imposing as considerable burden to human and veterinary public health. In Mozambique over one hundred species of potential arbovirus mosquito vectors have been identified, although their precise role in maintaining such viruses in circulation in the country remains to be elucidated. The aim of this study was to screen for the presence of flaviviruses, alphaviruses and bunyaviruses in mosquitoes from different regions of Mozambique. RESULTS: Our survey analyzed 14,519 mosquitoes, and the results obtained revealed genetically distinct insect-specific flaviviruses, detected in multiple species of mosquitoes from different genera. In addition, smaller flavivirus-like NS5 sequences, frequently detected in Mansonia seemed to correspond to defective viral sequences, present as viral DNA forms. Furthermore, three lineages of putative members of the Phenuiviridae family were also detected, two of which apparently corresponding to novel viral genetic lineages. CONCLUSION: This study reports for the first-time novel insect-specific flaviviruses and novel phenuiviruses, as well as frequent flavivirus-like viral DNA forms in several widely known vector species. This unique work represents recent investigation of virus screening conducted in mosquitoes from Mozambique and an important contribution to inform the establishment of a vector control program for arbovirus in the country and in the region.

Genomic and phylogenetic characterization of a bunya-like virus from the freshwater Chinese mitten crab Eriocheir sinensis.

The freshwater Chinese mitten crab (Eriocheir sinensis), an indigenous crustacean in China, has been cultured for more than 30 years. It was reported that the bunya-like virus from Eriocheir sinensis (EsBV) was associated with the tremor disease (TD), which causes high mortality and has a serious impact on production. In this study, full-length genome sequences of EsBV were pursued using next generation sequencing; the genome of EsBV was found to be composed of 6.7 kb L, 3.3 kb M, and 0.8 kb S segments, respectively. PCR detection based genomic sequences showed that the positive rate of EsBV reached 40% in crabs from farming ponds. EsBV had the highest similarity with the Wenling crustacean virus 9, an unassigned, negative sense ssRNA virus. EsBV clustered with the Wenling crustacean virus 9 firstly, and then the branch clustered with Peribunyaviridae clade in every phylogenetic tree - based on L, M and S encoded sequences, respectively, indicating that EsBV can be classified in the family Peribunyaviridae, to which the orthobunyaviruses belongs, but not belonging to any known genera in the family Peribunyaviridae. There were unique complimentary terminal sequences for EsBV, with only partial consensus with members from the orthobunyaviruses. We believe that the findings of this research will be vital for future research about EsBV and will also go a long way in illuminating its relationship with TD. Keywords: Eriocheir sinensis; tremor disease; bunyavirus; EsBV; genome sequences.

Tamdy Virus in Ixodid Ticks Infesting Bactrian Camels, Xinjiang, China, 2018.

We isolated Tamdy virus (TAMV; strain XJ01/TAMV/China/2018) from Hyalomma asiaticum ticks infesting Bactrian camels in Xinjiang, China, in 2018. The genome of the strain showed high nucleotide similarity with previously described TAMV strains from Asia. Our study highlights the potential threat of TAMV to public health in China.

Wheat Virus Identification Within Infected Tissue Using Nanopore Sequencing Technology.

Viral diseases are a limiting factor to wheat production. Viruses are difficult to diagnose in the early stages of disease development and are often confused with nutrient deficiencies or other abiotic problems. Immunological methods are useful to identify viruses, but specific antibodies may not be available or require high virus titer for detection. In 2015 and 2017, wheat plants containing Wheat streak mosaic virus (WSMV) resistance gene, Wsm2, were found to have symptoms characteristic of WSMV. Serologically, WSMV was detected in all four samples. Additionally, High Plains wheat mosaic virus (HPWMoV) was also detected in one of the samples. Barley yellow dwarf virus (BYDV) was not detected, and a detection kit was not readily available for Triticum mosaic virus (TriMV). Initially, cDNA cloning and Sanger sequencing were used to determine the presence of WSMV; however, the process was time-consuming and expensive. Subsequently, cDNA from infected wheat tissue was sequenced with single-strand, Oxford Nanopore sequencing technology (ONT). ONT was able to confirm the presence of WSMV. Additionally, TriMV was found in all of the samples and BYDV in three of the samples. Deep coverage sequencing of full-length, single-strand WSMV revealed variation compared with the WSMV Sidney-81 reference strain and may represent new variants which overcome Wsm2. These results demonstrate that ONT can more accurately identify causal virus agents and has sufficient resolution to provide evidence of causal variants.

Characterization of Ti Ringspot-Associated Virus, a Novel Emaravirus Associated with an Emerging Ringspot Disease of Cordyline fruticosa.

Ti ringspot is an emerging foliar disease of the ti plant (Cordyline fruticosa) in Hawaii that is quickly spreading throughout the islands. Symptoms include small chlorotic ringspots on leaves that often coalesce to form larger lesions. Although several virus species have been discovered in symptomatic plants, none have been associated with these symptoms. Here, we report and characterize a novel virus closely associated with ti ringspot symptoms in Hawaii. The presence of double membrane bodies approximately 85 nm in diameter in symptomatic cells and sequence analyses of five genomic RNA segments obtained by high-throughput sequencing indicate that this virus is most closely related to members of the plant virus genus Emaravirus. Phylogenetic and sequence homology analyses place this virus on a distinct clade within the Emaravirus genus along with High Plains wheat mosaic emaravirus, blue palo verde broom virus, and Raspberry leaf blotch emaravirus. Sequence identity values with taxonomically relevant proteins indicate that this represents a new virus species, which we are tentatively naming ti ringspot-associated virus (TiRSaV). TiRSaV-specific reverse transcription PCR assays detected the virus in several experimental herbaceous host species following mechanical inoculation. TiRSaV was also detected in eriophyid mites collected from symptomatic ti plants, which may represent a putative arthropod vector of the virus.

Characterization of a new bunyavirus and its derived small RNAs in the brown citrus aphid, Aphis citricidus.

High-throughput sequencing is widely used for virus discovery, and many RNA viruses have been discovered and identified. A new negative-sense single-stranded RNA virus was identified in the brown citrus aphid and named Aphis citricidus bunyavirus. The genome consists of large (7037 nt), medium (3462 nt), and small (1163 nt) segments. Phylogenetic analysis and amino acid sequences identities of this virus with other bunyaviruses suggest that it is a new species belonging to the family Phenuiviridae. The small interfering RNA pathway could be involved against the infection of this virus in brown citrus aphid as supported by the viral derived small RNAs. The discovery of this virus illustrates the diversity of RNA viruses and contributes to the classification of bunyaviruses.

Taxonomy of the order Bunyavirales: update 2019.

In February 2019, following the annual taxon ratification vote, the order Bunyavirales was amended by creation of two new families, four new subfamilies, 11 new genera and 77 new species, merging of two species, and deletion of one species. This article presents the updated taxonomy of the order Bunyavirales now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Determination of the complete genome sequence of European mountain ash ringspot-associated emaravirus from Sorbus intermedia reveals two additional genome segments.

We report the complete genome sequence, comprising six single-stranded RNAs of negative orientation, of a European mountain ash ringspot-associated virus (EMARaV) isolate identified in a diseased Sorbus intermedia (Swedish whitebeam) tree exhibiting prominent chlorotic ringspots, mottle and line pattern on leaves. Since the first observation of EMARaV-like symptoms and detection of the virus in whitebeam in 2012, the tree displayed leaf symptoms every year in at least one third of its canopy, developed dieback symptoms, and showed signs of decline. Two previously unrecorded genome segments of the virus were identified, each encoding a single protein in a negative orientation. RNA5 is 1629 nucleotides long and encodes the putative movement protein (MP) of EMARaV with a molecular mass of 42.4 kDa. RNA6 (1362 nucleotides) encodes a small protein (26.8 kDa) exhibiting some sequence similarity to the P4 protein encoded by EMARaV RNA4. However, its biological function remains to be elucidated. Both novel genome segments are systematically present in EMARaV-infected Sorbus spp., and no additional genome segments could be identified by two independent methods. It is concluded that the six RNAs represent the complete genome of EMARaV.

Viromes in Xylariaceae fungi infecting avocado in Spain.

Four isolates of Entoleuca sp., family Xylariaceae, Ascomycota, recovered from avocado rhizosphere in Spain were analyzed for mycoviruses presence. For that, the dsRNAs from the mycelia were extracted and subjected to metagenomics analysis that revealed the presence of eleven viruses putatively belonging to families Partitiviridae, Hypoviridae, Megabirnaviridae, and orders Tymovirales and Bunyavirales, in addition to one ourmia-like virus plus other two unclassified virus species. Moreover, a sequence with 98% nucleotide identity to plant endornavirus Phaseolus vulgaris alphaendornavirus 1 has been identified in the Entoleuca sp. isolates. Concerning the virome composition, the four isolates only differed in the presence of the bunyavirus and the ourmia-like virus, while all other viruses showed common patterns. Specific primers allowed the detection by RT-PCR of these viruses in a collection of Entoleuca sp. and Rosellinia necatrix isolates obtained from roots of avocado trees. Results indicate that intra- and interspecies horizontal virus transmission occur frequently in this pathosystem.

Thailand orthohantavirus infection in patients with chronic kidney disease of unknown aetiology in Sri Lanka.

Chronic kidney disease of unknown aetiology (CKDu) reported in Sri Lanka and other countries is a mysterious and serious disease. Recently, we reported a high seroprevalence of antibodies to a hantavirus antigen among CKDu patients in Girandurukotte, Badulla district, Sri Lanka. However, the type of hantavirus with which the residents were infected was not determined. In this study, a total of 89 seropositive sera were examined to identify their serotypes using an indirect immunofluorescent antibody assay, a truncated-N-protein-based enzyme-linked immunosorbent assay, and a cross-neutralization test. These results indicated that the residents in this area were frequently infected with Thailand orthohantavirus or an antigenically related virus.

Animal models for viral haemorrhagic fever.

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.

Discovery and high prevalence of Phasi Charoen-like virus in field-captured Aedes aegypti in South China.

Arboviruses have caused significant global health concerns during the past decade. In this regard, continuous viral surveillance is essential to timely identify emerging arboviruses and other novel viruses. Here, a novel isolate of Phasi Charoen-like virus (PCLV Zhanjiang01) was identified from field-captured Aedes aegypti mosquitoes in Zhanjiang by next generation sequencing. Phylogenetic analysis suggested that PCLV Zhanjiang01 belonged to the genus Phasivirus in the family Phenuiviridae. The presence of PCLV in three batches of Aedes aegypti confirmed its high prevalence in nature. Further detection of PCLV in progenies and adult males suggested vertical transmission in mosquitoes. In parallel, PCLV was detected from multiple organs indicating its broad tissue distribution in the infected mosquitoes. To the best of our knowledge, this is the first report of PCLV in China. Our results expanded the global biogeographic distribution of PCLV. Further investigations of PCLV on the arboviral transmission and control strategies are warranted.

Virome of > 12 thousand Culex mosquitoes from throughout California.

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.

Bunyavirus Taxonomy: Limitations and Misconceptions Associated with the Current ICTV Criteria Used for Species Demarcation.

The International Committee on Taxonomy of Viruses (ICTV) has implemented numerous changes to the taxonomic classification of bunyaviruses over the years. Whereas most changes have been justified and necessary because of the need to accommodate newly discovered and unclassified viruses, other changes are a cause of concern, especially the decision to demote scores of formerly recognized species to essentially strains of newly designated species. This practice was first described in the seventh taxonomy report of the ICTV and has continued in all subsequent reports. In some instances, viruses that share less than 75% nucleotide sequence identity across their genomes, produce vastly different clinical presentations, possess distinct vector and host associations, have different biosafety recommendations, and occur in nonoverlapping geographic regions are classified as strains of the same species. Complicating the matter is the fact that virus strains have been completely eliminated from ICTV reports; thus, critically important information on virus identities and their associated biological and epidemiological features cannot be readily related to the ICTV classification. Here, we summarize the current status of bunyavirus taxonomy and discuss the adverse consequences associated with the reclassification and resulting omission of numerous viruses of public health importance from ICTV reports. As members of the American Committee on Arthropod-borne Viruses, we encourage the ICTV Bunyavirus Study Group to reconsider their stance on bunyavirus taxonomy, to revise the criteria currently used for species demarcation, and to list additional strains of public and veterinary importance.

A novel virus (order Bunyavirales) from stressed redclaw crayfish (Cherax quadricarinatus) from farms in northern Australia.

Athtabvirus, a bunya-like virus and chequa iflavirus infect redclaw crayfish (Cherax quadricarinatus) and they may cause mortality reaching 20-40% after about three weeks following transportation stress. Lesions were seen in the muscles of broodstock and juveniles and nerve cords of craylings. Using NextGen sequencing, the whole transcriptomes of a farmed case crayfish and a tank-reared, unaffected crayfish were assembled producing over 500,000 contigs. The average depth of reads was 18 replicates with a range from 15 to 44. The near complete sequences of the large and middle genome segments of a bunya-like virus were detected along with chequa iflavirus. The internal bunya-like motifs; RNA-dependent RNA polymerase on the L segment, and glycoprotein n (Gn) on the M segment were easily identified. In the opposite, positive-sense direction on the M segment, another presumed glycoprotein (glycoprotein c) with a low-density lipoprotein receptor (cysteine-rich) motif was identified by position specific iterated (psi)-BLASTp. The athtabvirus was related to Whenzhou Shrimp Virus 2 (E = 0.0, 43% amino acid identity), an unassigned, -ve sense ssRNA virus, and to peribunyaviruses (E = 10-50-20). In descending order of the number of RNA copies/0.2 mg of tissue, the organs most heavily infected were muscle (9.4 × 106), nerve cord (5.24 × 106), heart (4.07 × 106), gills (3.96 × 106), hepatopancreas (1.58 × 106) and antennal gland (6.6 × 105). Given the tissue tropism (muscle and nerves) of athtabvirus and the original lesions, this virus is implicated in being involved in the mortalities in crayfish after transportation.