Multiple conformations of trimeric spikes visualized on a non-enveloped virus.

Many viruses utilize trimeric spikes to gain entry into host cells. However, without in situ structures of these trimeric spikes, a full understanding of this dynamic and essential process of viral infections is not possible. Here we present four in situ and one isolated cryoEM structures of the trimeric spike of the cytoplasmic polyhedrosis virus, a member of the non-enveloped Reoviridae family and a virus historically used as a model in the discoveries of RNA transcription and capping. These structures adopt two drastically different conformations, closed spike and opened spike, which respectively represent the penetration-inactive and penetration-active states. Each spike monomer has four domains: N-terminal, body, claw, and C-terminal. From closed to opened state, the RGD motif-containing C-terminal domain is freed to bind integrins, and the claw domain rotates to expose and project its membrane insertion loops into the cellular membrane. Comparison between turret vertices before and after detachment of the trimeric spike shows that the trimeric spike anchors its N-terminal domain in the iris of the pentameric RNA-capping turret. Sensing of cytosolic S-adenosylmethionine (SAM) and adenosine triphosphate (ATP) by the turret triggers a cascade of events: opening of the iris, detachment of the spike, and initiation of endogenous transcription.

Type II Grass Carp Reovirus Infects Leukocytes but Not Erythrocytes and Thrombocytes in Grass Carp (Ctenopharyngodon idella).

Grass carp reovirus (GCRV) causes serious losses to the grass carp industry. At present, infectious tissues of GCRV have been studied, but target cells remain unclear. In this study, peripheral blood cells were isolated, cultured, and infected with GCRV. Using quantitative real-time polymerase chain reaction (qRT-PCR), Western Blot, indirect immunofluorescence, flow cytometry, and transmission electron microscopy observation, a model of GCRV infected blood cells in vitro was established. The experimental results showed GCRV could be detectable in leukocytes only, while erythrocytes and thrombocytes could not. The virus particles in leukocytes are wrapped by empty membrane vesicles that resemble phagocytic vesicles. The empty membrane vesicles of leukocytes are different from virus inclusion bodies in C. idella kidney (CIK) cells. Meanwhile, the expression levels of IFN1, IL-1ß, Mx2, TNFα were significantly up-regulated in leukocytes, indicating that GCRV could cause the production of the related immune responses. Therefore, GCRV can infect leukocytes in vitro, but not infect erythrocytes and thrombocytes. Leukocytes are target cells in blood cells of GCRV infections. This study lays a theoretical foundation for the study of the GCRV infection mechanism and anti-GCRV immunity.

Identification, Virulence, and Molecular Characterization of a Recombinant Isolate of Grass Carp Reovirus Genotype I.

The hemorrhagic disease of grass carp (HDGC) caused by grass carp reovirus (GCRV) still poses a great threat to the grass carp industry. Isolation and identification of the GCRV genotype I (GCRV-I) has been rarely reported in the past decade. In this study, a new GCRV was isolated from diseased fish with severe symptoms of enteritis and mild hemorrhages on the body surface. The isolate was further identified by cell culture, transmission electron, indirect immunofluorescence, and SDS-PAGE electrophoretic pattern analysis of genomic RNA. The results were consistent with the new isolate as a GCRV-I member and tentatively named GCRV-GZ1208. Both grass carp and rare minnow infected by the GCRV-GZ1208 have no obvious hemorrhagic symptoms, and the final mortality rate was ≤10%, indicating that it may be a low virulent isolate. GZ1208 possessed highest genomic homology to 873/GCHV (GCRV-I) and golden shiner reovirus (GSRV). Additionally, it was found a 90.7-98.3% nucleotide identity, a 96.4-100% amino acid identity, and <50% identity with GCRV-II and III genotypes. Interestingly, the sequences of some segments of GZ1208 were similar to GCRV-8733/GCHV, whereas the remaining segments were more closely related to GSRV, suggesting that a recombination event had occurred. Bootscan analysis of the complete genomic sequence confirmed this hypothesis, and recombination events between 873/GCHV and other GSRV-like viruses were also accompanied by gene mutations.

Effect of mutations in capsid shell protein on the assembly of BmCPV virus-like particles.

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a typical single-layer capsid dsRNA virus belonging to the genus Cypovirus in the family Reoviridae. The results of cryo-electron microscopy showed that the BmCPV capsid consists of 60 asymmetric units, and each asymmetric unit contains one turret protein (TP), two large protrusion proteins (LPP) and two capsid shell proteins (CSP). CSP has the ability to self-assemble into virus-like particles (VLPs), and the small protrusion domain (SPD) in CSP may play an essential role in the assembly of viral capsids. In this study, three critical amino acid sites, D828, S829 and V945, in the SPD were efficiently mutated (point mutation) based on the principle of PCR circular mutagenesis. Moreover, a multi-gene expression system, Ac-MultiBac baculovirus, was used to produce eight different recombinant VLPs in vitro. Transmission electron microscopy showed that the single site and double site mutations had little effect on the efficiency and morphology of the assembly of VLPs. Still, the simultaneous mutation of the three sites had a significant impact. The experimental results demonstrate that the SPD of CSP plays an essential role in assembly of the viral capsid, which lays the foundation for further analysis of the molecular and structural mechanism of BmCPV capsid assembly.

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 modified lysosomal organelle mediates nonlytic egress of reovirus.

Mammalian orthoreoviruses (reoviruses) are nonenveloped viruses that replicate in cytoplasmic membranous organelles called viral inclusions (VIs) where progeny virions are assembled. To better understand cellular routes of nonlytic reovirus exit, we imaged sites of virus egress in infected, nonpolarized human brain microvascular endothelial cells (HBMECs) and observed one or two distinct egress zones per cell at the basal surface. Transmission electron microscopy and 3D electron tomography (ET) of the egress zones revealed clusters of virions within membrane-bound structures, which we term membranous carriers (MCs), approaching and fusing with the plasma membrane. These virion-containing MCs emerged from larger, LAMP-1-positive membranous organelles that are morphologically compatible with lysosomes. We call these structures sorting organelles (SOs). Reovirus infection induces an increase in the number and size of lysosomes and modifies the pH of these organelles from ∼4.5-5 to ∼6.1 after recruitment to VIs and before incorporation of virions. ET of VI-SO-MC interfaces demonstrated that these compartments are connected by membrane-fusion points, through which mature virions are transported. Collectively, our results show that reovirus uses a previously undescribed, membrane-engaged, nonlytic egress mechanism and highlights a potential new target for therapeutic intervention.

Arrangement of the Polymerase Complexes inside a Nine-Segmented dsRNA Virus.

Members of the family Reoviridae package several copies of the viral polymerase complex into their capsid to carry out replication and transcription within viral particles. Classical single-particle reconstruction encounters difficulties resolving structures such as the intraparticle polymerase complex because refinement can converge to an incorrect map and because the map could depict a nonrepresentative subset of particles or an average of heterogeneous particles. Using the nine-segmented Fako virus, we tested hypotheses for the arrangement and number of polymerase complexes within the virion by measuring how well each hypothesis describes the set of cryoelectron microscopy images of individual viral particles. We find that the polymerase complex in Fako virus binds at ten possible sites despite having only nine genome segments. A single asymmetric configuration describes the arrangement of these complexes in both virions and genome-free capsids. Similarities between the arrangements of Reoviridae with 9, 10, and 11 segments indicate the generalizability of this architecture.

Structure of RdRps Within a Transcribing dsRNA Virus Provides Insights Into the Mechanisms of RNA Synthesis.

RNA-dependent RNA polymerases (RdRps) catalyze RNA synthesis of RNA viruses. During initiation of RNA synthesis, the RdRp catalyzes the formation of the first dinucleotide, acting as primer for subsequent processive RNA elongation. Here, we present the structure of the RdRp complexes in the dinucleotide primed state in situ within a transcribing cypovirus under near physiological conditions using cryo-electron microscopy. The 3' end of RNA templates, paired RNA dinucleotide primer, incoming nucleotide, and catalytic divalent cations in the RdRp were resolved at 3.8 Å resolution. The end of the RNA template and the dinucleotide is buttressed by the aromatic tyrosine in a loop from the RdRp bracelet domain. Our structure reveals the interactions between the nucleotide substrates and the conserved residues during the RdRp initiation, and the coordinated structural changes preceding the elongation stage. In addition, it provides the direct evidence for existence of the slow step of the dinucleotide primed state in the viral RdRp transcription.

Conservative transcription in three steps visualized in a double-stranded RNA virus.

Endogenous RNA transcription characterizes double-stranded RNA (dsRNA) viruses in the Reoviridae, a family that is exemplified by its simple, single-shelled member cytoplasmic polyhedrosis virus (CPV). Because of the lack of in situ structures of the intermediate stages of RNA-dependent RNA polymerase (RdRp) during transcription, it is poorly understood how RdRp detects environmental cues and internal transcriptional states to initiate and coordinate repeated cycles of transcript production inside the capsid. Here, we captured five high-resolution (2.8-3.5 Å) RdRp-RNA in situ structures-representing quiescent, initiation, early elongation, elongation and abortive states-under seven experimental conditions of CPV. We observed the 'Y'-form initial RNA fork in the initiation state and the complete transcription bubble in the elongation state. These structures reveal that de novo RNA transcription involves three major conformational changes during state transitions. Our results support an ouroboros model for endogenous conservative transcription in dsRNA viruses.

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.

Synthesis and Translation of Viral mRNA in Reovirus-Infected Cells: Progress and Remaining Questions.

At the end of my doctoral studies, in 1988, I published a review article on the major steps of transcription and translation during the mammalian reovirus multiplication cycle, a topic that still fascinates me 30 years later. It is in the nature of scientific research to generate further questioning as new knowledge emerges. Our understanding of these fascinating viruses thus remains incomplete but it seemed appropriate at this moment to look back and reflect on our progress and most important questions that still puzzle us. It is also essential of being careful about concepts that seem so well established, but could still be better validated using new approaches. I hope that the few reflections presented here will stimulate discussions and maybe attract new investigators into the field of reovirus research. Many other aspects of the viral multiplication cycle would merit our attention. However, I will essentially limit my discussion to these central aspects of the viral cycle that are transcription of viral genes and their phenotypic expression through the host cell translational machinery. The objective here is not to review every aspect but to put more emphasis on important progress and challenges in the field.

In Situ Structures of the Polymerase Complex and RNA Genome Show How Aquareovirus Transcription Machineries Respond to Uncoating.

Reoviruses carry out genomic RNA transcription within intact viruses to synthesize plus-sense RNA strands, which are capped prior to their release as mRNA. The in situ structures of the transcriptional enzyme complex (TEC) containing the RNA-dependent RNA polymerase (RdRp) and NTPase are known for the single-layered reovirus cytoplasmic polyhedrosis virus (CPV), but not for multilayered reoviruses, such as aquareoviruses (ARV), which possess a primed stage that CPV lacks. Consequently, how the RNA genome and TEC respond to priming in reoviruses is unknown. Here, we determined the near-atomic-resolution asymmetric structure of ARV in the primed state by cryo-electron microscopy (cryo-EM), revealing the in situ structures of 11 TECs inside each capsid and their interactions with the 11 surrounding double-stranded RNA (dsRNA) genome segments and with the 120 enclosing capsid shell protein (CSP) VP3 subunits. The RdRp VP2 and the NTPase VP4 associate with each other and with capsid vertices; both bind RNA in multiple locations, including a novel C-terminal domain of VP4. Structural comparison between the primed and quiescent states showed translocation of the dsRNA end from the NTPase to the RdRp during priming. The RNA template channel was open in both states, suggesting that channel blocking is not a regulating mechanism between these states in ARV. Instead, the NTPase C-terminal domain appears to regulate RNA translocation between the quiescent and primed states. Taking the data together, dsRNA viruses appear to have adapted divergent mechanisms to regulate genome transcription while retaining similar mechanisms to coassemble their genome segments, TEC, and capsid proteins into infectious virions.IMPORTANCE Viruses in the family Reoviridae are characterized by the ability to endogenously synthesize nascent RNA within the virus. However, the mechanisms for assembling their RNA genomes with transcriptional enzymes into a multilayered virion and for priming such a virion for transcription are poorly understood. By cryo-EM and novel asymmetric reconstruction, we determined the atomic structure of the transcription complex inside aquareoviruses (ARV) that are primed for infection. The transcription complex is anchored by the N-terminal segments of enclosing capsid proteins and contains an NTPase and a polymerase. The NTPase has a newly discovered domain that translocates the 5' end of plus-sense RNA in segmented dsRNA genomes from the NTPase to polymerase VP2 when the virus changes from the inactive (quiescent) to the primed state. Conformation changes in capsid proteins and transcriptional complexes suggest a mechanism for relaying information from the outside to the inside of the virus during priming.

Genome packaging of reovirus is mediated by the scaffolding property of the microtubule network.

Reovirus replication occurs in the cytoplasm of the host cell, in virally induced mini-organelles called virus factories. On the basis of the serotype of the virus, the virus factories can manifest as filamentous (type 1 Lang strain) or globular structures (type 3 Dearing strain). The filamentous factories morphology is dependent on the microtubule cytoskeleton; however, the exact function of the microtubule network in virus replication remains unknown. Using a combination of fluorescent microscopy, electron microscopy, and tomography of high-pressure frozen and freeze-substituted cells, we determined the ultrastructural organisation of reovirus factories. Cells infected with the reovirus microtubule-dependent strain display paracrystalline arrays of progeny virions resulting from their tiered organisation around microtubule filaments. On the contrary, in cells infected with the microtubule-independent strain, progeny virions lacked organisation. Conversely to the microtubule-dependent strain, around half of the viral particles present in these viral factories did not contain genomes (genome-less particles). Complementarily, interference with the microtubule filaments in cells infected with the microtubule-dependent strain resulted in a significant increase of genome-less particle number. This decrease of genome packaging efficiency could be rescued by rerouting viral factories on the actin cytoskeleton. These findings demonstrate that the scaffolding properties of the microtubule, and not biochemical nature of tubulin, are critical determinants for reovirus efficient genome packaging. This work establishes, for the first time, a functional correlation between ultrastructural organisation of reovirus factories with genome packaging efficiency and provides novel information on how viruses coordinate assembly of progeny particles.

Filamentous Structures Induced by a Phytoreovirus Mediate Viral Release from Salivary Glands in Its Insect Vector.

Numerous viral pathogens are persistently transmitted by insect vectors and cause agricultural or health problems. These viruses circulate in the vector body, enter the salivary gland, and then are released into the apical plasmalemma-lined cavities, where saliva is stored. The cavity plasmalemma of vector salivary glands thus represents the last membrane barrier for viral transmission. Here, we report a novel mechanism used by a persistent virus to overcome this essential barrier. We observed that the infection by rice gall dwarf virus (RGDV), a species of the genus Phytoreovirus in the family Reoviridae, induced the formation of virus-associated filaments constructed by viral nonstructural protein Pns11 within the salivary glands of its leafhopper vector, Recilia dorsalis Such filaments attached to actin-based apical plasmalemma and induced an exocytosis-like process for viral release into vector salivary gland cavities, through a direct interaction of Pns11 of RGDV and actin of R. dorsalis Failure of virus-induced filaments assembly by RNA interference with synthesized double-stranded RNA targeting the Pns11 gene inhibited the dissemination of RGDV into salivary cavities, preventing viral transmission by R. dorsalis For the first time, we show that a virus can exploit virus-induced inclusion as a vehicle to pass through the apical plasmalemma into vector salivary gland cavities, thus overcoming the last membrane barrier for viral transmission by insect vectors.IMPORTANCE Understanding how persistent viruses overcome multiple tissue and membrane barriers within the insect vectors until final transmission is the key for viral disease control. The apical plasmalemma of the cavities where saliva is stored in the salivary glands is the last barrier for viral transmission by insect vectors; however, the mechanism is still poorly understood. Here we show that a virus has evolved to exploit virus-induced filaments to perform an exocytosis-like process that enables viral passage through the apical plasmalemma into salivary cavities. This mechanism could be extensively exploited by other persistent viruses to overcome salivary gland release barriers in insect vectors, opening new perspectives for viral control.

Structure and components of the globular and filamentous viroplasms induced by Rice black-streaked dwarf virus.

Viroplasms of members of the family Reoviridae are considered to be viral factories for genome replication and virion assembly. Globular and filamentous phenotypes have different components and probably have different functions. We used transmission electron microscopy and electron tomography to examine the structure and components of the two viroplasm phenotypes induced by Rice black-streaked dwarf virus (RBSDV). Immuno-gold labeling was used to localize each of the 13 RBSDV encoded proteins as well as double-stranded RNA, host cytoskeleton actin-11 and α-tubulin. Ten of the RBSDV proteins were localized in one or both types of viroplasm. P5-1, P6 and P9-1 were localized on both viroplasm phenotypes but P5-1 was preferentially associated with filaments and P9-1 with the matrix. Structural analysis by electron tomography showed that osmiophilic granules 6-8nm in diameter served as the fundamental unit for constructing both of the viroplasm phenotypes but were more densely packed in the filamentous phenotype.

Virus-mediated suppression of host non-self recognition facilitates horizontal transmission of heterologous viruses.

Non-self recognition is a common phenomenon among organisms; it often leads to innate immunity to prevent the invasion of parasites and maintain the genetic polymorphism of organisms. Fungal vegetative incompatibility is a type of non-self recognition which often induces programmed cell death (PCD) and restricts the spread of molecular parasites. It is not clearly known whether virus infection could attenuate non-self recognition among host individuals to facilitate its spread. Here, we report that a hypovirulence-associated mycoreovirus, named Sclerotinia sclerotiorum mycoreovirus 4 (SsMYRV4), could suppress host non-self recognition and facilitate horizontal transmission of heterologous viruses. We found that cell death in intermingled colony regions between SsMYRV4-infected Sclerotinia sclerotiorum strain and other tested vegetatively incompatible strains was markedly reduced and inhibition barrage lines were not clearly observed. Vegetative incompatibility, which involves Heterotrimeric guanine nucleotide-binding proteins (G proteins) signaling pathway, is controlled by specific loci termed het (heterokaryon incompatibility) loci. Reactive oxygen species (ROS) plays a key role in vegetative incompatibility-mediated PCD. The expression of G protein subunit genes, het genes, and ROS-related genes were significantly down-regulated, and cellular production of ROS was suppressed in the presence of SsMYRV4. Furthermore, SsMYRV4-infected strain could easily accept other viruses through hyphal contact and these viruses could be efficiently transmitted from SsMYRV4-infected strain to other vegetatively incompatible individuals. Thus, we concluded that SsMYRV4 is capable of suppressing host non-self recognition and facilitating heterologous viruses transmission among host individuals. These findings may enhance our understanding of virus ecology, and provide a potential strategy to utilize hypovirulence-associated mycoviruses to control fungal diseases.

Near-Atomic Resolution Structure Determination of a Cypovirus Capsid and Polymerase Complex Using Cryo-EM at 200kV.

Single-particle cryo-electron microscopy (cryo-EM) allows the high-resolution structural determination of biological assemblies in a near-native environment. However, all high-resolution (better than 3.5Å) cryo-EM structures reported to date were obtained by using 300kV transmission electron microscopes (TEMs). We report here the structures of a cypovirus capsid of 750-Å diameter at 3.3-Å resolution and of RNA-dependent RNA polymerase (RdRp) complexes within the capsid at 3.9-Å resolution using a 200-kV TEM. The newly resolved structure revealed conformational changes of two subdomains in the RdRp. These conformational changes, which were involved in RdRp's switch from non-transcribing to transcribing mode, suggest that the RdRp may facilitate the unwinding of genomic double-stranded RNA. The possibility of 3-Å resolution structural determinations for biological assemblies of relatively small sizes using cryo-EM at 200kV was discussed.

Preliminary characterization of Tasmanian aquareovirus (TSRV) isolates.

In an attempt to determine whether or not genetic variants of the Tasmanian strain of Atlantic salmon aquareovirus (TSRV) exist, 14 isolates of TSRV, originating from various locations in Tasmania, covering a 20-year period (1990-2010), obtained from various host species and tissues, and isolated on different cell lines, were selected for this study. Two categories, termed "typical" and "atypical", of variants of TSRV were identified based on preliminary genotypic and phenotypic characterization carried out on these 14 different isolates. In addition, electron microscopic examination indicated the existence of at least three variants based on viral particle size. Finally, this study demonstrated the existence of at least one new variant of TSRV isolates, other than the more commonly isolated typical TSRV isolates, in farmed Tasmanian Atlantic salmon.