Discussion
China is the largest country for pig farming in the world, with an
annual pig slaughter in 2020 about 600 million. The re-emergence of PEDV
especially the variant strains since 2010 has brought a great threat to
Chinese pig industry. PEDV was the major causative agent that lead to
the diarrheal disease and death in piglets in a comparison with other
coronaviruses, such as TGEV, PDCoV, and SADS-CoV
(Su et al., 2020). Although a variety of
commercial vaccines based on classical GI strain CV777 and the variant
GII strain AJ1102 have been employed to control the outbreak of PEDV,
their efficacies have been always poor. The immunity failure was
associated with the constant variation of virus and the an insufficient
mucosal immunity induced by the vaccines
(Guo et al., 2019). Therefore,
understanding the prevalence and variation characterization of PEDV
genome is of importance in preventing PEDV prevalence. In a recent
study, 49 complete S genes were sequenced in China from 2014 to 2016,
and novel insertions, deletions, and multiple S gene recombination
events were found in PEDV variant strains
(Fan et al., 2020). Lei et al. collected
184 specimens from pig farms in China in 2017–2018 and detected an
average PEDV-positive rate of 38.04% (Tan
et al., 2020). Zhang et al. made a survey of molecular characteristics
of PEDV in Henan province, China in 2015–2019 and found PEDV existed
widely in both PEDV-vaccine immunized (25.00%) and non-immunized swine
herd (62.29%). Sixteen of the sequenced PEDV Henan strains were located
in the GII clade (H. Zhang et al., 2021).
However, there is no nationwide epidemiological survey of PEDV in the
past two years. Furthermore, African swine fever (ASF) outbreaks were
declared in China since 2018 and quickly swept through the whole country
(Bao, Qiu, Luo, Rodriguez, & Qiu, 2021).
Management and prevention measures of epidemic diseases have been
altered in pig farms. It is still unknown about if the prevalence
characterization of PEDV has changed under the background of ASF. In
this study, 115 PEDV positive samples were collected in 17 different
provinces throughout China, including large swine-raising provinces,
such as Sichuan, Henan, Hunan, and Shandong province. Our data showed
that the positive rate of PEDV was from 10.77% to 25.10% from March
2020 to April 2021, indicating that PEDV was still one of the major
pathogenic agents in Chinese swine herd. Our data also showed that the
positive rate of PEDV was the highest in October and December. Thus, we
should pay more attention on the prevention of PEDV when the weather
turns cold, such as a turn of autumn and winter and the winter.
The full-length nucleotide sequences of S genes of 91 field strains were
successfully sequenced. Based on the phylogenetic analysis for the S
genes, all of the PEDV strains could be classified into two main
genotypes and five subgroups, including GI-a (classical strain), GI-b
(attenuated classical strain), GI-c (S-INDEL strain), GII-a, and GII-b
(variant strain). All strains detected in the study shared 94.2%–100%
homology with each other. A majority of them belonged to GII genotype
(84 out of 91) which represented the pandemic strains of PEDV in recent
years. GII genotype strains shared 93.2%–93.8% and 96.7%–99.1%
homology with attenuated Chinese vaccine strains CV777 and AJ1102,
respectively. Vaccines based on CV777 strain was widely used and
contributed to a good control of PEDV in China before 2010
(Yang et al., 2013). However, the variant
strains which were clustered into different subgroup (GII) shared lower
homology with CV777 (GI). The different genotypes between CV777 and PEDV
epidemic strains may lead to an incomplete protection provided by the
vaccines in China since 2010.
The detected strains CH/JSXZ/202012, CH/SCYB/202012, CH/SCGA/202103,
CH/SCST/202004, CH/HNBR/202101, CH/JXXG/202101, and CH/HNSQ/202102 were
more related to S-INDEL strains. They were obtained from four provinces
of China and classified into GI-c subgroup, thus suggesting that S-INDEL
like strains have spread and circulated in China. Seven of S-INDEL like
strains shared a high homology of 98.6%–99.3% with each other.
Although the presence of S-INDEL strains of PEDV have been described to
induce less severe symptoms and low fatality rate as compared with
non-INDEL strains (Lin et al., 2015;
Vlasova et al., 2014), the pathogenicity
of this subgroup is still controversial. Severe diarrhea and vomiting,
along with high mortality rates were associated with the S-INDEL PEDV
strains in some European countries
(Stadler et al., 2015). Therefore, the
pathogenicity and infection rate of S-INDEL like strains in China should
be constantly monitored.
To date, S-INDEL strains have been detected widely in the world
including America, Asia, and Europe. The S-INDEL strains could be
separated into two distant clades (INDEL 1 and INDEL 2) based on the
characteristics of the S-INDEL genotype (de
Nova et al., 2020). INDEL 2 contained prototype American (OH851) and
Europe strains (FR/001/2014), as well as the first identified Chinese
S-INDEL strain ZL29. Some other Asia S-INDEL strains belonged to the
INDEL 2 clade. A further phylogenetic analysis using the seven S-INDEL
like strains and reference S-INDEL strains retrieved from the NCBI
nucleotide database showed that all of the detected S-INDEL like strains
were categorized into INDEL 2. However, they were clearly clustered
within a new cluster in INDEL 2 clade (Figure 5). Although the
identified seven S-INDEL like strains had the common four aa deletions
(58QGVN61) in line with reference S-INDEL strains, they had novel three
amino acid mutations and one deletion located at the positions 156-160
aa. As previously described, INDEL 2 clade might originate from virulent
strains DR13, Italy/7239/2009 or other field NON-INDEL strains, whereas
INDEL 1 showed a common ancestor including CV777 or other PEDV strains
detected in China before 2010 (Guo et al.,
2019; J. Zhang et al., 2018). According
to our results, the novel S-INDEL like strains detected in this study
were originated from FR/001/2014 in INDEL 2 clade and vaccine strain
AJ1102. This indicated that natural recombinant events might exist among
variant strains and vaccine strains in China. Therefore, the use of live
vaccine strains should be carefully considered especially in PEDV
positive pig farms.
Spike protein is the most variable protein of PEDV. The variation of S
protein is considered to be responsible for changes of viral
antigenicity, determination of the genetic diversity for PEDV and
affected the viral virulence (Suzuki,
Terada, Enjuanes, Ohashi, & Kamitani, 2018;
Van Diep et al., 2020). To evaluate
whether the antigenicity of PEDV field strains has changed as compared
with vaccine strains used in China, the neutralizing antigenic epitopes
on S protein were analyzed. Several single nucleotide polymorphisms
(SNPs) were observed on the S1A,SS6,2C10 epitopes
and COE domain. Five main serine substitution (520A-S,
552T-S, 597G-S,
764L-S, 766D-S) and one Glutamate
substitution (636Q-E) were found in the field strains
as compared with the vaccine strain CV777. One common substitution in
S1A (479S-A) and multiple mutations
(499I-T, 502V-I,
524H-S/L/Y, 539F-L,
566K-N, 569D-A/N,
606Y-H, 612G-V/S and
634P-S) in individual sequences were detected in the
field stains as compared with the vaccine strain AJ1102. These mutations
might involve in an immune escape and the antigenicity of virus,
resulting in the less effective immune protection provided by the
commercial vaccines CV777 and AJ1102. Notably, five PEDV samples with
new insertion in the S protein sequences were detected. CH/GDMM/202012
had a continuous 5 aa insertion (360QGRKS364) located in S1 domain while
the other four (CH/GXDX/202010, CH/AHBZ/202010, CH/AHLA/202010 and
CH/HNLY/202003) had three or four amino acid insertions (1279DVF1281 or
1278VDVF1281) located in S2 domain. To the best of our knowledge, this
is the first report for these novel S-insertion variants. The
S-INDEL-variants were proved to have decreased virulence in host,
whereas one strain with a novel four-amino-acid insertion in the COE
domain was highly pathogenic to neonatal pigs
(Ji et al., 2021). Although we did not
obtain the isolated novel S-insertion strains, it could be predicted
that the conformational structure of S protein might have changed,
resulting in a change for the pathogenicity of these strains.
Collectively, this study described the nationwide investigation of the
PEDV prevalence in China in recent years. The major causative virus
strains were GII genotype variants with the ratio of 92.3%. Notably,
seven S-INDEL like strains were detected in four provinces in China.
Alignment of S deduced aa sequences revealed novel mutations and
deletion compared with prototype S-INDEL strain. Based on recombination
analyses, the novel S-INDEL like strains were originated from
FR/001/2014 in INDEL 2 clade and vaccine strain AJ1102. Moreover,
variant PEDV strains with novel insertions (360QGRKS364 and
1278VDVF1281) in S protein sequences were detected and needed to be
addressed on the specific function of insertions. In addition, we also
identified multiple mutations in the aa sequences of S proteins of the
variant strains compared to those of the vaccine strains. These PEDV
mutants derived from genetic mutations, deletions, insertions, and
recombination of the S genes might be the major cause of antigenic drift
and immune failure. The molecular characterization of S protein should
be investigated continuously and would work in the control and
prevention of PED in China.