Novel mutation N588 residue in the NS1 protein of feline parvovirus greatly augments viral replication.

Feline parvovirus (FPV) infection is highly fatal in felines. NS1, which is a key nonstructural protein of FPV, can inhibit host innate immunity and promote viral replication, which is the main reason for the severe pathogenicity of FPV. However, the mechanism by which the NS1 protein disrupts host immunity and regulates viral replication is still unclear. Here, we identified an FPV M1 strain that is regulated by the NS1 protein and has more pronounced suppression of innate immunity, resulting in robust replication. We found that the neutralization titer of the FPV M1 strain was significantly lower than that of the other strains. Moreover, FPV M1 had powerful replication ability, and the FPV M1-NS1 protein had heightened efficacy in repressing interferon-stimulated genes (ISGs) expression. Subsequently, we constructed an FPV reverse genetic system, which confirmed that the N588 residue of FPV M1-NS1 protein is a key amino acid that bolsters viral proliferation. Recombinant virus containing N588 also had stronger ability to inhibit ISGs, and lower ISGs levels promoted viral replication and reduced the neutralization titer of the positive control serum. Finally, we confirmed that the difference in viral replication was abolished in type I IFN receptor knockout cell lines. In conclusion, our results demonstrate that the N588 residue of the NS1 protein is a critical amino acid that promotes viral proliferation by increasing the inhibition of ISGs expression. These insights provide a reference for studying the relationship between parvovirus-mediated inhibition of host innate immunity and viral replication while facilitating improved FPV vaccine production.IMPORTANCEFPV infection is a viral infectious disease with the highest mortality rate in felines. A universal feature of parvovirus is its ability to inhibit host innate immunity, and its ability to suppress innate immunity is mainly accomplished by the NS1 protein. In the present study, FPV was used as a viral model to explore the mechanism by which the NS1 protein inhibits innate immunity and regulates viral replication. Studies have shown that the FPV-NS1 protein containing the N588 residue strongly inhibits the expression of host ISGs, thereby increasing the viral proliferation titer. In addition, the presence of the N588 residue can increase the proliferation titer of the strain 5- to 10-fold without affecting its virulence and immunogenicity. In conclusion, our findings provide new insights and guidance for studying the mechanisms by which parvoviruses suppress innate immunity and for developing high-yielding FPV vaccines.

A potential dual protection vaccine: Recombinant feline herpesvirus-1 expressing feline parvovirus VP2 antigen.

Recently, herpesvirus viral vectors that stimulate strong humoral and cellular immunity have been demonstrated to be the most promising platforms for the development of multivalent vaccines, because they contain various nonessential genes and exhibit long-life latency characteristics. Previously, we showed that the feline herpesvirus-1 (FHV-1) mutant WH2020-ΔTK/gI/gE, which was safe for felines and provided efficacious protection against FHV-1 challenge, can be used as a vaccine vector. Moreover, previous studies have shown that the major neutralizing epitope VP2 protein of feline parvovirus (FPV) can elicit high levels of neutralizing antibodies. Therefore, to develop a bivalent vaccine against FPV and FHV-1, we first generated a novel recombinant virus by CRISPR/Cas9-mediated homologous recombination, WH2020-ΔTK/gI/gE-VP2, which expresses the VP2 protein of FPV. The growth characteristics of WH2020-ΔTK/gI/gE-VP2 were similar to those of WH2020-ΔTK/gI/gE, and WH2020-ΔTK/gI/gE-VP2 was stable for at least 30 generations in CRFK cells. As expected, we found that the felines immunized with WH2020-ΔTK/gI/gE-VP2 produced FPV-neutralizing antibody titers (27.5) above the positive cutoff (26) on day 14 after single inoculation. More importantly, recombinant WH2020-ΔTK/gI/gE-VP2 exhibited severely impaired pathogenicity in inoculated and cohabiting cats. The kittens immunized with WH2020-ΔTK/gI/gE and WH2020-ΔTK/gI/gE-VP2 produced similar levels of FHV-specific antibodies and IFN-ß. Furthermore, felines immunized with WH2020-ΔTK/gI/gE-VP2 were protected against challenge with FPV and FHV-1. These data showed that WH2020-ΔTK/gI/gE-VP2 appears to be a potentially safe, effective, and economical bivalent vaccine against FPV and FHV-1 and that WH2020-ΔTK/gI/gE can be used as a viral vector to develop feline multivalent vaccines.

Feline infectious peritonitis virus ORF7a is a virulence factor involved in inflammatory pathology in cats.

A hyperinflammatory response is a prominent feature of feline infectious peritonitis (FIP), but the mechanisms behind the feline infectious peritonitis virus (FIPV)-induced cytokine storm in the host have not been clarified. Studies have shown that coronaviruses encode accessory proteins that are involved in viral replication and associated with viral virulence, the inflammatory response and immune regulation. Here, we found that FIPV ORF7a gene plays a key role in viral infection and host proinflammatory responses. The recombinant FIPV strains lacking ORF7a (rQS-79Δ7a) exhibit low replication rates in macrophages and do not induce dramatic upregulation of inflammatory factors. Furthermore, through animal experiments, we found that the rQS-79Δ7a strain is nonpathogenic and do not cause symptoms of FIP in cats. Unexpectedly, after three vaccinations with rQS-79Δ7a strain, humoral and cellular immunity was increased and provided protection against virulent strains in cats, and the protection rate reaches 40%. Importantly, our results demonstrated that ORF7a is a key virulence factor that exacerbates FIPV infection and inflammatory responses. Besides, our findings will provide novel implications for future development of live attenuated FIPV vaccines.

Safety and immunogenicity of a TK/ gI/gE gene-deleted feline herpesvirus-1 mutant constructed via CRISPR/Cas9 in feline.

Feline herpesvirus-1 (FHV-1) is the aetiological agent of feline viral rhinotracheitis, which accounts for approximately 50 % of all viral upper respiratory diseases in cats. Commercially available modified live vaccines containing FHV-1 are generally safe and effective, but these FHV-1 vaccines retain full virulence genes and can establish latency and reactivate to cause infectious rhinotracheitis in vaccine recipients, raising safety concerns. To address this shortcoming, we constructed a novel TK/gI/gE -gene-deleted recombinant FHV-1 (WH2020-ΔTK/gI/gE) through CRISPR/Cas9-mediated homologous recombination. The growth kinetics of WH2020-ΔTK/gI/gE were slightly delayed compared to those of the parent strain WH2020. Recombinant FHV-1 had severely impaired pathogenicity in cats. Felines immunized with WH2020-ΔTK/gI/gE produced high levels of gB-specific antibodies, neutralizing antibodies and IFN-ß. Additionally, WH2020-ΔTK/gI/gE provided greater protection against challenge with FHV-1 field strain WH2020 than did the commercial modified live vaccine. After challenge, the cats vaccinated with WH2020-ΔTK/gI/gE showed significantly fewer clinical signs, pathological changes, viral shedding, and viral loads in the lung and trigeminal ganglia than those vaccinated with the commercial vaccine or unvaccinated. Our results suggest that WH2020-ΔTK/gI/gE is a promising candidate as a safer and more efficacious live FHV-1 vaccine, with a decreased risk of vaccine-related complications, and could inform the design of other herpesvirus vaccines.

Adaptive Mutation in the Main Protease Cleavage Site of Feline Coronavirus Renders the Virus More Resistant to Main Protease Inhibitors.

The rapid global emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused serious health problems, highlighting the urgent need for antiviral drugs. The viral main protease (Mpro) plays an important role in viral replication and thus remains the target of choice for the prevention or treatment of several viral diseases due to high sequence and structural conservation. Prolonged use of viral protease inhibitors can lead to the development of mutants resistant to those inhibitors and to many of the available antiviral drugs. Here, we used feline infectious peritonitis virus (FIPV) as a model to investigate its development of resistance under pressure from the Mpro inhibitor GC376. Passage of wild-type (WT) FIPV in the presence of GC376 selected for a mutation in the nsp12 region where Mpro cleaves the substrate between nsp12 and nsp13. This mutation confers up to 3-fold resistance to GC376 and nirmatrelvir, as determined by EC50 assay. In vitro biochemical and cellular experiments confirmed that FIPV adapts to the stress of GC376 by mutating the nsp12 and nsp13 hydrolysis site to facilitate cleavage by Mpro and release to mediate replication and transcription. Finally, we demonstrate that GC376 cannot treat FIP-resistant mutants that cause FIP in animals. Taken together, these results suggest that Mpro affects the replication of coronaviruses (CoVs) and the drug resistance to GC376 by regulating the amount of RdRp from a distant site. These findings provide further support for the use of an antiviral drug combination as a broad-spectrum therapy to protect against contemporary and emerging CoVs. IMPORTANCE CoVs cause serious human infections, and antiviral drugs are currently approved to treat these infections. The development of protease-targeting therapeutics for CoV infection is hindered by resistance mutations. Therefore, we should pay attention to its resistance to antiviral drugs. Here, we identified possible mutations that lead to relapse after clinical treatment of FIP. One amino acid substitution in the nsp12 polymerase at the Mpro cleavage site provided low-level resistance to GC376 after selection exposure to the GC376 parental nucleoside. Resistance mutations enhanced FIPV viral fitness in vitro and attenuated the therapeutic effect of GC376 in an animal model of FIPV infection. Our research explains the evolutionary characteristics of coronaviruses under antiviral drugs, which is helpful for a more comprehensive understanding of the molecular basis of virus resistance and provides important basic data for the effective prevention and control of CoVs.