Genetic Variation And Pathogenicity Analysis Of PRRSV NADC30 Strain HNLY1707 At Different Passage Levels

Porcine reproductive and respiratory syndrome（PRRS）is a porcine infectious disease caused by porcine reproductive and respiratory syndrome virus（PRRSV）,which is characterized by sow reproductive disorders and respiratory diseases of growing pigs.Since2012,strains with high homology of NADC30 gene from the United States have been isolated in China.Due to the close genetic evolution relationship with NADC30 strain,these viruses are now collectively referred to as NADC30-like strain in China.Since 2014,the PRRSV NADC30-like strain has shown an intensified trend in China,and the epidemic situation involves a wide range of regions.Studies have shown that existing commercial blue ear vaccines have limited protection against NADC30-like strains,so it is urgent to develop a vaccine with stronger protection against NADC30-like strains.In order to analyze the pathogenicity of a previously isolated PRRSV NADC30-like strain HNLY1707 isolated by our laboratory,thirty healthy piglets（with double negative PRRSV）at four weeks of age were randomly divided into three groups and infected with nasal drip at a dose of 2×10⁵TCID₅₀ for twenty-one days.The results showed that the pigs infected with HNLY1707 strain showed clinical symptoms of increased body temperature,depression,dyspnea,runny nose and decreased daily weight gain.The pulmonary edema and consolidation,infiltration of inflammatory cells around the lung bronchi,detachment of bronchial color membrane epithelium,and intravascular congestion,but compared with the JXA1group,the piglets infected HNLY1707 strain were relatively late and mild symptom.Moreover,all the experimental pigs in the JXA1 group died on the 10th day after challenge,but no death occurred in the HNLY1707 group.It is indicated that HNLY1707 strain has a certain pathogenicity to piglets,and it will not cause piglets’death under the premise of the challenge dose and infected method.In addition,the standard of HNLY1707-infected pigs was:the body temperature was above 40℃for at least three days;Mental appetite loss,cough,wheeze and other respiratory symptoms;There was spot consolidation in the gross anatomy of the lung.Histopathological changes of interstitial pneumonia.One of the above three can be determined as sick pigs.In this study,the laboratory-isolated NADC30-like strain HNLY1707 was continuously propagated to one-hundred passages on Marc-145 cells,and the whole genome sequencing and plaque purification were conducted every ten passages.The sequence of parent strain HNLY1707 and its different passage were compared,and the nucleotide sequence of HNLY1707 strain had different degrees of variation during the passage.After one-hundred generations,a total of thirty-three amino acid sites and forty synonymous nucleotides were mutated,and the mutation sites were changed and distributed in each structural protein and non-structural protein,and some sites showed the same mutation as the PRRSV vaccine strain.During the passage,the titer was measured every ten passage,and it was found that the titer increased from 10^6.5TCID₅₀/mL in the 10th passage to 10^8.5TCID₅₀/mL in the 100th passage.This indicated that the adaptability of the strain increased during continuous cell passage and its replication ability in Marc-145 cells increased.Whether the amino acid changes in the passage process are related to the virulence of the virus remains to be further verified by future scientific experiments.This study provides a certain theoretical basis for the development of an ideal PRRSV NADC30-like attenuated vaccine.