Panax Notoginseng Saponins Suppress Type 2 Porcine Reproductive and Respiratory Syndrome Virus Replication in vitro and Enhance the Immune Effect of the Live Vaccine JXA1-R in Piglets.

Porcine reproductive and respiratory syndrome virus (PRRSV) suppresses the innate immune response in the host, reducing and delaying neutralizing antibody production against PRRSV infection and promoting viral infection. Here, we aimed to assess the potential of Panax notoginseng saponins (PNS) for improving the immune response exerted upon PRRSV-2-modified live virus (MLV) vaccine administration. Thirty piglets were randomly divided into six groups. Group 1 piglets were injected with medium 0 days post vaccination (dpv). Group 2 piglets were fed PNS 0-28 dpv. Group 3 and group 4 piglets were administered the JXA1-R vaccine 0 dpv. Group 4 piglets were also fed PNS 0-28 dpv. Group 1-4 piglets were challenged intranasally with the PRRSV JXA1 strain 28 dpv. Group 5 piglets were fed with PNS without challenge. Group 6 piglets served as controls. During the experiment, the samples were collected regularly for 49 days. Compared with group 1 piglets, group 3 piglets showed significantly reduced viremia and clinical scores, and significantly increased average daily gain (ADWG). Compared with group 3 piglets, group 4 piglets showed significantly improved neutralizing antibody titers, IFN-α and IFN-ß mRNA expression, and significantly decreased viremia and viral load in the lungs and lymph nodes, but did not demonstrate any further improvement in PRRSV-specific antibody titer, rectal temperature, ADWG, or clinical scores. PNS upregulates neutralizing antibodies against PRRSV-2 and enhances the expression of IFN-α and IFN-ß, which may reduce PRRSV viremia upon PRRSV-2 MLV vaccine administration. PNS may serve as an effective immunomodulator for boosting the immune defense against PRRSV.

A Method for the Analysis of African Swine Fever by Viral Metagenomic Sequencing.

In 2018, there was an outbreak of African swine fever (ASF) in China, which spread to other provinces in the following 3 years and severely damaged China's pig industry. ASF is caused by the African swine fever virus (ASFV). Given that the genome of the African swine fever virus is very complex and whole genome information is currently inadequate, it is important to efficiently obtain virus genome sequences for genomic and epidemiological studies. The prevalent ASFV strains have low genetic variability; therefore, whole genome sequencing analysis provides a basis for the study of ASFV. We provide a method for the efficient sequencing of whole genomes, which requires only a small number of tissues. The database construction method was selected according to the genomic types of ASFV, and the whole ASFV genome was obtained through data filtering, host sequence removal, virus classification, data assembly, virus sequence identification, statistical analysis, gene prediction, and functional analysis. Our proposed method will facilitate ASFV genome sequencing and novel virus discovery.

Ser-Leu substitution at P2 position of the hemagglutinin cleavage site attenuates replication and pathogenicity of Eurasian avian-like H1N2 swine influenza viruses.

Swine influenza viruses not only constitute a potential economic problem for livestock, but also pose a substantial threat to human health. Mutation in the proteolytic cleavage site of hemagglutinin (HA) is recognized as an essential factor of tissue tropism and viral pathogenicity. However, the molecular properties of the cleavage site of Eurasian avian-like swine (EA) H1N2 virus remain largely unknown. In this study, we found a serine-leucine (Ser-Leu) substitution at the P2 position of the HA cleavage site (S328 L) in naturally occurring EA H1N2 virus. To study the effect of this substitution, we used reverse genetics to generate recombinant wild-type and mutant viruses containing a single amino acid mutation at the P2 position in A/swine/Guangdong/YJ28/2014 (YJ28) or A/swine/Guangdong/DG2/2015 (DG2) background. In vitro experiments showed that the Ser-Leu substitution at the P2 position attenuated the viral replication and HA cleavage efficiency. In vivo analyses revealed that, while all mice inoculated with r/DG2-S328 L or r/YJ28 viruses survived, the survival rates of r/DG2- and r/YJ28-L328S-inoculated animals were 20 % and 40 %, respectively. Furthermore, the Ser-Leu substitution at the P2 position attenuated the replication in nasal turbinate and lungs. In summary, this amino acid change may be useful to understand the molecular properties of the cleavage site and be valuable for vaccine development.

The R251K Substitution in Viral Protein PB2 Increases Viral Replication and Pathogenicity of Eurasian Avian-like H1N1 Swine Influenza Viruses.

The Eurasian avian-like swine (EA) H1N1 virus has affected the Chinese swine industry, and human infection cases have been reported occasionally. However, little is known about the pathogenic mechanism of EA H1N1 virus. In this study, we compared the mouse pathogenicity of A/swine/Guangdong/YJ4/2014 (YJ4) and A/swine/Guangdong/MS285/2017 (MS285) viruses, which had similar genotype to A/Hunan/42443/2015 (HuN-like). None of the mice inoculated with 106 TCID50 of YJ4 survived at 7 days post infection, while the survival rate of the MS285 group was 100%. Therefore, a series of single fragment reassortants in MS285 background and two rescued wild-type viruses were generated by using the reverse genetics method, and the pathogenicity analysis revealed that the PB2 gene contributed to the high virulence of YJ4 virus. Furthermore, there were 11 amino acid differences in PB2 between MS285 and YJ4 identified by sequence alignment, and 11 single amino acid mutant viruses were generated in the MS285 background. We found that the R251K mutation significantly increased the virulence of MS285 in mice, contributed to high polymerase activity and enhanced viral genome transcription and replication. These results indicate that PB2-R251K contributes to the virulence of the EA H1N1 virus and provide new insight into future molecular epidemiological surveillance strategies.

Ginsenoside Rg1 Suppresses Type 2 PRRSV Infection via NF-κB Signaling Pathway In Vitro, and Provides Partial Protection against HP-PRRSV in Piglet.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a huge threat to the modern pig industry, and current vaccine prevention strategies could not provide full protection against it. Therefore, exploring new anti-PRRSV strategies is urgently needed. Ginsenoside Rg1, derived from ginseng and notoginseng, is shown to exert anti-inflammatory, neuronal apoptosis-suppressing and anti-oxidant effects. Here we demonstrate Rg1-inhibited PRRSV infection both in Marc-145 cells and porcine alveolar macrophages (PAMs) in a dose-dependent manner. Rg1 treatment affected multiple steps of the PRRSV lifecycle, including virus attachment, replication and release at concentrations of 10 or 50 µM. Meanwhile, Rg1 exhibited broad inhibitory activities against Type 2 PRRSV, including highly pathogenic PRRSV (HP-PRRSV) XH-GD and JXA1, NADC-30-like strain HNLY and classical strain VR2332. Mechanistically, Rg1 reduced mRNA levels of the pro-inflammatory cytokines, including IL-1ß, IL-8, IL-6 and TNF-α, and decreased NF-κB signaling activation triggered by PRRSV infection. Furthermore, 4-week old piglets intramuscularly treated with Rg1 after being challenged with the HP-PRRSV JXA1 strain display moderate lung injury, decreased viral load in serum and tissues, and an improved survival rate. Collectively, our study provides research basis and supportive clinical data for using Ginsenoside Rg1 in PRRSV therapies in swine.