Results and Discussion
In total, 412 pigs died over the course of 150 days. A total of 46 of 412 piglets died in the first 15 days (Fig 1). The main clinical symptoms were loss of appetite and fever, followed by acute death, and the main pathological changes observed during piglet necropsy were intestinal hemorrhage, abdominal hemorrhage or peritoneal effusion. Symptoms were preliminarily presumed to be caused by bacterial infection. Symptoms subsided after emergency antibiotics were administered for prevention and treatment on the 10th day. The peak mortality period (287/412) occurred from the 16th day to the 45th day (Fig 1). Most pigs showed obvious clinical respiratory symptoms, such as cough, wheezing or diaphragmatic breathing. Necropsy of dead pigs showed lung consolidation, partial intestinal bleeding, and abdominal hemorrhage. Antibiotics administered after 35 days, the number of dead pigs (79/412) decreased (Fig 1), and the majority of the deaths occurred in pigs previously isolated for respiratory symptoms. To explore the causes of death in the pigs, a total of 283 samples were collected from dead piglets and tested for AFSV, CFSV, PRRSV, PRV and PCV2. PRRSV and PCV2 were detected, while ASFV, CSFV and PRV were not. During the three different stages noted above, the detection rates of PRRSV and PCV2 were 17.39% (8/46), 33.13% (53/160), and 29.87% (23/77); and 89.13% (41/46), 81.88% (131/160), and 80.52% (62/77), respectively (Fig 1). The above results demonstrate that death in the early stage may have been mainly due to bacterial infection and that PRRSV contributed to the death curve but not PCV2 did not on this pig farm.
To explore the relationship between the PRRSV subtype and the death of pigs on the farm, we sequenced the NSP2 and ORF5 genes for all PRRSV positive samples. A total of 78 NSP2 sequences and 69 ORF5 sequences were obtained. Of these, 54 samples were NADC30-like PRRSV (64.29%), 17 were NADC34-like PRRSV (20.24%), 11 were HP-PRRSV-like (13.10%), 1 was CH-1a-like PRRSV (1.19%), and 1 was QYYZ-like PRRSV (1.19%) (Fig 3a). Therefore, NADC30-like PRRSV, NADC34-like PRRSV and HP-PRRSV-like were the main epidemic strains on this farm. Furthermore, the detection rates of the main epidemic strains were 12.50%, 71.70%, and 65.22% (NADC30-like PRRSV); 0.00%, 26.42%, and 13.04% (NADC34-like PRRSV); and 87.50%, 1.89%, and 17.39% (HP-PRRSV-like) in the three different stages, respectively. Surprisingly, the outbreak times of NADC30-like and NADC34-like PRRSVs were consistent with the peak periods of pig deaths on the pig farm (Fig 1). The above results demonstrated that NADC30-like PRRSV and NADC34-like PRRSV but not HP-PRRSV-like PRRSV were closely related to the deaths of pigs on this farm. In addition, a number of NADC34-like strains (14/17) were detected in the subsequent 10 days after initial detection in stage 2. The spread of NADC34-like PRRSV seemed to be faster than that of NADC30-like PRRSV. The pathogenicity of NADC34-like PRRSV on this farm remains to be studied.
NADC34-like PRRSV emergence was first reported in Liaoning Province in 2017 (Zhang et al., 2018). This strain subsequently emerged in other provinces of China (Bao and Li, 2021; Liu et al., 2019; Sun et al., 2020; Xie et al., 2020a; Xie et al., 2020b; Xu et al., 2020). All the NSP2 sequences of the NADC34-like strains shared the same 100 consecutive amino acid deletions between 328 and 427 as previously reported, compared with ATCC_VR2332 (accession number: U87392) (Fig 2). These deletions can be used as molecular markers to distinguish NADC34-like strains from other type 2 PRRSV strains in China, similar to the consistent Nsp2 protein deletion pattern in NADC30-like PRRSV (Brockmeier et al., 2012; Xu et al., 2020). The amino acid identities of NSP2 of the NADC34-like strains on this farm were between 99.2% and 99.9%. In the NCBI library, the highest identity was with IA/2014/NADC34 (accession number: MF326985), at 92.6%.
The nucleotide identity of the NADC34-like PRRSV ORF5 gene on this pig farm was 99.2%-100%, which also has the highest identity with the IA/2014/NADC34 strain in the NCBI, and the nucleotide similarity was 96.9%-97.2%. The consistency between these strains and the first NADC34-like strain, LNWK130, reported in China was 94.9%-95.0%. These indicate that NADC34-like PRRSV has evolved in China. Combined with the NSP2 analysis of NADC34-like PRRSV, NADC34-like PRRSV infection on this pig farm was caused by a single strain; this provides a good platform for studying the evolution rate of the NADC34-like strain (Barba-Montoya et al., 2020). Many methodological approaches previously used to study PRRSV ignored the fact that the evolutionary and epidemiological dynamics of rapidly evolving pathogens, such as PRRSV, occur on approximately the same timescale. Thus, they must be studied under a unified methodological setting to be properly understood and to prevent biased conclusions, subsequently improving related decision-making processes (Alkhamis et al., 2016; Pybus et al., 2013). The NADC34-like strains on this farm showed a strong time signal (the correlation between the genetic difference and sampling time r2was 0.52) and was thus suitable for phylogenetic analysis involving a molecular clock. The estimated viral substitution rates were 3.1×10−2 substitutions/site/year, which was higher than the evolution rate, which ranged from 6.6 × 10−3to 1.3 × 10−2 substitutions/site/year for all subtypes of lineage 1 previously reported in the U.S. (Alkhamis et al., 2016; Paploski et al., 2021). This is a dangerous signal that indicates that the time from the appearance of NADC34-like PRRSV in China to its peak in the population will be shorter than that in the U.S. (4.5 years on average) (Paploski et al., 2021). Moreover, surveillance of PRRSV showed that the number of NADC34-like PRRSVs has obviously increased since 2020, especially in 2021 (unpublished data). Therefore, we speculate that NADC34-like PRRSV has become dominant in parts of China.
We further classified the NADC34-like PRRSV strains on this farm according to restriction fragment length polymorphism (RFLP) of the ORF5 gene (Brar et al., 2011; Cha et al., 2004; Trevisan et al., 2021). The RFLP pattern of ORF5 of TZJ1277 is 1-4-4, while the others are 1-7-4. Compared with the newly emerged PRRSV lineage 1C variant (MW887655) in the U.S., TZJ1277 has a closer relationship with the earlier reported NADC34-like PRRSV (Fig 3a). RFLP typing has recognized shortcomings, which include an inability to represent genetic relationships between different RFLP types, the potential for distantly related viruses to share the same RFLP type, and instability of RFLP types over as few as 10 animal passages (Cha et al., 2004; Paploski et al., 2019). Partially due to these ambiguities in the interpretation of RFLP types, researchers in many countries have formulated their own naming conventions based on the epidemic situation in their countries (Paploski et al., 2019; Paploski et al., 2021), so the classification of virus strains in their home countries is crucial.
Whole-genome sequencing (WGS) can reveal more information about PRRSV than traditional Sanger sequencing analysis of ORF5 (Frias-De-Diego et al., 2021; Risser et al., 2021). WGS phylogenetic tree analysis showed that the lineage 1C variant (MW887655) and the two full-length sequences measured (TZJ864 and TZJ921) were clustered into the same branch (Fig 3b). The United States 1-4-4 lineage1C PRRSV variant is based on IA14737-2016 (NADC34-like) as the parent strain, and IA/2014/NADC34 and NADC30 strains provide recombinant fragmented recombinant virus. PRRSV recombination events have been the focus of researchers (van Geelen et al., 2018; Wang et al., 2019; Yu et al., 2020). Recombinant PRRSVs have been increasingly isolated since NADC30-like PRRSVs emerged in China (Chen et al., 2018; Ramirez et al., 2019; Sun et al., 2020). To explore whether the NADC34-like PRRSV isolated from this farm was a recombinant virus, we sequenced two complete genomes from NADC34-like PRRSVs from this farm. Recombination analysis and sequence alignment showed that they were not recombinant viruses and had relatively high identity (98.9%). However, five types of PRRSVs coexisting on the same farm will certainly increase the likelihood of recombination. Coincidentally, we have detected many recombinant NADC34-like PRRSVs from other pig farms since June 2021 (data unpublished). The characteristics and virulence of these recombinant NADC34-like PRRSVs need to be further studied.
Concerningly, the U.S. has reported high economic losses due to NADC34-like reorganization (van Geelen et al., 2018). Importantly, NADC34-like and NADC30-like PRRSVs recombine with strains of different subtypes, resulting in inconsistent virulence among the recombinant strains and causing great obstacles in the prevention of PRRSV (Chen et al., 2018; Chen et al., 2021). Considering that NADC30-like PRRSV has become the main epidemic strain in China (Jiang et al., 2020), outbreaks of NADC34-like PRRSVs in pig farms will inevitably lead to more frequent fragment exchanges between them. Therefore, we need to increase awareness of the importance of continuous monitoring of NADC34-like PRRSV strains, strictly control the selection of breeding pigs, and prevent the occurrence of multiple subtypes of PRRSV on pig farms.