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.