3 Results and discussion
A total of 1,174 samples, 179 samples were positive for the PRV gE
detection, yielding an average positive rate of 15.25% (179/1174). The
positive rates of PRV detection from 2012 to 2019 were 17.08% (41/240),
20.41% (30/147), 25.00% (45/180), 13.69% (23/168), 13.68% (13/95),
9.26% (10/108), 7.83% (9/115) and 6.61% (8/121), respectively, with
the peak at 2014, showing that the positive rate gradually decreased
after 2014, which might be related to the development and use of several
new vaccines based on the epidemic strains and the Chinese government
proposed the eradication program based on PR in 2012 (Wang et al., 2014,
Hu et al., 2015). As for seasons, the overall PRV-positivity rates over
the 2012-2019 study period were 14.02% (46/328) in the spring (March,
April and May), 9.83% (17/173) in summer (June, July and August),
16.04% (60/374) in autumn (September, October and November) and 18.73%
(56/299) in winter (December, January and February), respectively. The
PRV-positive rate of summer was lower than that of the winter and autumn
every year, and the positive rate was even 0% (0/21) in summer in 2019
(Fig.2A). The winter and autumn were the seasons with higher
PRV-positive rate, suggesting that the morbidity of PR was relatively
high in cold season, it might be interrelated with the epidemic
characteristics of the disease. The result was consistent with the Sun’s
study that winter, spring and autumn were the seasons with the high
positive rate in mainland China between 2012 and 2017 (Sun et al.,
2018).
Henan province of China is divided into five parts including Eastern
Henan, Western Henan, Southern Henan, Northern Henan and Middle Henan
(Fig.1). Among regions, the PRV-positive rates for eight-year-total were
15.25% (43/282), 13.11% (24/183), 17.62% (40/227), 16.84% (50/297)
and 12.09% (22/182) in Eastern Henan, Western Henan, Southern Henan,
Northern Henan and Middle Henan respectively (Fig.2B). The highest
PRV-positive rate of 40% was observed in Middle Henan in 2014. The
positive rates of PRV detection during 2012 and 2019 were 15.38%
(6/39), 28.13% (9/32), 17.54% (10/57), 16.98% (9/53), 20.00% (5/25),
12.50% (3/24), 13.33% (4/30) and 10.81% (4/37) in Southern Henan,
respectively, which were higher than that of other regions, except for
2012 and 2014. Western Henan or Northern Henan was the region with the
lowest PRV-positive rate among 2012 to 2019 (except for 2016), with the
positive rates of 0% observed in 2018 for Northern Henan and 2019 for
Western Henan. In addition, the detection rate of PRV in different
regions between different years displayed diversity. For instance, the
detection rates of PRV in Northern Henan during 2013 to 2016 were
averaged approximately 15.00% (3/20), and 9.09% (3/33) in 2012, and
8.70% (2/23) in 2017, and 4.35% (1/23) in 2019, but zero in 2018; in
Western Henan, the positivity rates in 2012 and 2014 were higher than
20.00%, but the positive rates in 2013, 2015, 2016, 2017 and 2018 were
lower than 10.00%, even zero in 2019; in Middle Henan, the positivity
rates in 2012, 2013, 2015, 2016 and 2017 were ranging from 11.11%
(2/18) to 21.43% (6/28), and approximately 40.00% in 2014, but 8.00%
(2/25) in 2018, but 9.52% (2/21) in 2019; in Eastern Henan, the
positive rates in 2012, 2013 and 2014 were higher than 18.00%, and
10.00% (2/20) in 2018, but those of 2015, 2016, 2017 and 2019 were
lower than 10.00%. These data demonstrated that PR remains in Henan
province, China, which is coincidence with the reports of high
prevalence of novel PR in China (An et al., 2013, Wu et al., 2013, Sun
et al., 2018, Tian et al., 2020). The PRV-positive rate might be related
to the different feeding and management methods, biological safety
measures and geographical location.
Hence, PRV strains were isolated, and a distinct CPE was observed after
three passages of virus on ST cells, which was characterized by cell
rounding, pyknosis, and degeneration of the cell monolayer (Fig. 3A and
Fig.3B).
The
gE gene in the infected cells was detected by PCR using the primers
gEp-F/R, and the result of electrophoresis showed that the product was
identical to the
predicted
DNA fragment size (429 bp, Fig.3C). Thus, these PRV isolates were
formally named as NY, GY, LGX, MZ1, MZ2, ZM, ZK, JY, M5, YY, WY, BP, YZ,
SMX, WZ and XC (Table 1). TCID50 of these PRV isolates
were NY 109.0/0.1 ml, GY 106.1/0.1
ml, LGX 106.0/0.1 ml, MZ1 105.6/0.1
ml, MZ2 105.3/0.1 ml, ZM 105.8/0.1
ml, ZK 106.0/0.1 ml, JY 107.4/0.1
ml, M5 106.0/0.1 ml, YY 106.0/0.1
ml, WY 106.0/0.1 ml, BP 105.0/0.1
ml, YZ 108.0/0.1 ml, SMX 106.0/0.1
ml, WZ 107/0.1 ml and XC 105.375/0.1
ml, respectively. The 16 isolates were highly pathogenic in mice,
causing skin inflammation, neural symptom and leading to death in all
experimentally-infected mice at 48-72 h after challenge. In contrast,
all mice survived in negative control groups, which were in agreement
with previous study (Laval et al., 2018).
The identification results of representative PRV isolate NY were shown
in Table 2. The NY isolate was sensitive to chloroform, trypsin,
formaldehyde and ultraviolet ray, revealing that it belonged to the
enveloped virus. The virus was not inactivated until the heating time
was above an hour at , showing that NY isolate had good heat resistance.
Otherwise, no detectable titers were observed when the culture medium pH
was adjusted to 3.0 or 11.0. These results were consistent with the
physicochemical properties of PRV. As shown in Fig. 3D, a circular
viral particle of about 110~150 nm was observed in the
ST cells infected with NY isolate. Furthermore, virus particles
exhibited envelope protein with a radially arranged spike. Thus, the
morphological features were basically consistent with those of
pseudorabies virus.
Sequencing analysis of the main PRV gE and gC genes for 16 PRV isolates
revealed the maximal amino acid (nucleotide) sequence divergences of
0.3% (0.1%) and 1.2% (0.5%) within the 16 isolates, and 4.7%
(2.2%) and 10.3% (5.2%) compared with those isolates from other
countries, respectively. The maximal amino acid (nucleotide) sequence
divergence of the two genes of the 16 isolates were 1.4% (0.9%) and
1.0% (0.4%) compared to those strains prevalent in China before 2012,
and were 0.7% (0.2%) and 0.4% (0.1%) after 2012. The phylogenetic
tree based on the sequences of gE gene revealed two distinct groups
(Fig. 4A): one formed by the four European-American PRV strains (Clade
2), and the other (Clade 1) formed by three subgroups: one (Clade 1-1)
formed by the 16 PRV strains in this study and the 11 Chinese variant
PRV strains (after 2012), and the other (Clade 1-2 and Clade 1-3) formed
by the 4 early Chinese PRV strains (before 2012), suggesting that gE
gene of 16 isolates were genetically closer to the variants. In the
deduced gE amino acid (aa) sequences of the 16 PRV isolates and 25
referent strains, compared to all strains of Clade 2, the complete gE
sequences containing two aa insertions at position 48 (D) and 497 (D)
were found in the strains of Clade 1-1, which were not observed in early
Chinese strains Fa-2002-China and GX-NL-2007-China. Furthermore, all the
16 isolates of Clade 1-1 had eighteen aa interspersed substitutions (at
positions 54, 59, 63, 106, 179, 181, 215, 216, 449, 472, 474, 504, 509,
512, 522, 526, 577 and 578) compared with the whole strains of Clade 2,
whereas gE aa of the isolates YY and BP in this study were not changed
at site 512 and 578 respectively. Remarkably, only four aa substitutions
in 16 isolates at position 449 (V to I), 512 (G to S, except for YY),
577 (N to M) and 578 (A to S, except for BP) were different from the
substitutions of 7 Chinese variant PRV strains. Compared with the Clade
1-2 and Clade 1-3, the 16 isolates had two aa substitutions at positions
449 (V to I) and 512 (G to S, except for YY). Compared with the
variants, two isolates NY and BP existed one aa change at position 386
(T to M), and the WZ and ZK had two aa substitutions at positions 329 (W
to R) and 532(S to G), respectively.
For the gC, phylogenetic tree revealed two distinct groups (Fig. 4B):
one formed by the four European-American PRV strains (Clade 2), and the
other (Clade 1) formed by three subgroups: one (Clade 1-1) formed by the
16 PRV isolates in this study and the 6 Chinese variant PRV strains, and
the other (Clade 1-2) formed by the 6 early Chinese PRV strains and 2
Chinese variant PRV strains, indicating that 16 isolates had the
relatively closely relationship with the variants. Compared to all
strains of Clade 2, gC protein containing seven aa deletions at sites 63
– 69 (AAASTPA) was found in the strains of Clade 1-1, which was also
observed in early Chinese PRV strains. Moreover, the strains of Clade
1-1 had 23 aa interspersed substitutions (at position 16, 43, 52, 55,
57, 59, 60, 61, 87, 90, 102, 130, 142, 188, 431, 437, 449, 457, 461,
467, 485, 486 and 487) compared with the strains of Clade 2.
Interestingly, eight strains MZ1, NY, LGX, MZ1, XY, ZK, ZM and GY in
this study harbored one aa substitution at sites 280 (F to L) compared
to other European-American strains of Clade 2 and Chinese strains of
Clade 1, which was the first reported that the characteristic aa
substitution
(at position 280) was existed in the Chinese variant PRV strains’ gC.
These might be the reasons that the virulence of PRV variants enhanced,
and should be paid more attention.
All mice of four groups did not display adverse reactions after
vaccination (data not shown). After challenge with PRV NY isolate, mice
in group 1 survived without typical PR symptoms, and the mortality was
0% (0/10). While the mortalities of groups 2-4 were 50% (5/10),
30% (3/10) and 100% (10/10). As
for neutralizing antibodies against PRV, mice in groups 1-3 induced
neutralizing antibodies with titers of 1:82, 1:24 and 1:22,
respectively. No neutralizing antibodies were detected in group 4 as
control. The neutralization titer between PRV NY isolate and its own
serum was the highest, followed by Bartha-K61 strain and Hubei 98
strain, which indicated that PRV NY strain might exist the cross
antigenicity with both Bartha-K61 and Hubei 98 strain, with the
different antigenicity. These results were consistent with the previous
finding (An et al., 2013), further indicating that Bartha-K61 vaccine
cannot provide the full-protection against the PRV variants. Therefore,
the development of novel vaccine based on the variants was urgent.
Live vaccines are currently used to control pseudorabies in many swine
farms in China, mainly based on strain Bartha-K61 which is an attenuated
strain of PRV produced by extensive in vitro passages and contains a
well-characterized deletion of the complete gE and partial gI genes
encoding proteins that attenuate virulence, and Bartha-K61 has played a
key role in the eradication of PR (Lomniczi et al., 1987). Thus,
detection of gE-specific antibodies is used to differentiate vaccinated
animals from those with wild-type virus infections. During 2005 to 2010,
the positive PRV gE antibodies were detected in only 3%–5% of serum
samples (Gu et al., 2015). Since 2012, severe PRV outbreaks have
occurred on several pig farms and spread rapidly to most of China (An et
al., 2013, Wu et al., 2013, Gu et al., 2015, Zhai et al., 2019).
Although the PRV infection has recently decreased since Chinese
government proposed the
eradication
program based on PR in 2011, it remains not completely eradicated (Wang
et al., 2015, Sun et al., 2018, Zhai et al., 2019). Some researches
demonstrated that the novel PR was caused by PRV variants (An et al.,
2013, Hu et al., 2015, Ye et al., 2015, Sun et al., 2018, Zhai et al.,
2019), and this study further confirmed PRV variants were prevalent in
China. To control and prevent the PRV infection in pig farms, we should
develop the effective labeled vaccines and combine with other integrated
control measures, such as serological and virological monitoring, and
biosafety procedures. In addition, the Chinese government should call on
all relevant practitioners (farmers, veterinarians, scientists, vaccine
manufacturers, officials and communities) to join hands for fighting the
disease, and learn from some countries in South America and North
America which successfully eliminated PR for a long time. Facing the
prevalence of PR, it is worthy of further study to evaluate whether the
current immunization procedures and biosafety measures are reasonable
and effective.
In conclusion, this study suggests that PR is not yet eradicated, and
even exists in nearly whole Henan province, China, and the new PRV
isolates identified by sequencing of the complete gE and gC genes may be
PRV variants. Further, it can be speculated that these variations may be
the cause of the re-emergence of PR in China. The pathogenicity of the
genetic variations seen in complete gE and gC genes of these PRV
isolates is currently under further investigation, and the complete
genome sequencing of the isolates is also necessary.