Bovine leukemia virus (BLV) causes enzootic bovine leukosis in cattle and leads to heavy economic losses in the husbandry industry. Heilongjiang Province, China, is rich in dairy cattle. However, its current BLV epidemiology and genotypes have still not been evaluated and confirmed. In this report, we investigated the BLV epidemiology in dairy cattle in the major regions of Heilongjiang Province via the nested PCR assay. A total of 730 blood samples were collected from nine different farms in six regions of Heilongjiang. The results showed that the infection rate of these regions ranged from null to 31%. With a clustering analysis of 60 published BLV env sequences, genotypes 1 and 6 were confirmed to be circulating in Heilongjiang. Importantly, a new genotype, 11, and a new subgenotype, 6E, were also identified in the Harbin and Daqing regions, respectively. An epitope analysis showed that a cluster of T-X-D-X-R-XXXX-A sequences in genotype 11 gp51 neutralizing domain 2 was unique among all currently known BLV isolates and was therefore a defining feature of this new genotype. BLV epidemics and genotypes were initially investigated in dairy cattle of Heilongjiang. A relatively high infection rate was found in some regions of this province. A new genotype, G11, with a highly specific motif, was identified and thus added as a new member to the current BLV genotype family. This report provides an initial reference for future investigations and subsequent control of BLV transmission and spread in this region.

As the only widely used live lentiviral vaccine, the equine infectious anima virus (EIAV) attenuated vaccine was developed by in vitro passaging of a virulent strain for 121 generations. In our previous study, we observed that the attenuated vaccine was gradually selected under increased environmental pressure at the population level (termed a quasispecies). To further elucidate the potential correlation between viral quasispecies evolution and pathogenesis, a systematic study was performed by sequencing env using several methods. Some key mutations were identified within Env, and we observed that increased percentages of these mutations were accompanied by an increased passage number and attenuated virulence. Phylogenetic analysis revealed that env mutations related to the loss of virulence might have occurred evolutionarily. Among these mutations, deletion of amino acid 236 in the V4 region of Env resulted in the loss of one N-glycosylation site that was crucial for virulence. Notably, the 236-deleted sequence represented a “vaccine-specific” mutation that was also found in wild EIAV_LN40 strains based on single genome amplification (SGA) analysis. Therefore, our results suggest that the EIAV attenuated vaccine may originate from a branch of quasispecies of EIAV_LN40. Generally, the presented results may increase our understanding of the attenuation mechanism of the EIAV vaccine and provide more information about the evolution of other lentiviruses.

Interferon-mediated host factors myxovirus (Mx) proteins are key features in regulating influenza A virus (IAV) infections. Viral polymerases are essential for viral replication. The Mx1 protein has been known to interact with viral nucleoprotein (NP) and PB2, resulting in the influence of polymerase activity and providing interspecies restriction. The equine influenza virus has evolved as an independent lineage to influenza viruses from other species. We estimated the differences in antiviral activities between human MxA (huMxA) and equine Mx1 (eqMx1) against a broad range of IAV strains. We found that huMxA has antiviral potential against IAV strains from non-human species, whereas eqMx1 could only inhibit the polymerase activity of non-equine species. Here, we demonstrated that NP is the main target of eqMx1. Subsequently, we found adaptive mutations in the NP of strains A/equine/Jilin/1/1989 (H3N8_JL89) and A/chicken/Zhejiang/DTID-ZJU01/2013 (H7N9_ZJ13) that confer eqMx1 resistance and sensitivity respectively. A substantial reduction in Mx1 resistance was observed for the two mutations G34S and H52N in H3N8_JL89 NP. Thus, eqMx1 is an important dynamic force in IAV nucleoprotein evolution. We, therefore, suggest that the amino acids responsible for Mx1 resistance should be regarded as a robust indicator for the pandemic potential of lately evolving IAVs.

Tetherin is an interferon-inducible type II transmembrane glycoprotein which inhibits the release of viruses, including retroviruses, through a “physical tethering” model. However, the role that the glycosylation of tetherin plays in its antiviral activity remains controversial. In this study, we found that mutation of N-glycosylation sites resulted in an attenuation of the antiviral activity of equine tetherin (eqTHN), as well as a reduction in the expression of eqTHN at the plasma membrane (PM). In addition, eqTHN N-glycosylation mutants colocalize obviously with ER, CD63, LAMP1 and endosomes, while WT eqTHN do not. Furthermore, we also found that N-glycosylation impacts the transport of eqTHN in the cell not by affecting the endocytosis, but rather by influencing the anterograde trafficking of the protein. These results suggest that the N-glycosylation of eqTHN is important for the antiviral activity of the protein through regulating its normal subcellular localization. This finding will enhance our understanding of the function of this important restriction factor.