An Expeditious Neutralization Assay for Porcine Reproductive and Respiratory Syndrome Virus Based on a Recombinant Virus Expressing Green Fluorescent Protein.

Due to the extensive genetic and antigenic variation in Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), as well as its rapid mutability and evolution, PRRS prevention and control can be challenging. An expeditious and sensitive neutralization assay for PRRSV is presented to monitor neutralizing antibodies (NAbs) in serum during vaccine research. Here, a PRRSV expressing eGFP was successfully rescued with reverse genetics based on the infectious clone HuN4-F112-eGFP which we constructed. The fluorescent protein expressions of the reporter viruses remained stable for at least five passages. Based on this reporter virus, the neutralization assay can be easily used to evaluate the level of NAbs by counting cells with green fluorescence. Compared with the classical CPE assay, the newly developed assay increases sensitivity by one- to four-fold at the early antibody response stage, thus saving 2 days of assay waiting time. By using this assay to unveil the dynamics of neutralizing antibodies against PRRSV, priming immunity through either a single virulent challenge or only vaccination could produce limited NAbs, but re-infection with PRRSV would induce a faster and stronger NAb response. Overall, the novel HuN4-F112-eGFP-based neutralization assay holds the potential to provide a highly efficient platform for evaluating the next generation of PRRS vaccines.

Potential Threats to Human Health from Eurasian Avian-Like Swine Influenza A(H1N1) Virus and Its Reassortants.

During 2018-2020, we isolated 32 Eurasian avian-like swine influenza A(H1N1) viruses and their reassortant viruses from pigs in China. Genomic testing identified a novel reassortant H3N1 virus, which emerged in late 2020. Derived from G4 Eurasian H1N1 and H3N2 swine influenza viruses. This virus poses a risk for zoonotic infection.

Recombination between NADC34-like and QYYZ-like strain of porcine reproductive and respiratory syndrome virus with high pathogenicity for piglets in China.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen that causes huge economic losses to the swine industry worldwide. Here, a novel variant of PRRSV strain named TJnh2021 was isolated from nursery piglets with morbidity rate (75%) and mortality rate (40%) in Tianjin Province of China in 2021. Phylogenetic and molecular evolutionary analyses revealed that TJnh2021 was highly similar to NADC34-like (lineage 1.5, isolated in North America in 2014) in the ORF1ab-ORF2 and ORF6-ORF7 coding regions, as well as to QYYZ-like (lineage 3, isolated in China in 2010) in the ORF3-ORF5, suggestive of a natural recombination event. Recombination analyses revealed that recombination events occurred in two interlineage recombination events between lineages 1.5 and 3, and two breakpoints in ORF2 (nt12196) and ORF5 (nt13628) (with reference to the VR-2332 strain). Animal experiments demonstrated that TJnh2021 caused mortality rates of 40% and exhibited higher pathogenicity in piglets compared to other lineage 1.5 strains reported in China. Taken altogether, NADC34-like PRRSV has undergone genetic exchange with Chinese local PRRSV strains and recombination might be responsible for the variations in pathogenicity and highlight the importance of surveillance of this lineage in China.

Immune efficacy of a candidate porcine reproductive and respiratory syndrome vaccine rHN-NP49 administered by a Needle-free intradermal delivery system in comparison with intramuscular injection.

Porcine reproductive and respiratory syndrome (PRRS) is one of the major drivers of economic loss in the swine industry worldwide. In commercial pig production, vaccination is the first option in an attempt to control infectious diseases. Pigs are therefore often immunized with different vaccines, and almost all of them are delivered via the intramuscular (IM) route. However, the IM injection may result in physical damage, stress reactions, and is labor demanding. An alternative route is urgently needed to reduce the disadvantages of conventional vaccination. In this study, a needle-free intradermal (ID) delivery system was evaluated for delivering a live PRRS vaccine as compared with the traditional needle-syringe method. Fifty-two 4-week-old piglets were divided into six groups: piglets in groups A-C were immunized using ID delivery system with 104, 105 and 106 TCID50 of PRRS candidate vaccine strain rHN-NP49, respectively; piglets in group D were immunized IM with 105 TCID50 of rHN-NP49; and group E and F were used as challenge and control groups, respectively. At 28 days post vaccination, piglets in group A to E were challenged with a lethal dose of highly-pathogenic PRRSV. Similar results were found in viremia and antibody response among the ID and IM groups during the immunization stage. After challenge, similar results were found in average body weight gain, viral shedding, serum viral load, and clinical score among the immunization groups, with a higher protection ratio in the ID group compared with IM group with the same immunization dose. These results demonstrated that the ID delivery system could provide similar or even better protection compared with IM route, and could be an effective route for PRRS vaccination.

The role of PA-X C-terminal 20 residues of classical swine influenza virus in its replication and pathogenicity.

PA-X is a fusion protein encoded by a +1 frameshifted open reading frame (X-ORF) in PA gene. The X-ORF can be translated in full-length (61 amino acids, aa) or truncated (41 aa) form. However, the role of C-Terminal 20 aa of PA-X in virus function has not yet been fully elucidated. To this end, we constructed the contemporary influenza viruses with full and truncated PA-X by reverse genetics to compare their replication and pathogenicity. The full-length PA-X virus in MDCK and human A549 cells conferred 10- to 100-fold increase in viral replication, and more virulent and caused more severe inflammatory responses in mice relative to corresponding truncated PA-X virus, suggesting that the terminal 20 aa could play a role in enhancing viral replication and contribute to virulence.

An epidemiological investigation of porcine circovirus type 2 and porcine circovirus type 3 infections in Tianjin, North China.

Novel porcine circovirus type 3 (PCV3), first identified in the United States, has been detected in many other countries. Porcine circovirus is associated with postweaning multisystemic wasting syndrome, reproductive failure, congenital tremors, and other clinical symptoms. In this study, we established a double polymerase chain reaction assay for detecting both porcine circovirus type 2 (PCV2) and PCV3. This is the first study to detect and characterize the PCV3 genome in the Tianjin region of North China. We collected a total of 169 tissue samples from seven farms between 2016 and 2018. The PCV3-positive rate of all tissue samples was 37.3% (63/169) and the rate of PCV2 and PCV3 coinfection was 14.8% (25/169). PCV2 and PCV3 coinfections with more serious clinical symptoms were found in only three farms. We sequenced three PCV3 strains selected from tissue samples that were positively identified. The complete genome sequences of the three strains shared 97.6-99.4% nucleotide identities with the PCV3 strains in GenBank. Our results showed the extent of PCV3's spread in Tianjin, and the need to further study PCV3's pathobiology, epidemiology, isolation, and coinfection.

Protective efficacy of a bivalent inactivated reassortant H1N1 influenza virus vaccine against European avian-like and classical swine influenza H1N1 viruses in mice.

The classical swine (CS) H1N1 swine influenza virus (SIVs) emerged in humans as a reassortant virus that caused the H1N1 influenza virus pandemic in 2009, and the European avian-like (EA) H1N1 SIVs has caused several human infections in European and Asian countries. Development of the influenza vaccines that could provide effective protective efficacy against SIVs remains a challenge. In this study, the bivalent reassortant inactivated vaccine comprised of SH1/PR8 and G11/PR8 arboring the hemagglutinin (HA) and neuraminidase (NA) genes from prevalent CS and EA H1N1 SIVs and six internal genes from the A/Puerto Rico/8/34(PR8) virus was developed. The protective efficacy of this bivalent vaccine was evaluated in mice challenged with the lethal doses of CS and EA H1N1 SIVs. The result showed that univalent inactivated vaccine elicited high-level antibody against homologous H1N1 viruses while cross-reactive antibody responses to heterologous H1N1 viruses were not fully effective. In a mouse model, the bivalent inactivated vaccine conferred complete protection against lethal challenge doses of EA SH1 virus or CS G11 virus, whereas the univalent inactivated vaccine only produced insufficient protection against heterologous SIVs. In conclusion, our data demonstrated that the reassortant bivalent inactivated vaccine comprised of SH1/PR8 and G11/PR8 could provide effective protection against the prevalent EA and CS H1N1 subtype SIVs in mice.

Protective efficacy of a high-growth reassortant H1N1 influenza virus vaccine against the European Avian-like H1N1 swine influenza virus in mice and pigs.

Swine influenza A viruses (SIVs) causing outbreaks of acute, highly contagious respiratory disease in pigs also pose a potential threat to public health. European avian-like H1N1 (EA H1N1) SIVs are the predominant circulating viruses in pigs in China and also occasionally cause human infection. In this study, a high-growth reassortant virus (SH1/PR8), with HA and NA genes from a representative EA H1N1 isolate A/Swine/Shanghai/1/2014 (SH1) in China and six internal genes from the high-growth A/Puerto Rico/8/34 (PR8) virus, was generated by plasmid-based reverse genetics and tested as a candidate seed virus for the preparation of inactivated vaccine. The protective efficacy of inactivated SH1/PR8 was evaluated in mice and pigs challenged with wild-type SH1 virus. After primer and boost vaccination, the SH1/PR8 vaccine induced high-level hemagglutination inhibiting (HI) antibodies, IgG antibodies, and neutralization antibodies in mice and pigs. Mice and pigs in the vaccinated group showed less clinical phenomena and pathological changes than those in the unvaccinated group. In conclusion, the inactivated high-growth reassortant vaccine SH1/PR8 could induce high antibody levels and complete protection is expected against SH1 wild type SIV, and protection against heterologous EA H1N1 SIV needs further evaluation.

Monoclonal Antibody Against HA Protein of the European Avian-Like H1N1 Swine Influenza Virus.

The purified whole-virus proteins derived from A/swine/Shanghai/1/2014 (H1N1) (SH1) were chosen to immunize BALB/c mice to prepare the monoclonal antibody (MAb) against hemagglutinin (HA) protein of an European avian-like (EA) H1N1 swine influenza virus (SIV). After cloning three times by limiting dilution, one strain of hybridoma cells named 3C7 secreting anti-HA protein MAb was obtained by hybridoma technique. The results of indirect immunofluorescence assay and western blot analyses showed that the MAb 3C7 specifically reacted with the HA protein of EA H1N1 SIV. This work indicated that the MAb 3C7 would be a valuable tool as a specific diagnostic reagent for SIV epidemiological surveys and identification of HA protein epitopes of the EA H1N1 SIVs in the future.

A meta-analysis of transcriptomic characterization revealed extracellular matrix pathway involved in the H5N1 and H7N9 infections.

Avian-origin H5N1 and H7N9 influenza A viruses are capable of causing lethal infection in humans, with serious lung pathology and leading to acute respiratory distress syndrome. The contribution of host response associated with the poor prognosis of H5N1 and H7N9 infections remains unclear. The aim of this study was to identify the host factors involved in the high pathogenicity of H5N1 and H7N9 by a systematical meta-analysis. The RNA-seq datasets related to H5N1, H7N9, and H1N1 infections with time series were retrieved from GEO. After merging the data from different series, ComBat was used to adjust the known variances from different batches. The transcription factors binding the genes in each cluster were predicted by PASTAA. We figured out the genes that were differentially expressed at any time point in samples infected with H5N1, H7N9, or H1N1. The analysis of biological function showed that genes related with cytokine were up-regulated in all three viruses. However, genes associated with carbon metabolism were found exclusively down-regulated in H7N9 and the extracellular matrix pathway were only enriched in H5N1 and H7N9. To summary, our study suggested that the extracellular matrix might be associated with the high fatality of H5N1 and H7N9 viruses in humans.

PA-X protein decreases replication and pathogenicity of swine influenza virus in cultured cells and mouse models.

Swine influenza viruses have been circulating in pigs throughout world and might be potential threats to human health. PA-X protein is a newly discovered protein produced from the PA gene by ribosomal frameshifting and the effects of PA-X on the 1918 H1N1, the pandemic 2009 H1N1, the highly pathogenic avian H5N1 and the avian H9N2 influenza viruses have been reported. However, the role of PA-X in the pathogenesis of swine influenza virus is still unknown. In this study, we rescued the H1N1 wild-type (WT) classical swine influenza virus (A/Swine/Guangdong/1/2011 (H1N1)) and H1N1 PA-X deficient virus containing mutations at the frameshift motif, and compared their replication properties and pathogenicity of swine influenza virus in vitro and in vivo. Our results show that the expression of PA-X inhibits virus replication and polymerase activity in cultured cells and decreases virulence in mouse models. Therefore, our study demonstrates that PA-X protein acts as a negative virulence regulator for classical H1N1 swine influenza virus and decreases virulence by inhibiting viral replication and polymerase activity, deepening our understanding of the pathogenesis of swine influenza virus.