Reovirus genomic diversity confers plasticity for protease utility during adaptation to intracellular uncoating.

IMPORTANCE: Reoviruses infect many mammals and are widely studied as a model system for enteric viruses. However, most of our reovirus knowledge comes from laboratory strains maintained on immortalized L929 cells. Herein, we asked whether naturally circulating reoviruses possess the same genetic and phenotypic characteristics as laboratory strains. Naturally circulating reoviruses obtained from sewage were extremely diverse genetically. Moreover, sewage reoviruses exhibited poor fitness on L929 cells and relied heavily on gut proteases for viral uncoating and productive infection compared to laboratory strains. We then examined how naturally circulating reoviruses might adapt to cell culture conditions. Within three passages, virus isolates from the parental sewage population were selected, displaying improved fitness and intracellular uncoating in L929 cells. Remarkably, selected progeny clones were present at 0.01% of the parental population. Altogether, using reovirus as a model, our study demonstrates how the high genetic diversity of naturally circulating viruses results in rapid adaptation to new environments.

Resistance Evaluation of Dominant Varieties against Southern Rice Black-Streaked Dwarf Virus in Southern China.

Southern rice black-streaked dwarf virus (SRBSDV), a Fijivirus in the Reoviridae family, is transmitted by the white-backed planthopper (Sogatella furcifera, WBPH), a long-distance migratory insect, and presents a serious threat to rice production in Asia. It was first discovered in China's Guangdong Province in 2001 and has been endemic in the south of China and north of Vietnam for two decades, with serious outbreaks in 2009, 2010, and 2017. In this study, we evaluated the resistance of 10 dominant rice varieties from southern China, where the virus overwinters and accumulates as a source of early spring reinfection, against this virus by artificial inoculation. The results showed that in all tested varieties there was no immune resistance, but there were differences in the infection rate, with incidence rates from 21% to 90.7%, and in symptom severity, with plant weight loss from 66.71% to 91.20% and height loss from 34.1% to 65.06%. Additionally, and valuably, the virus titer and the insect vector virus acquisition potency from diseased plants were significantly different among the varieties: an over sixfold difference was determined between resistant and susceptible varieties, and there was a positive correlation between virus accumulation and insect vector virus acquisition. The results can provide a basis for the selection of rice varieties in southern China to reduce the damage of SRBSDV in this area and to minimize the reinfection source and epidemics of the virus in other rice-growing areas.

Novel duck reovirus exhibits pathogenicity to specific pathogen-free chickens by the subcutaneous route.

To study the pathogenicity of new duck reovirus (NDRV) to chickens, eighty 3-day-old SPF chickens were equally divided into two groups. The experimental group was inoculated with a NDRV challenge strain of 100 µL (10-5.00 ELD50/0.1 mL) by the subcutaneous (s.c.) route, and the control group was inoculated with 100 µL of sterile phosphate-buffered saline (PBS) by the same route. In the experimental group, chickens exhibited introflexion of claws, performing of splits, stunting syndrome, weight loss and death. Gross lesions such as enlargement and yellowish-white focal necroses were observed in the liver and spleen. Microscopic changes were typical including varying degrees of hepatocyte steatosis and necrosis, splenic lymphocyte necrosis, interstitial pneumonia. Viral loads were detected in lung, liver, heart, spleen, duodenum, burse and kidney. The liver and spleen viral loads remained a much higher level and maintained for a longer time, suggesting that these tissues might be the target organs. In summary, NDRV can cause systemic infections and death in chickens, which indicated that chickens may be infected by NDRV in poultry production.

RNA virome abundance and diversity is associated with host age in a bird species.

Despite the ongoing interest in virus discovery, little is known about the factors that shape communities of viruses within individual hosts. Here, we address how virus communities might be impacted by the age of the hosts they infect, using total RNA sequencing to reveal the RNA viromes of different age groups of Ruddy Turnstones (Arenaria interpres). From oropharyngeal and cloacal swabs we identified 14 viruses likely infecting birds, 11 of which were novel, including members of the Reoviridae, Astroviridae, and Picornaviridae. Strikingly, 12 viruses identified were from juvenile birds sampled in the first year of their life, compared to only two viruses in adult birds. Both viral abundance and alpha diversity were marginally higher in juvenile than adult birds. As well as informing studies of virus ecology, that host age might be associated with viral composition is an important consideration for the future surveillance of novel and emerging viruses.

Characterization of taro reovirus and its status in taro (Colocasia esculenta) germplasm from the Pacific.

Taro reovirus (TaRV) has been reported infecting taro (Colocasia esculenta) in the South Pacific, but information on the virus is limited. Here, we report the genome sequence of a reovirus infecting taro in Papua New Guinea that had 10 genomic segments ranging from 1.1 to 3.9 kilobase pairs (kbp) in length with a total genome length of 26.3 kbp. TaRV was most closely related to rice ragged stunt virus (RRSV) but did not cross-react with RRSV polyclonal antisera. TaRV was not detected in 82 germplasm accessions of taro in Hawaii, or samples collected in American Samoa, Fiji, Guam, Palau, or Vanuatu.

Isolation, Identification, and Genomic Analysis of a Novel Reovirus from Healthy Grass Carp and Its Dynamic Proliferation In Vitro and In Vivo.

A new grass carp reovirus (GCRV), healthy grass carp reovirus (HGCRV), was isolated from grass carp in 2019. Its complete genome sequence was determined and contained 11 dsRNAs with a total size of 23,688 bp and 57.2 mol% G+C content, encoding 12 proteins. All segments had conserved 5' and 3' termini. Sequence comparisons showed that HGCRV was closely related to GCRV-873 (GCRV-I; 69.57-96.71% protein sequence identity) but shared only 22.65-45.85% and 23.37-43.39% identities with GCRV-HZ08 and Hubei grass carp disease reovirus (HGDRV), respectively. RNA-dependent RNA-polymerase (RdRp) protein-based phylogenetic analysis showed that HGCRV clustered with Aquareovirus-C (AqRV-C) prior to joining a branch common with other aquareoviruses. Further analysis using VP6 amino acid sequences from Chinese GCRV strains showed that HGCRV was in the same evolutionary cluster as GCRV-I. Thus, HGCRV could be a new GCRV isolate of GCRV-I but is distantly related to other known GCRVs. Grass carp infected with HGCRV did not exhibit signs of hemorrhage. Interestingly, the isolate induced a typical cytopathic effect in fish cell lines, such as infected cell shrank, apoptosis, and plague-like syncytia. Further analysis showed that HGCRV could proliferate in grass carp liver (L28824), gibel carp brain (GiCB), and other fish cell lines, reaching a titer of up to 7.5 × 104 copies/µL.

Isolation and characterization of hirame aquareovirus (HAqRV): A new Aquareovirus isolated from diseased hirame Paralichthys olivaceus.

We isolated a novel Aquareovirus (hirame aquareovirus: HAqRV) from Japanese flounder Paralichthys olivaceus suffering from reovirus-like infection. In electron microscopy, the spherical virion (75 nm in diameter) was observed with multi-layered capsid structure. The viral genome consisted of 11 segments and regions encoding 7 virion structural proteins and 5 non-structural proteins were predicted. The deduced amino acid sequences of those proteins were highly similar to those of the aquareoviruses. However, the similarity of complete genome sequence between the HAqRV and other aquareoviruses was less than 60%. Phylogenetic analyses based on the deduced amino acid sequences suggested that the HAqRV is not classified into the known species of Aquareovirus. Pathogenicity of HAqRV was clearly demonstrated in accordance with Koch's postulates by experimental infection using Japanese flounder. The results suggest that the HAqRV is a new Aquareovirus species which is highly virulent for the Japanese flounder at early life stages.

Diversity and classification of reoviruses in crustaceans: A proposal.

A variety of reoviruses have been described in crustacean hosts, including shrimp, crayfish, prawn, and especially in crabs. However, only one genus of crustacean reovirus - Cardoreovirus - has been formally recognized by ICTV (International Committee on Taxonomy of Viruses) and most crustacean reoviruses remain unclassified. This arises in part from ambiguous or incomplete information on which to categorize them. In recent years, increased availability of crustacean reovirus genomic sequences is making the discovery and classification of crustacean reoviruses faster and more certain. This minireview describes the properties of the reoviruses infecting crustaceans and suggests an overall classification of brachyuran crustacean reoviruses based on a combination of morphology, host, genome organization pattern and phylogenetic sequence analysis.

Origin and evolution of emerging Liao ning Virus (genus Seadornavirus, family Reoviridae).

BACKGROUND: Liao ning virus (LNV) is a member of the genus Seadornavirus, family Reoviridae and has been isolated from kinds of vectors in Asia and Australia. However, there are no systematic studies describe the molecular genetic evolution and migration of LNVs. With the development of bioinformatics, viral genetic data combining the information of virus isolation time and locations could be integrated to infer the virus evolution and spread in nature. METHODS: Here, a phylogenetic and phylogeographic analysis using Bayesian Markov chain Monte Carlo simulations was conducted on the LNVs isolated from a variety of vectors during 1990-2014 to identify the evolution and migration patterns of LNVs. RESULTS: The results demonstrated that the LNV could be divided into 3 genotypes, of which genotype 1 mainly composed of LNVs isolated from Australia during 1990 to 2014 and the original LNV strain (LNV-NE97-31) isolated from Liaoning province in northern China in 1997, genotype 2 comprised of the isolates all from Xinjiang province in western China and genotype 3 consisted the isolates from Qinghai and Shanxi province of central China. LNVs emerged about 272 years ago and gradually evolved into three lineages in the order genotype 1, genotype 2 and genotype 3. Following phylogeographic analysis, it shows genotype 1 LNVs transmitted from Australia (113°E-153°E,10°S-42°S) to Liaoning province (118°E-125°E,38°N-43°N) in Northeast Asian continent then further spread across the central part of China to western China (75°E-95°E,35°N-50°N). CONCLUSION: LNVs were initially isolated from Liaoning province of China in the Northeast Asia, however, the present study revealed that LNVs were first appeared in Australia in the South Pacific region and transmitted to mainland China then rapidly spread across China and evolved three different genotypes. The above results suggested that LNV had the characteristics of long-distance transmission and there were great genetic diversity existed in the LNV population. Notably, current information of 80 strains of LNVs are limited. It is of great importance to strengthen the surveillance of LNVs to explore its real origin in nature and monitoring of the LNVs' population variation and maintain vigilance to avoid LNV breaking through the species barrier and further clarify its relationship to human and animal infection.

Diaphorina citri reovirus is most closely related to fijiviruses.

The Asian citrus psyllid, Diaphorina citri Kuwayama, is an important insect vector of Candidatus Liberibacter asiaticus, the causal agent of Huanglongbing, which is the most destructive disease of citrus worldwide. Sequences for putative Diaphorina citri reovirus (DcRV) were identified from some worldwide populations of D. citri. Here, field surveys indicated that the virus was common in D. citri populations from Hawaii and Fuzhou of PR China. Electron microscopy showed that DcRV virions possessed a typical reovirus-like morphology. The U. S. and Chinese DcRV isolates both showed 10 segments of double-stranded RNA sharing >96% nucleotide sequence identity, and encoding 11 deduced proteins. All genome segments contained conserved 5' and 3' terminal nucleotide sequences and inverted repeats that are hallmarks of reovirus sequence. Phylogenetic analysis showed that DcRV may be considered a new species of the genus Fijivirus sharing a most recent common ancestor with the insect-specific fijivirus Nilaparvata lugens reovirus.

A new cypovirus from the Japanese peppered moth, Biston robustus.

A cypovirus was isolated from larvae of the Japanese peppered moth, Biston robustus. The viral genome is 23,954 bp comprising 10 segmented double-stranded RNAs with a new electropherotype among cypoviruses. Each segment encodes one putative protein and has non-coding regions that contain conserved sequences at their 5' and 3' termini, 5'-AGAA(U/A)U-3' and 5'-UGC-3', respectively. Seven proteins encoded in the genome are homologous to those of other cypoviruses at a cut-off E-value of 1 × 10-5. The maximal sequence identities of these proteins with cypovirus homologs are 24.30%-39.40%. These results indicate that the virus isolated is a novel cypovirus; herein designated as Biston robustus cypovirus 24 (BrCPV-24). This newly isolated BrCPV-24 infects the larvae of the silkworm Bombyx mori.

A real-time reverse-transcription isothermal recombinase polymerase amplification assay for the rapid detection of genotype III grass carp (Ctenopharyngodon idella) reovirus.

Grass carp (Ctenopharyngodon idella) hemorrhagic disease, which is characterized by external and internal hemorrhage, is a serious infectious disease affecting grass carp production. Strains of the causative agent, grass carp reovirus (GCRV), are divided into genotypes I, II and III, which are represented by the isolates GCRV-873, GCRV-HZ08 and GCRV-104, respectively. In this study, a real-time reverse-transcription recombinase polymerase amplification (real-time RT-RPA) assay was developed to detect the genotype III grass carp reovirus GCRV-104. The assay was based on the detection of the vp55 gene which encodes the outer fiber protein of the virus. A portable ESE-Quant Tube scanner, with a dimension of 17.4 × 18.8 cm, weighing about 1 kg, and equipped with temperature settings to amplify the DNA isothermally and spectral devices to detect the amplified products using fluorescence, was used to complete the assay. Under the optimal conditions, the assay took approximately 10 min to complete at 37 °C and showed no cross-reactions with other aquatic viruses. Consequently, this rapid real-time RT-RPA assay is a useful method for the simple, rapid and reliable detection of genotype III GCRV strains in resource-limited diagnostic laboratories.

Emergence of Southern Rice Black-Streaked Dwarf Virus in the Centuries-Old Chinese Yuanyang Agrosystem of Rice Landraces.

Southern rice black-streaked dwarf virus (SRBSDV), which causes severe disease symptoms in rice (Oriza sativa L.) has been emerging in the last decade throughout northern Vietnam, southern Japan and southern, central and eastern China. Here we attempt to quantify the prevalence of SRBSDV in the Honghe Hani rice terraces system (HHRTS)-a Chinese 1300-year-old traditional rice production system. We first confirm that genetically diverse rice varieties are still being cultivated in the HHRTS and categorize these varieties into three main genetic clusters, including the modern hybrid varieties group (MH), the Hongyang improved modern variety group (HY) and the traditional indica landraces group (TIL). We also show over a 2-year period that SRBSDV remains prevalent in the HHRTS (20.1% prevalence) and that both the TIL (17.9% prevalence) and the MH varieties (5.1% prevalence) were less affected by SRBSDV than were the HY varieties (30.2% prevalence). Collectively we suggest that SRBSDV isolates are freely moving within the HHRTS and that TIL, HY and MH rice genetic clusters are not being preferentially infected by particular SRBSDV lineages. Given that SRBSDV can cause 30-50% rice yield losses, our study emphasizes both the need to better monitor the disease in the HHRTS, and the need to start considering ways to reduce its burden on rice production.

Endangered wild salmon infected by newly discovered viruses.

The collapse of iconic, keystone populations of sockeye (Oncorhynchus nerka) and Chinook (Oncorhynchus tshawytscha) salmon in the Northeast Pacific is of great concern. It is thought that infectious disease may contribute to declines, but little is known about viruses endemic to Pacific salmon. Metatranscriptomic sequencing and surveillance of dead and moribund cultured Chinook salmon revealed a novel arenavirus, reovirus and nidovirus. Sequencing revealed two different arenavirus variants which each infect wild Chinook and sockeye salmon. In situ hybridisation localised arenavirus mostly to blood cells. Population surveys of >6000 wild juvenile Chinook and sockeye salmon showed divergent distributions of viruses, implying different epidemiological processes. The discovery in dead and dying farmed salmon of previously unrecognised viruses that are also widely distributed in wild salmon, emphasizes the potential role that viral disease may play in the population dynamics of wild fish stocks, and the threat that these viruses may pose to aquaculture.

Complete genome sequence and phylogenetic analysis of a novel aquareovirus isolated from a diseased marbled eel (Anguilla marmorata).

Marbled eel reovirus (MERV) is an aquareovirus (AQRV) isolated from diseased marbled eels (Anguilla marmorata) with petechial skin hemorrhage. In this study, we propagated MERV in a cell line derived from the brain of Aequidens rivulatus and purified viral particles by using a discontinuous cesium chloride gradient. Genomic RNA sequences were obtained through next-generation sequencing. MERV, similar to most other AQRVs, showed the presence of 11 double-stranded RNA segments encoding 12 proteins; however, the genome sequence displayed very little similarity to known AQRV sequences. Furthermore, the structural proteins of MERV were most closely related to American grass carp reovirus with sequence identity values of no more than 64.89%. Phylogenetic analysis based on the sequences of structural proteins indicated that MERV shows an evolutionary history between AQRV-B and -G, which belong to the saline and freshwater environment subgroups, respectively. We also observed that MERV showed a closer relationship to orthoreoviruses based on the protein sequences of NS38 and NS73. In summary, MERV is a novel AQRV that could be classified as a member of the new proposed AQRV species "Aquareovirus H". The taxonomic assignments and evolution of AQRVs thus warrant further investigation.

Fusogenic Reoviruses and Their Fusion-Associated Small Transmembrane (FAST) Proteins.

With no limiting membrane surrounding virions, nonenveloped viruses have no need for membrane fusion to gain access to intracellular replication compartments. Consequently, nonenveloped viruses do not encode membrane fusion proteins. The only exception to this dogma is the fusogenic reoviruses that encode fusion-associated small transmembrane (FAST) proteins that induce syncytium formation. FAST proteins are the smallest viral membrane fusion proteins and, unlike their enveloped virus counterparts, are nonstructural proteins that evolved specifically to induce cell-to-cell, not virus-cell, membrane fusion. This distinct evolutionary imperative is reflected in structural and functional features that distinguish this singular family of viral fusogens from all other protein fusogens. These rudimentary fusogens comprise specific combinations of different membrane effector motifs assembled into small, modular membrane fusogens. FAST proteins offer a minimalist model to better understand the ubiquitous process of protein-mediated membrane fusion and to reveal novel mechanisms of nonenveloped virus dissemination that contribute to virulence.

Characterization of Novel Reoviruses Wad Medani Virus (Orbivirus) and Kundal Virus (Coltivirus) Collected from Hyalomma anatolicum Ticks in India during Surveillance for Crimean Congo Hemorrhagic Fever.

In 2011, ticks were collected from livestock following an outbreak of Crimean Congo hemorrhagic fever (CCHF) in Gujarat state, India. CCHF-negative Hyalomma anatolicum tick pools were passaged for virus isolation, and two virus isolates were obtained, designated Karyana virus (KARYV) and Kundal virus (KUNDV), respectively. Traditional reverse transcription-PCR (RT-PCR) identification of known viruses was unsuccessful, but a next-generation sequencing (NGS) approach identified KARYV and KUNDV as viruses in the Reoviridae family, Orbivirus and Coltivirus genera, respectively. Viral genomes were de novo assembled, yielding 10 complete segments of KARYV and 12 nearly complete segments of KUNDV. The VP1 gene of KARYV shared a most recent common ancestor with Wad Medani virus (WMV), strain Ar495, and based on nucleotide identity we demonstrate that it is a novel WMV strain. The VP1 segment of KUNDV shares a common ancestor with Colorado tick fever virus, Eyach virus, Tai Forest reovirus, and Tarumizu tick virus from the Coltivirus genus. Based on VP1, VP6, VP7, and VP12 nucleotide and amino acid identities, KUNDV is proposed to be a new species of Coltivirus Electron microscopy supported the classification of KARYV and KUNDV as reoviruses and identified replication morphology consistent with other orbi- and coltiviruses. The identification of novel tick-borne viruses carried by the CCHF vector is an important step in the characterization of their potential role in human and animal pathogenesis.IMPORTANCE Ticks and mosquitoes, as well Culicoides, can transmit viruses in the Reoviridae family. With the help of next-generation sequencing (NGS), previously unreported reoviruses such as equine encephalosis virus, Wad Medani virus (WMV), Kammavanpettai virus (KVPTV), and, with this report, KARYV and KUNDV have been discovered and characterized in India. The isolation of KUNDV and KARYV from Hyalomma anatolicum, which is a known vector for zoonotic pathogens, such as Crimean Congo hemorrhagic fever virus, Babesia, Theileria, and Anaplasma species, identifies arboviruses with the potential to transmit to humans. Characterization of KUNDV and KARYV isolated from Hyalomma ticks is critical for the development of specific serological and molecular assays that can be used to determine the association of these viruses with disease in humans and livestock.

Isolation and genomic characterization of a cypovirus from the oleander hawk moth, Daphnis nerii.

The oleander hawk moth, Daphnis nerii, is a serious pest of plants belonging to the family Apocynaceae. Thus far, pathogen infection has not been reported in D. nerii. In this study, a new cytoplasmic polyhedrosis virus (cypovirus; CPV) was isolated from naturally diseased D. nerii larvae and named DnCPV-23. Virions were observed in ultrathin sections of DnCPV polyhedral bodies. Electrophoretic analysis revealed that the DnCPV genome consisted of 10 segments of double-stranded RNA (dsRNA). cDNA copies of these dsRNA segments were amplified using the method of full-length amplification of cDNAs (FLAC), cloned, and sequenced. Sequencing results showed that all segments contained one open reading frame (ORF); They shared the conserved terminal sequences AGUCAAA and AGC at 5' and 3' ends respectively, except segment 4, which is different from previously reported 22 cypoviruses. Phylogenetic analysis based on amino acid sequences of polyhedrin (encoded by segment 10) indicated that this CPV was closely related to CPV type 19. Altogether, DnCPV-23 is a new type of cypovirus.

Identification of a novel membrane-associated protein from the S7 segment of grass carp reovirus.

Aquareovirus is a genus of viruses in family Reoviridae, subfamily Spinareovirinae, members of which infect fish, shellfish and crustaceans. Grass carp reovirus (GCRV), a genotype 1 reovirus isolated from grass carp, has served as a model strain for investigating aquareovirus-host interactions. Herein, we report a neglected open reading frame (ORF), tentatively named NS12, residing between NS16 and NS31 in segment 7 (S7) of the virus genome. With an additional reading frame, the nucleotide sequence of NS12 partially overlaps with the 3' expressible nucleotide sequence of NS16. NS12 is not a pseudogene during virus replication, as confirmed in fish cells infected with GCRV and based on amino acid sequence analysis and protein expression pattern. Bioinformatics analysis indicated that NS12 is a transmembrane protein, which was confirmed by its exclusive presence in the membrane-associated fraction of the cell lysate. However, unlike fusion protein NS16, NS12 alone could not induce visible syncytium formation in fish cells. Thus, NS12 is functionally distinct from known aquareovirus membrane-associated protein NS16. NS12-like ORFs (with an AUG or non-AUG initiator codon) are also present in the S7 segment of other aquareoviruses, suggesting that NS12 homologues may be widely distributed in the genus Aquareovirus.

ITGB1b-Deficient Rare Minnows Delay Grass Carp Reovirus (GCRV) Entry and Attenuate GCRV-Triggered Apoptosis.

Integrin ß-1 (ITGB1) is a transmembrane protein belonging to the integrin family and it plays an important role in viral entry. In this study, the itgb1b gene of the rare minnow, Gobiocypris rarus, was cloned and analyzed. To investigate the possible role of itgb1b on grass carp reovirus (GCRV) infection, we generated an ITGB1b-deficient rare minnow (ITGB1b-/-) using the CRISPR/Cas9 system. Following stimulation with GCRV, the survival time of the -ITGB1b-/- rare minnows was extended in comparison to the wild-type minnows. Moreover, the relative copy number of GCRV and the level of clathrin-mediated endocytosis-associated and apoptosis-related gene expression in the ITGB1b-/- rare minnows was significantly lower than that of the wild-type minnows. These results suggested that the absence of itgb1b reduced viral entry efficiency and the expression of apoptosis-related genes. Moreover, the data suggested that itgb1b played an important role in mediating the entry of viruses into the cells via clathrin. Therefore, these findings provide novel insight into the function of itgb1b in the process of GCRV infection.