Isolation, Pathogenicity, and Comparative Phylogenetic Characteristics of an Intralineage Recombinant NADC34-Like PRRSV in China

Abstract

Porcine reproductive and respiratory syndrome (PRRS), which causes reproductive failure in sows and respiratory symptoms in piglets, poses a significant threat to the global pig industry. PRRS virus (PRRSV) variants continue to emerge and spread among pigs. NADC34-like PRRSV has been imported into China in recent years and has shown potential as an endemic strain, which is of great concern. In this study, a NADC34-like PRRSV, named HLJ13 strain, was isolated from a farm where pigs experienced respiratory symptoms and abortions. Genomic analysis revealed that the HLJ13 strain was a potential recombinant of NADC34-like and NADC30-like strains, and the restriction fragment length polymorphism of HLJ13 was a novel pattern that was not yet listed. In the PRRSV HLJ13-inoculated group, the piglets showed mild clinical symptoms, such as persistent fever, and showed histopathological lesions in the lungs, and the virus was detectable at 3 and 7 days postinoculation in anal and nasal swabs, respectively. Recombination analysis revealed that interlineage recombinant events were detected in 8 out of 27 Chinese NADC34-like PRRSVs. Phylogenetic analysis showed that Chinese NADC34-like PRRSVs were distributed in two clades of lineage 1, and Chinese NADC34-like PRRSVs showed different N-glycosylation modifications in glycoproteins, especially in GP3 and GP5. These findings shed light on the genomic characteristics and pathogenicity of the NADC34-like PRRSV in China.

Figure 1

Phylogenetic analysis of HLJ13 and other NADC34-like strains isolated in China. The phylogenetic tree was constructed based on ORF5. The red circle represents the HLJ13 strain, while the blue triangle shows other NADC34-like strains in China collected in GenBank. In the L1 phylogenetic tree, the branches of sublineage 1.5 are depicted in blue and sublineage 1.8 in yellow.

Figure 2

Recombination analysis of HLJ13 strain. (a) The similarity plots were generated by Simplot. The recombination breakpoint was determined in nt 1,561 (referenced to VR-2332 genome). Phylogenetic trees were constructed based on the two parts of 1–1,561 (b) and 1,562–15,467 (c), which were divided by the breakpoint.

Figure 3

Clinical symptoms of piglets in inoculated and control groups. (a) Rectal temperature. The daily rectal temperature was measured, and ≥40.0°C was defined as a fever. (b) Body weight gain rate. The body weight of the HLJ13-inoculated and control group was recorded at 0 and 21 dpi. ns, no significant difference. (c) Scores of clinical symptoms. (d) The ratio of thymus/body weight (g/kg) ratio. At 21 dpi, the weight of the thymus and body was determined, and the ratio of thymus/body weight (g/kg) was measured (ns, p > 0.05;  ^∗, p < 0.05;  ^∗∗, p < 0.01;  ^∗∗∗, p < 0.001;  ^∗∗∗∗, p < 0.0001).

Figure 4

Pathological lesion of thymus and lung. (a), (c), and (e) show the thymus, lung, and histological assessment of the lung in the HLJ13-inoculated group, respectively. (b), (d), and (f) Indicate thymus, lungs, and histological lesions of the lung from the control group, respectively.

Figure 5

Detection of PRRSV antibody. Serum was used to detect PRRSV N protein antibodies using the IDEXX HerdCheck ELISA kit and S/P > 0.4 as the threshold of seroconversion to positive.

Figure 6

Viremia, viral tissue distribution, and shedding. Viremia in serum (a) and viral distribution in tissues (b) of piglets were detected. (c) (d) Show the virus shedding in nasal swabs and anal swabs, respectively. The virus in the HLJ13-inoculated and control groups was detected by RT-qPCR (ns, p > 0.05;  ^∗, p < 0.05;  ^∗∗, p < 0.01;  ^∗∗∗, p < 0.001;  ^∗∗∗∗, p < 0.0001).

Figure 7

Geographical distribution and RFLP analysis of NADC34-like PRRSVs in China. (a) Geographical distribution. The number of NADC34-like PRRSV in different provinces was counted and illustrated by the shade of color. HLJ, Heilongjiang n = 14, LN, Liaoning n = 4, TJ, Tianjin n = 1, SD, Shandong n = 1, HN, Henan n = 1, JS, Jiangsu n = 1, SC, Sichuan n = 4, FJ, Fujian n = 1. (b) RFLP analysis based on ORF5 of NADC34-like PRRSVs.

Figure 8

Alignment of GP5 of NADC34-like PRRSV in China. Epitopes A, B, and C were indicated by shades of yellow. In the alignment, the residues and names of Chinese NADC34-like strains were marked with red.

Figure 9

N-glycosylation site prediction in PRRSV glycoprotein. The color indicates the probability of N-glycosylation in different positions. When the probability of prediction is >0.5, there is a potential N-glycan at that site.