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.

MARCO Inhibits Porcine Reproductive and Respiratory Syndrome Virus Infection through Intensifying Viral GP5-Induced Apoptosis.

Studying viral glycoprotein-host membrane protein interactions contributes to the discovery of novel cell receptors or entry facilitators for viruses. Glycoprotein 5 (GP5), which is a major envelope protein of porcine reproductive and respiratory syndrome virus (PRRSV) virions, is a key target for the control of the virus. Here, the macrophage receptor with collagenous structure (MARCO), which is a member of the scavenger receptor family, was identified as one of the host interactors of GP5 through a DUALmembrane yeast two-hybrid screening. MARCO was specifically expressed on porcine alveolar macrophages (PAMs), and PRRSV infection downregulated MARCO expression both in vitro and in vivo. MARCO was not involved in viral adsorption and internalization processes, indicating that MARCO may not be a PRRSV-entry facilitator. Contrarily, MARCO served as a host restriction factor for PRRSV. The knockdown of MARCO in PAMs enhanced PRRSV proliferation, whereas overexpression suppressed viral proliferation. The N-terminal cytoplasmic region of MARCO was responsible for its inhibitory effect on PRRSV. Further, we found that MARCO was a proapoptotic factor in PRRSV-infected PAMs. MARCO knockdown weakened virus-induced apoptosis, whereas overexpression aggravated apoptosis. MARCO aggravated GP5-induced apoptosis, which may result in its proapoptotic function in PAMs. The interaction between MARCO and GP5 may contribute to the intensified apoptosis induced by GP5. Additionally, the inhibition of apoptosis during PRRSV infection weakened the antiviral function of MARCO, suggesting that MARCO inhibits PRRSV through the regulation of apoptosis. Taken together, the results of this study reveal a novel antiviral mechanism of MARCO and suggest a molecular basis for the potential development of therapeutics against PRRSV. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) has been one of the most serious threats to the global swine industry. Glycoprotein 5 (GP5) exposed on the surface of PRRSV virions is a major glycoprotein, and it is involved in viral entry into host cells. A macrophage receptor with collagenous structure (MARCO), which is a member of the scavenger receptor family, was identified to interact with PRRSV GP5 in a DUALmembrane yeast two-hybrid screening. Further investigation demonstrated that MARCO may not serve as a potential receptor to mediate PRRSV entry. Instead, MARCO was a host restriction factor for the virus, and the N-terminal cytoplasmic region of MARCO was responsible for its anti-PRRSV effect. Mechanistically, MARCO inhibited PRRSV infection through intensifying virus-induced apoptosis in PAMs. The interaction between MARCO and GP5 may contribute to GP5-induced apoptosis. Our work reveals a novel antiviral mechanism of MARCO and advances the development of control strategies for the virus.

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.

Clonal Spread of Escherichia coli ST93 Carrying mcr-1-Harboring IncN1-IncHI2/ST3 Plasmid Among Companion Animals, China.

The purpose of this study was to investigate the occurrence of plasmid-mediated colistin resistance gene mcr-1 in Enterobacteriaceae isolates from companion animals in Guangzhou, China. Enterobacteriaceae isolated from 180 samples collected from cats and dogs were screened for mcr-1 by PCR and sequencing. MCR-1-producing isolates were further characterized by multilocus sequence typing and pulsed-field gel electrophoresis (PFGE). Plasmid characterization was performed by conjugation, replicon typing, S1-PFGE, and Southern blot hybridization. Plasmid pHN6DS2 as a representative IncN1-IncHI2/ST3 plasmid from ST93 E. coli was fully sequenced. pHN6DS2-like plasmids were screened by PCR-mapping and sequencing. The mcr-1 gene was detected in 6.25% (8/128) Escherichia coli isolates, of which, five belonged to E. coli ST93 and had identical PFGE patterns, resistance profiles and resistance genes. mcr-1 genes were located on ∼244.4 kb plasmids (n = 6), ∼70 kb plasmids, and ∼60 kb plasmids, respectively. Among them, five mcr-1-carrying plasmids were successfully transferred to recipient by conjugation experiments, and were classified as IncN1-IncHI2/ST3 (∼244.4 kb, n = 4, all obtained from E. coli ST93), and IncI2 (∼70 kb, n = 1), respectively. Plasmid pHN6DS2 contained a typical IncHI2-type backbone, with IncN1 segment (ΔrepA-Iterons I-gshB-ΔIS1294) inserted into the multiresistance region, and was similar to other mcr-1-carrying IncHI2/ST3 plasmids from Enterobacteriaceae isolates of various origins in China. The remaining five mcr-1-bearing plasmids with sizes of ∼244.4 kb were identified to be pHN6DS2-like plasmids. In conclusion, clonal spread of ST93 E. coli isolates was occurred in companion animals in Guangzhou, China.