Epidemiological Investigation And Pathogenicity Of Newly NADC34-like PRRSV In China

Porcine Reproductive and Respiratory Syndrome?PRRS?is caused by porcine reproductive and respiratory syndrome virus?PRRSV?,which is characterized by sow abortion,stillbirth,mummified fetuses and respiratory disorders in piglets.It has caused huge losses to the pig industry in China and around the world.Since the first PRRSV isolated in mainland China in 1996,there have emerged many new types of PRRSV.After more than 20 years of evolution,the genetic diversity of PRRSV has become more complicated.In 2017,China reported NADC34-like PRRSV for the first time,speculating that this type of strain was imported from the United States.This will further increase the complexity of China's PRRSV and challenge China's current prevention and control.In order to understand the prevalence and evolution characteristics of NADC34-like PRRSV in China,we examined 650 tissue samples collected from 16 Provinces from 2018 to 2019.Six NADC34-like PRRSV strains were detected in samples from 3 Provinces,and the complete genomes of four of these strains were sequenced.?HLJZD22-1812,HLJDZD32-1901,HLJZD30-1902,LNDZD10-1806?,HNLCL82-1811 and HNLCL58-1812 can only obtain the sequence of Nsp2 region.Combined with the earliest domestic NADC34-like PRRSV?LNWK96,LNWK130?and other strains submitted to NCBI in China after 2017,phylogenetic analysis showed that these novel PRRSV strains belong to sublineage1.5.Sequence alignment suggested that these novel strains share the same 100-aa deletion in the Nsp2 protein.Recombination analysis show that these novel strains have higher nucleotide similarity with the American strains.As the number and distribution of NADC34-like PRRSV increased in China in the past 3 years and this type of strain has many features similar to NADC30-like PRRSV,we speculated that NADC34-like PRRSV may become potential epidemic strain in China.Related reports indicate that NADC34-like PRRSV can cause a large number of sow abortion in the United States.Interestingly,under experimental conditions,NADC34-like PRRSVs demonstrate a broad range of virulence in piglets.However,the pathogenicity of NADC34-like PRRSV in China is unclear.In this study,NADC34-like PRRSV HLJDZD32-1901 was successfully isolated from PAM cells.The titer of HLJDZD32-1901 on primary PAM cells was 1×10⁴ TCID₅₀/mL determined by indirect immunofluorescence.Seven 5-week-old SPF piglets were randomly divided into 2 groups s:a challenge group?n=5?and a control group?n=2?.The piglets in the challenge group and control group were inoculated intramuscularly?2 ml?and intranasally?2 ml?with HLJDZD32-1901(1×10⁴ TCID₅₀/ml,with 4 ml per pig)and RPMI 1640,respectively.Blood was collected at 0,3,5,7,10and 14 days post infection?dpi?for viremia detection via Real-time Quantitative PCR?qPCR?.Anti-PRRSV antibodies were detected using an ELISA kit?IDEXX PRRS X3,USA?.Rectal temperature and clinical features were recorded every day.The body weights of piglets were measured at 0,7 and 14 dpi.All piglets were euthanized at 14 dpi.Animal experiments showed that the weight loss of HLJDZD32-1901-infected piglets was significantly different from that of control piglets at8-14 dpi.In addition,the challenge group had obvious histopathological lesions including interstitial pneumonia and enlarged lymph nodes,and increased viremia and viral loads in three tissues.However,piglets in the challenge group had only mild clinical symptoms,with no death or fever.The above results indicate that HLJDZD32-1901 can cause typical PRRSV infection characteristics and is moderately pathogenic to piglets.This study shows that NADC34-like PRRSV may become a potential epidemic strain in China,and HLJDZD32-1901 can cause typical PRRSV infection characteristics and and is moderately pathogenic to piglets.The above results laid the foundation for the further spread of our national defense-controlled NADC34-like PRRSV and vaccine development.