Genetic Characterization and Pathogenicity of a Novel Recombined Porcine Reproductive and Respiratory Syndrome Virus 2 among Nadc30-Like, Jxa1-Like, and Mlv-Like Strains

Abstract

Recombination among porcine reproductive and respiratory syndrome viruses (PRRSVs), coupled with point mutations, insertions, and deletions occurring in the genome, is considered to contribute to the emergence of new variants. Here, we report the complete genome sequences of a PRRSV field strain, designated SCN17, isolated from a RespPRRS MLV-vaccinated piglet in China in 2017. Sequence alignment revealed that SCN17 had discontinuous 131-amino acid (111 + 1 + 19-aa) deletion in the NSP2-coding region identical to that of NADC30 when compared to VR-2332. Notably, the strain, SCN17, contained an additional 1-aa deletion in NSP2, a 1-aa deletion in ORF5, and a unique 3-nt deletion in the 3'-UTR. Phylogenetic analysis showed that SCN17 clustered into NADC30-like lineage based on ORF5 genotyping, whereas it belonged to an inter-lineage between the NADC30-like and VR-2332-like lineages as established based on the full-length genome. Importantly, the SCN17 was identified as a novel virus recombined between a NADC30-like (moderately pathogenic), a JXA1-like (highly pathogenic), and an attenuated vaccine strain, RespPRRS MLV (parental strain VR-2332). Furthermore, we tested its pathogenicity in piglets. SCN17 infection caused a persistent fever, moderate interstitial pneumonia, and increased the viremia and antibody levels in the inoculated piglets. Of note, all SCN17-infected piglets survived throughout the study. The new virus was showed to be a moderately virulent isolate and have lower pathogenicity than HP-PRRSV strain, SCwhn09CD. Our results provide evidence for the continuing evolution of PRRSV field strain by genetic recombination and mutation leading to outbreaks in the vaccinated pig populations in China.

Keywords: modified live virus (MLV); mutation; pathogenicity; porcine reproductive and respiratory syndrome virus (PRRSV); recombination.

Figure 1

Multiple sequences alignment of NSP2, GP5, and 3′-UTR from representative PRRSV strains and SCN17 isolate. (a) Three discontinuous amino acid deletions at positions 322–432, 483, and 504–522 (yellow regions), and an additional 1-aa deletion at position 831 (green region) in NSP2-cording region are observed in SCN17. The blue regions (positions 481, 553–562) indicate the classical HP-PRRSV deletion in NSP2-cording region; (b) one amino acid deletion in GP5 at position 33 of SCN17 and SCcd17 (yellow region); (c) three continuous nucleotides deletion in 3′-UTR at positions 118 to 120 of SCN17 (yellow region). Five representative PRRSVs, including NADC30 (JN654459), JXA1 (EF112445), JXwn06 (EF641008), VR-2332 (AY150564), and CH-1a (AY032626), are included in the analysis.

Figure 2

Phylogenetic trees based on ORF5 (a) and complete genomic sequences (b) of strain SCN17 and 40 reference PRRSV strains. The new recombinant virus, SCN17, is marked with a “red triangle”. PRRSV-2 strains in China are clustered into four lineages (lineages 1, 3, 5, and 8), and the representative strains of each lineage are marked with “black squares”, while the MLV vaccines were labeled with “black circles”. The phylogenetic trees are computed using the neighbor-joining method with 1000 replicates using the MEGA6 program. Scale bar indicates nucleotide substitute per site.

Figure 3

Genome recombination analyses of the PRRSV isolate, SCN17. The y-axis indicates the percentage similarity between the query sequence (SCN17) and four representative sequences. (a) Genome scale similarity comparisons of SCN17 with NADC30 (blue), RespPRRS MLV (red), JXA1 (green), and CH-1a (yellow). The eight supposed recombination regions (regions A-H) and the recombination breakpoints are shown at the bottom with nucleotide sites. A complete viral genome structure is shown under the similarity plot with reference to VR-2332; (b) phylogenetic trees based on each recombinant region (region A-H) of SCN17.

Figure 4

The rectal temperature, survival rate, weight gain, viremia, and antibody level of piglets during the challenge study. (a) Rectal temperatures of piglets inoculated with SCwhn09CD, SCN17, and RPMI-1640 medium. Mean ± SD (error bars) temperatures (°C) are shown. The fever cut off value was set at 40.0 °C; (b) the survival and mortality curves of the inoculated piglets; (c) average daily weight gain of the inoculated piglets. Mean ± SD (error bars) weight gain (kg) for the survival pigs of each group is shown; (d) the PRRSV RNA copies in sera of pigs at different days post challenge were detected by one-step Taq-Man RT-qPCR. Mean ± SD (error bars) viral RNA copies (lg copies/mL) are shown; (e) PRRSV-specific antibodies in sera of pigs at different days post challenge. Pig serum was assayed for PRRSV-specific antibodies using ELISA kit, IDEXX HerdCheck ELISA. ORF5 gene was amplified with specific primers, the sense primer was 5’-AGCCTGTCTTTTTGCCATTCT-3’, and the reverse primer was 5’-CTTTTGTGGAGCCGTGCTATC-3’. Mean ± SD (error bars) S/P values (lg copies/mL) are shown. The significant difference is marked with the asterisk (*).

Figure 5

Gross and microscopic lung lesions observation of the inoculated piglets. (a) Severe interstitial pneumonia with hemorrhage, consolidation, and pulmonary edema were observed in SCwhn09CD-inoculated pigs; (b) moderate interstitial pneumonia with consolidation was observed in SCN17-inoculated pigs; (c) no gross lung changes were observed in the RPMI-1640-inoculated pigs; (d) interstitial pneumonia with infiltration of inflammatory cells, foci of necrosis (blue arrow), necrotic cell mass (red arrow), and exfoliated epithelial cells (black arrow) in the bronchiole could be observed in SCwhn09CD-inoculated pigs; (e) interstitial pneumonia with infiltration of inflammatory cells, foci of necrosis (blue arrow), and alveolar septal thickening (yellow arrow) could be observed in SCN17-inoculated pigs; (f) no pathological lesions were identified in the RPMI-1640-inoculated pigs. Original magnification, 200×.