Phylogeographic Genetic Diversity in the White Sucker Hepatitis B Virus across the Great Lakes Region and Alberta, Canada.

Hepatitis B viruses belong to a family of circular, double-stranded DNA viruses that infect a range of organisms, with host responses that vary from mild infection to chronic infection and cancer. The white sucker hepatitis B virus (WSHBV) was first described in the white sucker (Catostomus commersonii), a freshwater teleost, and belongs to the genus Parahepadnavirus. At present, the host range of WSHBV and its impact on fish health are unknown, and neither genetic diversity nor association with fish health have been studied in any parahepadnavirus. Given the relevance of genomic diversity to disease outcome for the orthohepadnaviruses, we sought to characterize genomic variation in WSHBV and determine how it is structured among watersheds. We identified WSHBV-positive white sucker inhabiting tributaries of Lake Michigan, Lake Superior, Lake Erie (USA), and Lake Athabasca (Canada). Copy number in plasma and in liver tissue was estimated via qPCR. Templates from 27 virus-positive fish were amplified and sequenced using a primer-specific, circular long-range amplification method coupled with amplicon sequencing on the Illumina MiSeq. Phylogenetic analysis of the WSHBV genome identified phylogeographical clustering reminiscent of that observed with human hepatitis B virus genotypes. Notably, most non-synonymous substitutions were found to cluster in the pre-S/spacer overlap region, which is relevant for both viral entry and replication. The observed predominance of p1/s3 mutations in this region is indicative of adaptive change in the polymerase open reading frame (ORF), while, at the same time, the surface ORF is under purifying selection. Although the levels of variation we observed do not meet the criteria used to define sub/genotypes of human and avian hepadnaviruses, we identified geographically associated genome variation in the pre-S and spacer domain sufficient to define five WSHBV haplotypes. This study of WSHBV genetic diversity should facilitate the development of molecular markers for future identification of genotypes and provide evidence in future investigations of possible differential disease outcomes.

Molecular detection of a novel cyprinid herpesvirus in roach (Rutilus rutilus) and asp (Leuciscus aspius) showing typical signs of carp pox disease.

In the early spring of 2018, in Lake Balaton (Hungary), a roach (Rutilus rutilus) and an asp (Leuciscus aspius) were found in an fish trap at the outlet of the river Sió showing typical signs of the so-called carp pox disease, such as foci of epidermal hyperplasia on the head and the whole body surface, including the fins. Molecular tests revealed the presence of the DNA of an unknown fish herpesvirus. Three genes encoding the DNA-dependent DNA polymerase, major capsid protein and ATPase subunit of terminase were amplified and sequenced from the alloherpesviral genome. The gene sequences of the viruses obtained from the two different fish species shared 94.4% nucleotide sequence identity (98.1% amino acid sequence identity), suggesting that they belong to the same virus species. Phylogenetic analysis based on the DNA polymerase (and the concatenated sequences of the amplified genes, as well) implied that the detected virus belongs to the genus Cyprinivirus within the family Alloherpesviridae. The sequences of the novel alloherpesvirus diverge from those of the five cyprinivirus species described previously, so it putatively represents the sixth virus species in the genus.

CMTV-like ranavirus infection associated with high mortality in captive catfish-like loach, Triplophysa siluorides, in China.

Ranaviruses are important emerging pathogens of ectothermic vertebrates that threaten aquaculture and wildlife worldwide. A mortality event occurred in a cultured population of catfish-like loach (Triplophysa siluorides) in Sichuan Province, China. Gross clinical signs of the affected fish included skin lesions and haemorrhagic ulcers, which are often associated with ranaviruses. Inoculation of liver, kidney and spleen tissue homogenates in epithelioma papulosum cyprini (EPC) cells at 25°C resulted in cytopathic effect within 24 hr. Transmission electron microscopy of infected EPC cells revealed hexagonal viral arrays in the cytoplasm and icosahedral geometry of the virions. Following exposure of T. siluroides to the isolated virus, similar clinical signs were observed and the fish experienced 40% and 90% mortality after 21 days at 103.58 and 107.8 TCID50 /0.1 ml doses, respectively, providing evidence the isolated virus was the main causative agent of the mortality event. Diagnostic PCR of the major capsid protein gene of ranavirus showed that all samples of diseased fish and isolated virus were positive. Phylogenetic analysis revealed that the isolated virus, designated as FYLl40220, was associated with the Common Midwife Toad Virus (CMTV)-like ranavirus clade. To our knowledge, this case represents the first report of CMTV-associated mortality in a fish species. Collectively, these results suggest that the host range of CMTV-like ranaviruses is greater than previously thought, and this clade of ranaviruses could have significant economic and biodiversity impacts.

ROS-induced HSP70 promotes cytoplasmic translocation of high-mobility group box 1b and stimulates antiviral autophagy in grass carp kidney cells.

Autophagy plays many physiological and pathophysiological roles. However, the roles and the regulatory mechanisms of autophagy in response to viral infections are poorly defined in teleost fish, such as grass carp (Ctenopharyngodon idella), which is one of the most important aquaculture species in China. In this study, we found that both grass carp reovirus (GCRV) infection and hydrogen peroxide (H2O2) treatment induced the accumulation of reactive oxygen species (ROS) in C. idella kidney cells and stimulate autophagy. Suppressing ROS accumulation with N-acetyl-l-cysteine significantly inhibited GCRV-induced autophagy activation and enhanced GCRV replication. Although ROS-induced autophagy, in turn, restricted GCRV replication, further investigation revealed that the multifunctional cellular protein high-mobility group box 1b (HMGB1b) serves as a heat shock protein 70 (HSP70)-dependent, pro-autophagic protein in grass carp. Upon H2O2 treatment, cytoplasmic HSP70 translocated to the nucleus, where it interacted with HMGB1b and promoted cytoplasmic translocation of HMGB1b. Overexpression and siRNA-mediated knockdown assays indicated that HSP70 and HMGB1b synergistically enhance ROS-induced autophagic activation in the cytoplasm. Moreover, HSP70 reinforced an association of HMGB1b with the C. idella ortholog of Beclin 1 (a mammalian ortholog of the autophagy-associated yeast protein ATG6) by directly interacting with C. idella Beclin 1. In summary, this study highlights the antiviral function of ROS-induced autophagy in response to GCRV infection and reveals the positive role of HSP70 in HMGB1b-mediated autophagy initiation in teleost fish.

Characterization of a Novel Hepadnavirus in the White Sucker (Catostomus commersonii) from the Great Lakes Region of the United States.

UNLABELLED: The white sucker Catostomus commersonii is a freshwater teleost often utilized as a resident sentinel. Here, we sequenced the full genome of a hepatitis B-like virus that infects white suckers from the Great Lakes Region of the United States. Dideoxy sequencing confirmed that the white sucker hepatitis B virus (WSHBV) has a circular genome (3,542 bp) with the prototypical codon organization of hepadnaviruses. Electron microscopy demonstrated that complete virions of approximately 40 nm were present in the plasma of infected fish. Compared to avi- and orthohepadnaviruses, sequence conservation of the core, polymerase, and surface proteins was low and ranged from 16 to 27% at the amino acid level. An X protein homologue common to the orthohepadnaviruses was not present. The WSHBV genome included an atypical, presumptively noncoding region absent in previously described hepadnaviruses. Phylogenetic analyses confirmed WSHBV as distinct from previously documented hepadnaviruses. The level of divergence in protein sequences between WSHBV and other hepadnaviruses and the identification of an HBV-like sequence in an African cichlid provide evidence that a novel genus of the family Hepadnaviridae may need to be established that includes these hepatitis B-like viruses in fishes. Viral transcription was observed in 9.5% (16 of 169) of white suckers evaluated. The prevalence of hepatic tumors in these fish was 4.9%, and only 2.4% of fish were positive for both virus and hepatic tumors. These results are not sufficient to draw inferences regarding the association of WSHBV and carcinogenesis in white sucker. IMPORTANCE: We report the first full-length genome of a hepadnavirus from fishes. Phylogenetic analysis of this genome indicates divergence from genomes of previously described hepadnaviruses from mammalian and avian hosts and supports the creation of a novel genus. The discovery of this novel virus may better our understanding of the evolutionary history of hepatitis B-like viruses of other hosts. In fishes, knowledge of this virus may provide insight regarding possible risk factors associated with hepatic neoplasia in the white sucker. This may also offer another model system for mechanistic research.

Identification and ultrastructural characterization of a novel virus from fish.

During routine investigations on fish, a virus (isolate DF 24/00) with novel morphological features and hitherto undescribed morphogenesis was isolated from a white bream (Blicca bjoerkna L.; Teleostei, order Cypriniformes). Cell-free virions consist of a rod-shaped nucleocapsid (120-150x19-22 nm) similar to that seen in baculoviruses. The virion has a bacilliform shape (170-200x75-88 nm) reminiscent of rhabdoviruses with an envelope containing coronavirus-like spikes (20-25 nm). DF 24/00 replicated well in various fish cell lines. Inhibitor studies with 5-iodo-2'-deoxyuridine indicated that the viral genome consists of RNA and chloroform sensitivity correlated with ultrastructural demonstration of enveloped virions. The buoyant density of the virus determined in sucrose was 1.17-1.19 g/ml. Preliminary biochemical characterization revealed the presence of six antigenic glycoproteins, three of which contain sugars with concanavalin-A specificity. Ultrastructurally, morphogenesis of virus progeny was detected only in the cytoplasm. Nucleocapsids were observed to bud through membranes of the endoplasmic reticulum and/or Golgi apparatus into dilated vesicles. Egress of mature virions occurs primarily by exocytosis and, only very rarely, by budding directly at the plasma membrane. Morphologically similar viruses had previously been isolated from grass carp (Ctenopharyngodon idella), blue crab (Callinectis sapidus), European shore crab (Carcinus maenas) and shrimp (Penaeus monodon). To date, none of them has been classified. In summary, the first characterization of a new virus that might represent a member of a novel virus family that has morphological features resembling those found in rhabdo-, corona- and baculoviruses is presented.