A widespread grass carp hemorrhagic disease (GCHD) caused by grass carp reovirus (GCRV) has been known in China since 1983. A virulent reovirus strain, HZ08, was isolated from diseased grass carp in Zhejiang Province, China. We sequenced and analyzed the complete genome of strain HZ08 and compared it with published GCRV genome sequences, contributing to the evidence of several genotypes of GCRV in China.

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Tilapia lake virus (TiLV) is a newly emerging pathogen responsible for high mortality and economic losses in the global tilapia industry. Currently, no antiviral therapy or vaccines are available for the control of this disease. The goal of the present study was to evaluate the immunological effects and protective efficacy of formaldehyde- and β-propiolactone-inactivated vaccines against TiLV in the presence and absence of the Montanide IMS 1312 VG adjuvant in tilapia. We found that β-propiolactone inactivation of viral particles generated a vaccine with a higher protection efficacy against virus challenge than did formaldehyde. The relative percent survivals of vaccinated fish at doses of 108, 107, and 106 50% tissue culture infectious dose (TCID50)/mL were 42.9%, 28.5%, and 14.3% in the absence of the adjuvant and 85.7%, 64.3%, and 32.1% in its presence, respectively. The vaccine generated specific IgM and neutralizing antibodies against TiLV at 3 weeks following immunization that were significantly increased after a second booster immunization. The steady state mRNA levels of the genes tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interferon γ (IFN-γ), cluster of differentiation 4 (CD4), major histocompatibility complex (MHC)-Ia, and MHC-II were all increased and indicated successful immune stimulation against TiLV. The vaccine also significantly lowered the viral loads and resulted in significant increases in survival, indicating that the vaccine may also inhibit viral proliferation as well as stimulate a protective antibody response. The β-propiolactone-inactivated TiLV vaccine coupled with the adjuvant Montanide IMS 1312 VG and booster immunizations can provide a high level of protection from virus challenge in tilapia.

A new grass carp reovirus, assigned HZ08, was isolated from a diseased grass carp case during routine examination in Huzhou City, Zhejiang Province, China. The complete nucleotide sequences of genomic segments S1-S3 and S5-S6 were obtained and comprised 3927, 3870, 3753, 2229, and 2030 bp, respectively. Each segment contained a single open reading frame which encoded putative proteins of 143.6, 143.1, 135.9, 80.5, and 68.4 kDa, respectively. Conserved motifs 5' (GUAAUUU...UUCAUC) 3' were found at the ends of each segment. At the amino acid level, HZ08 S1-S3 and S5-S6 showed similarity to the corresponding segments of Aquareovirus. Further phylogenetic analysis using the amino acid sequences of the RNA-dependent RNA polymerases protein encoded by S2 revealed that HZ08 formed a cluster close to the aquareovirus, but was far from the other isolates, which indicated that HZ08 is likely to be a new member of Aquareovirus.

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Herpesvirus of turkey (HVT) is being widely used as a vector for development of recombinant vaccines and US2 and US10 genes are often chosen as insertion sites for targeted gene expression. However, the different effects of the two genes for generation of recombinant HVT vaccines were unknown. In order to compare the effects of inserted genes in the two sites on the efficacy of the recombinant vaccines, host-protective haemagglutinin (HA) gene of the highly pathogenic avian influenza virus (HPAIV) H5N1 was inserted into either US2 or US10 gene locus of the HVT. The resulting US2 (rHVT-US2-HA) or US10 (rHVT-US10-HA) recombinant HVT viruses were used to infect chicken embryo fibroblasts. Plaques and the growth kinetics of rHVT-US2-HA-infected chicken embryo fibroblasts were similar to those of parental HVT whereas rHVT-US10-HA infected chicken embryo fibroblasts had different growth kinetics and plaque formation. The viremia levels in rHVT-US10-HA virus-infected chickens were significantly lower than those of rHVT-US2-HA group on 28 days post infection. The vaccine efficacy of the two recombinant viruses against H5N1 HPAIV and virulent Marek's disease virus was also evaluated in 1-day-old vaccinated chickens. rHVT-US2-HA-vaccinated chickens were better protected with reduced mortality than rHVT-US10-HA-vaccinated animals following HPAIV challenge. Furthermore, the overall hemaglutination inhibition antibody titers of rHVT-US2-HA-vaccinated chickens were higher than those of rHVT-US10-HA-vaccinated chickens. Protection levels against Marek's disease virus challenge following vaccination with either rHVT-US2-HA or rHVT-US10-HA, however, were similar to those of the parental HVT virus. These results, for the first time, indicate that US2 gene provides a favorable foreign gene insertion site for generation of recombinant HVT vaccines.

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Tilapia lake virus (TiLV) is an emerging disease threatening tilapia culture in many parts of the world. A cell line from the brain of tilapia, which was named TiB, was established, characterized and subcultured with more than 100 passages. The TiB cell line was optimally maintained at 27°C using medium 199 (M199) supplemented with 10% foetal bovine serum (FBS). Chromosome analysis revealed that 60% of TiB cells at passage 5 maintained the modal chromosome number 2n = 44, while at passage 60, there were 43% of TiB cells with the diploid chromosome number 2n = 50. A significant cytopathic effect was observed in TiB cells after infection with tilapia lake virus (TiLV‐2017A), and the viral replication in the cells was confirmed by transmission electron microscopy, immunofluorescence assays and viral titres, indicating the susceptibility of TiB cells to TiLV‐2017A. The viral titres of TiLV‐2017A in TiB cells reached 107.43 TCID50/ml within 10 days. The stable growth and susceptibility to fish viruses make TiB cells a useful tool for fish virus–host cell interaction and for immune response of fish.