VP2 mediates the release of the feline calicivirus RNA genome by puncturing the endosome membrane of infected cells.

Feline calicivirus (FCV) is one of the few members of the Caliciviridae family that grows well in cell lines and, therefore, serves as a surrogate to study the biology of other viruses in the family. Conley et al. (14) demonstrated that upon the receptor engagement to the capsid, FCV VP2 forms a portal-like assembly, which might provide a channel for RNA release. However, the process of calicivirus RNA release is not yet fully understood. Our findings suggest that the separation of the FCV capsid from its genome RNA (gRNA) occurs rapidly in the early endosomes of infected cells. Using a liposome model decorated with the FCV cell receptor fJAM-A, we demonstrate that FCV releases its gRNA into the liposomes by penetrating membranes under low pH conditions. Furthermore, we found that VP2, which is rich in hydrophobic residues at its N-terminus, functions as the pore-forming protein. When we substituted the VP2 N-terminal hydrophobic residues, the gRNA release efficacy of the FCV mutants decreased. In conclusion, our results suggest that in the acidic environment of early endosomes, FCV VP2 functions as the pore-forming protein to mediate gRNA release into the cytoplasm of infected cells. This provides insight into the mechanism of calicivirus genome release.IMPORTANCEResearch on the biology and pathogenicity of certain caliciviruses, such as Norovirus and Sapovirus, is hindered by the lack of easy-to-use cell culture system. Feline calicivirus (FCV), which grows effectively in cell lines, is used as a substitute. At present, there is limited understanding of the genome release mechanism in caliciviruses. Our findings suggest that FCV uses VP2 to pierce the endosome membrane for genome release and provide new insights into the calicivirus gRNA release mechanism.

Metavirome Analysis Reveals a High Prevalence of Porcine Hemagglutination Encephalomyelitis Virus in Clinically Healthy Pigs in China.

Six swine coronaviruses (SCoVs), which include porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine hemagglutination encephalomyelitis virus (PHEV), porcine respiratory coronavirus (PRCV), swine acute diarrhea syndrome coronavirus (SADS-CoV), and porcine delta coronavirus (PDCoV), have been reported as infecting and causing serious diseases in pigs. To investigate the genetic diversity and spatial distribution of SCoVs in clinically healthy pigs in China, we collected 6400 nasal swabs and 1245 serum samples from clinically healthy pigs at slaughterhouses in 13 provinces in 2017 and pooled them into 17 libraries by type and region for next-generation sequencing (NGS) and metavirome analyses. In total, we identified five species of SCoVs, including PEDV, PDCoV, PHEV, PRCV, and TGEV. Strikingly, PHEV was detected from all the samples in high abundance and its genome sequences accounted for 75.28% of all coronaviruses, while those belonging to TGEV (including PRCV), PEDV, and PDCoV were 20.4%, 2.66%, and 2.37%, respectively. The phylogenetic analysis showed that two lineages of PHEV have been circulating in pig populations in China. We also recognized two PRCVs which lack 672 nucleotides at the N-terminus of the S gene compared with that of TGEV. Together, we disclose preliminarily the genetic diversities of SCoVs in clinically healthy pigs in China and provide new insights into two SCoVs, PHEV and PRCV, that have been somewhat overlooked in previous studies in China.

Identification of MYH9 Key Domain Involved in the Entry of PRRSV Into Permissive Cells.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen that causes huge losses economically to the pig industry worldwide. Previous research suggested that receptor dependence is necessary for PRRSV infection. MYH9 and CD163 are indispensable for PRRSV entry into a porcine alveolar macrophage. In the present study, human MYH9 (hMYH9) and mouse MYH9 (mMYH9), similar to swine MYH9, could also accelerate PRRSV infection in pCD163-mediated cell lines. Knockdown of MYH9 activity using the specific small interfering RNA or inhibitor (blebbistatin) concomitantly decreased PRRSV infection. C-terminal fragment of MYH9 (PRA) proteins from different mammalian species contains a conserved binding domain (aa1676-1791) for PRRSV binding, since the recombinant MYH91676-1791protein could inhibit the PRRSV infection significantly. Furthermore, the specific polyclonal antibody of MYH91676-1791 could block PRRSV infection in host cells. These data strongly supported that MYH9, a very important cofactor, participated in PRRSV entry into target cells, which may facilitate the development of a new therapeutic agent to control PRRSV infection.

Structural Insights into Alphavirus Assembly Revealed by the Cryo-EM Structure of Getah Virus.

Getah virus (GETV) is a member of the alphavirus genus, and it infects a variety of animal species, including horses, pigs, cattle, and foxes. Human infection with this virus has also been reported. The structure of GETV has not yet been determined. In this study, we report the cryo-EM structure of GETV at a resolution of 3.5 Å. This structure reveals conformational polymorphism of the envelope glycoproteins E1 and E2 at icosahedral 3-fold and quasi-3-fold axes, which is believed to be a necessary organization in forming a curvature surface of virions. In our density map, three extra densities are identified, one of which is believed a "pocket factor"; the other two are located by domain D of E2, and they may maintain the stability of E1/E2 heterodimers. We also identify three N-glycosylations at E1 N141, E2 N200, and E2 N262, which might be associated with receptor binding and membrane fusion. The resolving of the structure of GETV provides new insights into the structure and assembly of alphaviruses and lays a basis for studying the differences of biology and pathogenicity between arthritogenic and encephalitic alphaviruses.

Recombinant MYH9 protein C-terminal domain blocks porcine reproductive and respiratory syndrome virus internalization by direct interaction with viral glycoprotein 5.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important infectious diseases impacting the swine industry worldwide. Prevention and control of PRRS have been problematic, as vaccination has achieved little success. MYH9 (encoded by the gene MYH9) is an essential cellular factor for PRRS virus (PRRSV) infection. The MYH9 C-terminal domain (designated PRA) interacts with viral glycoprotein 5 (GP5), a major PRRSV envelope protein. In this study, we investigated whether soluble PRA could serve as a novel blocking agent of PRRSV infection. Our data showed that preincubation of PRRSV with PRA inhibited virus infection of susceptible cells in a dose-dependent manner. Notably, PRA also exhibited broad-spectrum ability to inhibit infection with diverse strains of both PRRSV genotype 1 and 2. Analysis of the interaction between PRA and PRRSV GP5 revealed that PRA is able to capture PRRSV virions. In conclusion, our data suggest that PRA could serve as a novel broad-spectrum inhibitor of infection by heterogeneous PRRSV strains in vivo.

Improved Vaccine against PRRSV: Current Progress and Future Perspective.

Porcine reproductive and respiratory syndrome virus (PRRSV), one of the most economically significant pathogens worldwide, has caused numerous outbreaks during the past 30 years. PRRSV infection causes reproductive failure in sows and respiratory disease in growing and finishing pigs, leading to huge economic losses for the swine industry. This impact has become even more significant with the recent emergence of highly pathogenic PRRSV strains from China, further exacerbating global food security. Since new PRRSV variants are constantly emerging from outbreaks, current strategies for controlling PRRSV have been largely inadequate, even though our understanding of PRRSV virology, evolution and host immune response has been rapidly expanding. Meanwhile, practical experience has revealed numerous safety and efficacy concerns for currently licensed vaccines, such as shedding of modified live virus (MLV), reversion to virulence, recombination between field strains and MLV and failure to elicit protective immunity against heterogeneous virus. Therefore, an effective vaccine against PRRSV infection is urgently needed. Here, we systematically review recent advances in PRRSV vaccine development. Antigenic variations resulting from PRRSV evolution, identification of neutralizing epitopes for heterogeneous isolates, broad neutralizing antibodies against PRRSV, chimeric virus generated by reverse genetics, and novel PRRSV strains with interferon-inducing phenotype will be discussed in detail. Moreover, techniques that could potentially transform current MLV vaccines into a superior vaccine will receive special emphasis, as will new insights for future PRRSV vaccine development. Ultimately, improved PRRSV vaccines may overcome the disadvantages of current vaccines and minimize the PRRS impact to the swine industry.