VP0 Myristoylation Is Essential for Senecavirus A Replication.

Many picornaviruses require the myristoylation of capsid proteins for viral replication. Myristoylation is a site-specific lipidation to the N-terminal G residue of viral proteins, which is catalyzed by the ubiquitous eukaryotic enzyme N-myristoyltransferase (NMT) by allocating the myristoyl group to the N-terminal G residue. IMP-1088 and DDD85646 are two inhibitors that can deprive NMT biological functions. Whether Senecavirus A (SVA) uses NMT to modify VP0 and regulate viral replication remains unclear. Here, we found that NMT inhibitors could inhibit SVA replication. NMT1 knock-out in BHK-21 cells significantly suppressed viral replication. In contrast, the overexpression of NMT1 in BHK-21 cells benefited viral replication. These results indicated that VP0 is a potential NMT1 substrate. Moreover, we found that the myristoylation of SVA VP0 was correlated to the subcellular distribution of this protein in the cytoplasm. Further, we evaluated which residues at the N-terminus of VP0 are essential for viral replication. The substitution of N-terminal G residue, the myristoylation site of VP0, produced a nonviable virus. The T residue at the fifth position of the substrates facilitates the binding of the substrates to NMT. And our results showed that the T residue at the fifth position of VP0 played a positive role in SVA replication. Taken together, we demonstrated that SVA VP0 myristoylation plays an essential role in SVA replication.

Recombinant lentogenic Newcastle disease virus expressing Ebola virus GP infects cells independently of exogenous trypsin and uses macropinocytosis as the major pathway for cell entry.

BACKGROUND: Using reverse genetics, we generated a recombinant low-pathogenic LaSota strain Newcastle disease virus (NDV) expressing the glycoprotein (GP) of Ebola virus (EBOV), designated rLa-EBOVGP, and evaluated its biological characteristic in vivo and in vitro. RESULTS: The introduction and expression of the EBOV GP gene did not increase the virulence of the NDV vector in poultry or mice. EBOV GP was incorporated into the particle of the vector virus and the recombinant virus rLa-EBOVGP infected cells and spread within them independently of exogenous trypsin. rLa-EBOVGP is more resistant to NDV antiserum than the vector NDV and is moderately sensitive to EBOV GP antiserum. More importantly, infection with rLa-EBOVGP was markedly inhibited by IPA3, indicating that rLa-EBOVGP uses macropinocytosis as the major internalization pathway for cell entry. CONCLUSIONS: The results demonstrate that EBOV GP in recombinant NDV particles functions independently to mediate the viral infection of the host cells and alters the cell-entry pathway.

Identification of avian polyomavirus and its pathogenicity to SPF chickens.

The research aimed to study an Avian polyomavirus strain that was isolated in Shandong, China. To study the pathogenicity of APV in SPF chickens, and provide references for epidemiological research and disease prevention and control of APV. The genetic characterization of APV strain (termed APV-20) was analyzed and the pathogenicity of APV was investigated from two aspects: different age SPF chickens, and different infection doses. The results revealed that the APV-20 exhibits a nucleotide homology of 99% with the other three APV strains, and the evolution of APV In China was slow. In addition, the APV-20 infection in chickens caused depression, drowsiness, clustering, and fluffy feathers, but no deaths occurred in the infected chickens. The main manifestations of necropsy, and Hematoxylin and Eosin staining (HE) showed that one-day-old SPF chickens were the most susceptible, and there was a positive correlation between viral load and infection dose in the same tissue. This study showed that SPF chickens were susceptible to APV, and an experimental animal model was established. This study can provide a reference for the pathogenic mechanism of immune prevention and control of APV.

Engineering His-Tagged Senecavirus A for One-Step Purification of Viral Antigens.

Senecavirus A (SVA) is a picornavirus that causes vesicular disease in swine, and the inactivated vaccine is used to prevent and control SVA infection. To develop a new chromatography strategy for the purification and concentration of SVA vaccine antigens, we inserted a 6×His-tag at the VP1 C-terminal of the SVA/HLJ/CHA/2016 in an infectious clone to rescue a His-tagged SVA. The constructed and rescued recombinant virus, named as rSVA-His, exhibited similar growth kinetics to that of its parental virus. In addition, the expression of a 6×His-tag on the surface of SVA showed genetic stability in cell passages in vitro, which allowed one-step purification of SVA antigens by Ni2+ affinity columns. Furthermore, the immunogenicity of the inactivated rSVA-His was evaluated by inoculating rabbits and detecting neutralizing antibodies. The animals receiving two doses of the inactivated rSVA-His emulsified with oil adjuvant developed a high titer of neutralizing antibodies, indicating that SVA VP1 is tolerant to His-tag insertion without detriment to its antigenicity. In summary, the constructed 6×His-tagged SVA may offer a feasible approach to the affinity purification and concentration of antigens in the process of SVA inactivated vaccine production.

Molecular basis of neurovirulence of flury rabies virus vaccine strains: importance of the polymerase and the glycoprotein R333Q mutation.

The molecular mechanisms associated with rabies virus (RV) virulence are not fully understood. In this study, the RV Flury low-egg-passage (LEP) and high-egg-passage (HEP) strains were used as models to explore the attenuation mechanism of RV. The results of our studies confirmed that the R333Q mutation in the glycoprotein (G(R333Q)) is crucial for the attenuation of Flury RV in mice. The R333Q mutation is stably maintained in the HEP genome background but not in the LEP genome background during replication in mouse brain tissue or cell culture. Further investigation using chimeric viruses revealed that the polymerase L gene determines the genetic stability of the G(R333Q) mutation during replication. Moreover, a recombinant RV containing the LEP G protein with the R333Q mutation and the HEP L gene showed significant attenuation, genetic stability, enhancement of apoptosis, and immunogenicity. These results indicate that attenuation of the RV Flury strain results from the coevolution of G and L elements and provide important information for the generation of safer and more effective modified live rabies vaccine.

Generation of a recombinant rabies Flury LEP virus carrying an additional G gene creates an improved seed virus for inactivated vaccine production.

The rabies Flury Low Egg Passage virus (LEP) has been widely used as a seed virus to generate inactive vaccine. Here, we established a reverse genetic system for LEP and generated a recombinant LEP virus (rLEP-G) that carries two identical G genes. This recombinant virus showed similar properties to those of LEP with respect to in vitro growth, neurotropism index, and virulence in mice. rLEP-G produced 4.3-fold more G protein than did LEP in BHK-21 cells. The inactivated vaccine generated from rLEP-G induced significantly higher virus neutralization titers in mice and dogs than those produced in response to LEP-derived vaccine. Our results suggest that rLEP-G is an improved seed virus candidate for inactivated rabies virus vaccine manufacture.

Newcastle disease virus-vectored Nipah encephalitis vaccines induce B and T cell responses in mice and long-lasting neutralizing antibodies in pigs.

Nipah virus (NiV), a member of the Paramyxoviridae family, causes deadly encephalitis in humans and huge economic losses to the pig industry. Here, we generated recombinant avirulent Newcastle disease virus (NDV) LaSota strains expressing the NiV G and F proteins respectively (designated as rLa-NiVG and rLa-NiVF), and evaluated their immunogenicity in mice and pigs. Both rLa-NiVG and rLa-NiVF displayed growth properties similar to those of LaSota virus in chicken eggs. Co-infection of rLa-NiVG and rLa-NiVF caused marked syncytia formation, while intracerebral co-inoculation of these viruses in mice showed they were safe in at least one mammalian species. Animal immunization studies showed rLa-NiVG and rLa-NiVF induced NiV neutralizing antibody responses in mice and pigs, and F protein-specific CD8+ T cell responses in mice. Most importantly, rLa-NiVG and rLa-NiVF administered alone or together, induced a long-lasting neutralizing antibody response in pigs. Recombinant rLa-NiVG/F thus appear to be promising NiV vaccine candidates for pigs and potentially humans.