Epidemiological Investigation And Screening Of Antigenic Epitopes Of NADC34-like PRRSV In China

Porcine reproductive and respiratory syndrome（PRRS）is a severe infectious disease caused by porcine reproductive and respiratory syndrome virus（PRRSV）with respiratory disorders of piglets and reproductive disorders of sows as the main clinical symptoms.PRRSV was isolated for the first time in 1996,which caused enormous economic losses to the pig industry in our country.The RFLP 1-7-4 strain（NADC34-like PRRSV）pandemic event was reported in the US in 2012 and was described as an"abortion storm".In 2020,RFLP 1-4-4 Lineage 1C PRRSV was reported in the US.NADC34-like PRRSV virus was first detected in China in 2017 and was considered a potential epidemic strain in China in 2019.Understanding the latest epidemic situation of NADC34-like PRRSV strain in our country and identifying its pathogenicity is of great significance.Antigenic epitopes have always been the focus of PRRSV research.The identification of antigenic epitopes of the latest circulating strains in China can provide the basis for preparation of antibody kits and DIVA vaccine.In this study,828 clinical samples suspected to be infected with PRRSV from 14 provinces,municipalities and autonomous regions from 2020 to 2021 were detected by RT-PCR.The results showed that 433 samples were PRRSV positive,with a positive rate of 52.29%.There were 82NADC34-like PRRSV samples.In recent years,NADC34-like PRRSV has gradually spread.In 2021,the proportion of positive NADC34-like PRRSV has reached 28.6%,and NSDC34-like PRRSV has become one of the main circulating strains in some parts of China.Genetic evolution analysis showed that all NADC34-like PRRSV belonged to sublineage 1.5 when constructing phylogenetic tree based on ORF5.In order to better understand the relationship between Chinese newly NADC34-like PRRSV and global sublineage 1.5 strains,ORF5 sequences of all global sublineage 1.5 strains were collected as reference strains to construct a phylogenetic tree.NADC34-like PRRSV prevalent in China did not form independent branches,but was embedded in sublineage 1.5 strain in the United States.Therefore,NAD34-like PRRSV prevalent in China at the present stage not only came from the spread of earlier strains,but also may have sustained input and independent evolution.In this study,15 NADC34-like PRRSV-positive samples were selected for further whole genome sequencing.Based on the genetic evolution analysis of the whole genome sequence,it was found that the newly NADC34-like PRRSV belonged to sublineage 1.5 or sublineage 1.8,which indicated the emergence of a new recombinant strain.The results of recombination analysis showed that 6 of the 15 strains with complete genome sequences had recombination,and the recombination pattern was complex and no obvious recombination hot spots were found.BLAST analysis found that NADC34-like PRRSV had recombined with the locally prevalent HP-PRRSV-like and NADC30-like PRRSV in China,and the time of emergence of new recombinant strains coincided with the rapid increase in the proportion of NADC34-like PRRSV.Therefore,the emergence of new recombinant strains may be the cause of the rapid spread of NADC34-like PRRSV.Deduced amino acid comparison showed that most new NADC34-like PRRSV had 100 aa continuous deletion consistent with IA/2014/NADC34 in the Nsp2 region,and some strains had the same deletion pattern as NADC30,which was consistent with the results of recombinant analysis.Moreover,the novel NADC34-like PRRSV has a large number of amino acid mutations in the GP5 linear antigen region.Homology comparison of unrecombinant NADC34-like PRRSV showed that the genomic homology of this strain decreased,and NADC34-like PRRSV was rapidly mutating.In October 2020 and April of the following year,RFLP 1-4-4 Lineage 1C PRRSV variant caused two widespread PRRSV epidemics,which seriously harmed the local pig industry.In order to understand the relationship between the PRRSV strain and the current PRRSV strain in China,sequence analysis showed that the American RFLP 1-4-4 lineage1C PRRSV variant belonged to the same subtype clade（sublineage 1.5）as the Chinese NADC34-like PRRSV.They all have the same molecular characteristics of 100 amino acid deletion in the Nsp2 region.The 1-4-4 lineage1C PRRSV variant in the United States was a recombinant virus with NADC34-like strain IA14737-2016 as its major parent strain,and IA/2014/NADC34 and NADC30 strains providing recombinant fragments.It was also found that HLJZD22-1812 NADC34-like strain from China and 1-4-4lineage1C PRRSV variant from the United States had similar recombination pattern,the same Nsp2deletion feature and the same 1-4-4 ORF5 RFLP pattern.But the homology is less than 90%,which is not consistent with the high homology within the lineage described by American practitioners.So,they did not evolve from the same strain.As a result,1-4-4 lineage1C PRRSV variant has not been found in our country so far.However,it is very important to be alert to the input of RFLP 1-4-4Lineage 1C PRRSV variant so as to avoid losses caused by this strain to our pig breeding industry.The prevalent NADC34-like PRRSV in the United States has a very complex pathogenicity.Therefore,it is necessary to determine the pathogenicity of the newly NADC34-like PRRSV in China and prepare the PRRSV positive serum required for subsequent experiments.In this study,15NADC34-like PRRSV-positive samples with whole genome sequence were isolated and LNTZJ1341-2012 was successfully isolated from Marc-145 and PAM cells.In this study,eight 2-month-old SPF piglets were selected and randomly divided into two groups,including 5 in the challenge group and 3 in the control group.The third generation of PAM strain LNTZJ1341-2012was selected,and 4 ml of virus culture supernatant was injected into the nose（2 ml）and neck（2 ml）with 1×10⁵ TCID₅₀/ml,respectively.Control piglets were injected with 1640 medium in the same way.The clinical symptoms and temperature were observed daily during the experiment.Blood samples were collected at 0 d,3 d,5 d,7 d,10 d,14 d,17 d and 21 d after the challenge,and the average daily weight gain was recorded at 7 d,14 d and 21 d.All piglets were euthanized at the end of the 21-day experiment,and 2 samples of each organ tissue were collected.Used to determine tissue viral load and histopathological tests.The experimental results showed that only part of the pigs in the LNTZJ1341-2012 challenge group showed the typical clinical symptoms of PRRSV,such as loss of appetite,lethargy and cough.At the same time,some pigs（2/5）showed transient fever lasting for 2 days,serum antibodies turned positive at 7-10 dpi,and average daily weight gain showed no significant difference from the control group.All pigs in the challenge group had higher viral loads detected in the lung,submaxillary lymph nodes and tonsil,among which the tonsil had the highest viral load.Meanwhile,LNTZJ1341-2012 caused high viremia,which lasted for more than 21 days.Some pigs had a small amount of lung consolidation and hemorrhagic spots in submaxillary lymph nodes.Under microscope observation,the lungs of pigs in the challenge group showed extensive inflammatory cell infiltration with alveolar epithelial cell proliferation,moderate alveolar diaphragm widening,and local bleeding in the submaxillary lymph nodes in the paracortem region.In conclusion,LNTZJ1341-2012 strain can cause typical PRRSV characteristics to some pigs,and has weak pathogenicity to piglets.Screening of NADC34-like PRRSV epitopes is of great significance for guiding the development of antibody detection kits and the construction of DIVA vaccines.In this study,the positive serum of LNTZJ1341-2012 strain was pretreated to remove the non-specific binding between the serum and yeast cells.The ORF1a and ORF2-7 fragments of LNTZJ1341-2012 were amplified,and digested by DNase I randomly into 100-250 bp DNA fragments.Then it was connected to yeast display vector p CTCON2,and the connected product was cloned into DH5αby electric transformation,and yeast random display library was obtained.The library was of good quality and could completely cover ORF1a and ORF2～7 fragments.The extracted library plasmid was electrotransferred into EBY100 yeast receptor state and induced to express.The pre-treated LNTZJ1341-2012 positive serum was used to stain the positive yeast.Positive yeast was sorted by flow cytometry,and after two rounds of enrichment,yeast plasmid was extracted and transformed into DH5α,and monoclonal sequencing was performed.A1（Nsp1 171-191 aa）,A2（Nsp2 504-522aa）,A3（Nsp2 695-713 aa）and A4（Nsp7β0-96 aa）were screened from LNTZJ1341-2012 strain ORF1a gene fragment.B1（GP3 66-80 aa）,B2（GP3 80-100 aa）,B3（GP5 162-174 aa）and B4（M0-33 aa）were screened from LNTZJ1341-2012 strain ORF2-7 gene fragment.A total of 8 epitopes were obtained.Finally,the obtained 8 epitopes were transferred to yeast and induced to show their expression on the surface.Flow cytometry was used to verify their antigenicity.The results showed that A1,A2,A4,B1 and B2 had strong antigenicity,and had the potential to be labeled sites of DIVA vaccine and develop antibody detection kits.In conclusion,NADC34-like PRRSV has become one of the main circulating strains in some areas of China during 2020-2021.The current circulating NADC34-like strain in China not only originates from the transmission of earlier strains,but also may have continuous input and a separate evolutionary trajectory.In this study,we found for the first time that NADC34-like PRRSV has recombined with local strains in China and shows a complex recombination pattern.The American RFLP 1-4-4 lineage1C PRRSV variant belongs to the same subtype clade as Chinese NADC34-like PRRSV（sublineage 1.5）.NADC34-like PRRSV with the same characteristics has been isolated in our country,but no PRRSV strain has been found that is highly homologous to the lineage1C variant in the United States.LNTZJ1341-2012 strain can cause typical PRRSV characteristics to some pigs,and has weak pathogenicity to piglets.In this study,the ORF1a and ORF2-7 antigen library of PRRSV constructed by yeast surface display technique was displayed on the surface of yeast for the first time,and five highly antigenic antigen epitopes were screened and identified.The above contents are of great significance not only to understand the epidemic situation of PRRSV in our country at present and guide the prevention and control of PRRSV.It also laid the foundation for PRRSV antibody detection kit and DIVA vaccine development.