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\begin{document}

\title{Re-emergence of porcine epidemic diarrhea virus in a piglet-producing
farm in Jiangxi province in China in 2020}



\author[1]{Qi Gao}%
\author[2]{Zheng Zezhong}%
\author[1]{Heng Wang}%
\author[1]{Guihong Zhang}%
\author[3]{Lang Gong}%
\affil[1]{South China Agricultural University}%
\affil[2]{South China Agricultural University, College of Veterinary Medicine South China Agricultural University No.483 Wushan Street, Tianhe District Guangzhou, Guangdong, P. R. China}%
\affil[3]{South China Agriculture University(SCAU)}%


\vspace{-1em}



  
  \date{June 11, 2024}


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\selectlanguage{english}
\begin{abstract}
On October 30, 2020, piglets and sows in the farrowing house of a pig
farm in Jiangxi showed clinical symptoms such as anorexia, watery
diarrhea, and vomiting. Epidemiological test, clinical necropsy, and
RT-PCR test were carried out on the pig farm for diagnosis. After
comprehensive considerations, the disease was judged as porcine epidemic
diarrhea virus infection. Thereafter, a series of comprehensive
prevention and control measures such as emergency vaccination with
autogenous vaccines were adopted. Half a month after inoculation with
autogenous vaccines for the farm, the mortality rate of newborn piglets
in the farrowing house began to decline, and production gradually
returned to being stable. The second-generation sequencing analysis and
phylogenetic analysis showed that the porcine epidemic diarrhea virus
(PEDV) sequence obtained from the stool and small intestine samples of
the diseased pigs on the farm was 97.8\% homologous to the vaccine
strain. At the same time, antibody testing found that the vaccinated
pigs on the pig farm had satisfactory immune response, indicating that
the PEDV outbreak on the pig farm might aggravate owing to the strain
being mutated and could escape the immune protection of the existing
vaccine. This case has accumulated technical data for the clinical
prevention and control of porcine epidemic diarrhea.%
\end{abstract}%



\sloppy


\textbf{Re-emergence of porcine epidemic diarrhea virus in a
piglet-producing farm in Jiangxi province in China in 2020}

Qi Gao\textsuperscript{1}, Zezhong Zheng\textsuperscript{1,4}, Heng
Wang\textsuperscript{1,2,3}, Guihong Zhang\textsuperscript{1,2,3,4}*,
Lang Gong\textsuperscript{1,2}*

\textsuperscript{1}College of Veterinary Medicine, South China
Agricultural University, Guangzhou 510462, China

\textsuperscript{2}Key Laboratory of Zoonosis Prevention and Control of
Guangdong Province, South China Agricultural University, Guangzhou
510462, China

\textsuperscript{3}National Engineering Research Center for Breeding
Swine Industry, South China Agricultural University, Guangzhou 510462,
China

\textsuperscript{4}Guangdong Laboratory for Lingnan Modern
Agriculture,Guangzhou, 510642, China

* Correspondence: gonglang@scau.edu.cn

\textbf{Summary:} On October 30, 2020, piglets and sows in the farrowing
house of a pig farm in Jiangxi showed clinical symptoms such as
anorexia, watery diarrhea, and vomiting. Epidemiological test, clinical
necropsy, and RT-PCR test were carried out on the pig farm for
diagnosis. After comprehensive considerations, the disease was judged as
porcine epidemic diarrhea virus infection. Thereafter, a series of
comprehensive prevention and control measures such as emergency
vaccination with autogenous vaccines were adopted. Half a month after
inoculation with autogenous vaccines for the farm, the mortality rate of
newborn piglets in the farrowing house began to decline, and production
gradually returned to being stable. The second-generation sequencing
analysis and phylogenetic analysis showed that the porcine epidemic
diarrhea virus (PEDV) sequence obtained from the stool and small
intestine samples of the diseased pigs on the farm was 97.8\% homologous
to the vaccine strain. At the same time, antibody testing found that the
vaccinated pigs on the pig farm had satisfactory immune response,
indicating that the PEDV outbreak on the pig farm might aggravate owing
to the strain being mutated and could escape the immune protection of
the existing vaccine. This case has accumulated technical data for the
clinical prevention and control of porcine epidemic diarrhea.

\textbf{Keywords:} cross protection, phylogenetic analysis, porcine
epidemic diarrhea virus, strain variation

Background

Porcine epidemic diarrhea is an acute and highly contagious intestinal
infectious disease caused by porcine epidemic diarrhea virus infection.
The disease is prevalent in the cold season and is transmitted through
fecal-oral transmission. Its clinical symptoms include elevated body
temperature, anorexia, vomiting, diarrhea, dehydration, etc (Lee, C.,
2015) Porcine epidemic diarrhea virus (PEDV) is a single-stranded RNA
virus belonging to genus Coronavirus, family Coronaviridae, with a
genome length of 27-31 kb (Jung, K et al., 2015). The disease was first
discovered in the UK in 1971 (Debouck, P. et al., 1980) and successively
discovered in China in the early 1980s (Xuanhu, 1984). Since October
2010, a new variant strain of porcine epidemic diarrhea has appeared,
which has been widespread in China and other major pig-raising countries
in the world, and its prevalence in East Asia and North America is
severe, causing large economic losses to the farms (Wan Liang et al.,
2020, Huang, Y.W. et al., 2013). PEDV infection has become one of the
main reasons for the high mortality of suckling piglets.

Vaccination is an important way to control infectious diseases. In 1994,
Ma et al. used the attenuated CV777 strain to prepare an inactivated
vaccine with aluminum gel adjuvant. After immunizing piglets, the active
and passive protection efficiency exceeded 85\% (Ma Siqi, 1994). In
1995, the study team successfully developed a commercial TGEV+PEDV
bivalent vaccine. In 1998, Tong et al. confirmed that the CV777 strain
obtained by continuous \emph{in vitro} passage for 90 generations is
suitable for the preparation of attenuated vaccines. In 1999, they
successfully developed a PEDV+TGEV bivalent vaccine. The ratio of virion
counts of TGEV to PEDV was 1:1. After immunization, the active and
passive protection rates against PEDV were as high as 97.7\% and 98\%,
respectively (Tong Youen, 1999, Tong Youen, 1998). The PEDV vaccine
strains in these two commercial vaccines were both classic CV777
strains. Before 2010, these two bivalent vaccines were widely used in
China, effectively controlling the spread of PEDV and TGEV.

After the outbreak of PED in the United States in 2013, Collin et al.
prepared an inactivated vaccine of the American isolate (G2 genotype)
and proved that inactivated PEDV can induce sufficient humoral immunity
level in immunized 4-week-old piglets (Collin, E.A., 2015). The PEDV
strain used in the PEDV-TGEV and PoRV trivalent attenuated live vaccine
produced by the Harbin Veterinary Research Institute subordinated to the
Chinese Academy of Agricultural Sciences in early 2015 was still CV777.
The PEDV strain in the bivalent vaccine marketed in November of the same
year was ZJ08, which belongs to the G1b subtype (Liu Hao, 2018). In
December 2017, the PEDV bivalent inactivated vaccine and attenuated
vaccine prepared by Prof. Xiao of Huazhong Agricultural University were
successfully marketed. The vaccine strain was AJ1102, which was isolated
in 2011, and belongs to the G2b type (Jing Bi, 2012).

In the winter of 2020, a new wave of PEDV epidemic swept across China.
The question is, why was there a large-scale outbreak of PEDV under the
conditions that biosafety control was so strict under a non-plague
background, and whether new strains have emerged. Here, we report a
clinical case of PEDV in Jiangxi, which indicates that China's existing
PED variant vaccine may not fully protect against the PEDV epidemic
strain.

\textbf{Case presentation}

Case farm characteristics

Recently, a case of PEDV infection occurred in a pig farm in Jiangxi.
There were 8082 sows on hand in the mating house of the pig farm, which
were divided into 10 groups, with 800 pigs in each group. There were 902
sows in the farrowing house, with 80 farrowing beds as a unit and five
units constituting a building. Before the onset of the disease, the farm
recorded 25 weaned piglets per sow per year, and the loss of farrowing
house was 8\%. Loss from conservation and fattening was less than 6\%.
The pig farm regularly tested for enteric pathogens, and TGEV/PDCOV and
PEDV had not occurred in the last two years. There had often been
sporadic cases of porcine reproductive and respiratory syndrome (PRRS)
on the pig farm.

Routinely used immunization regimens include vaccination of sows with
live PRRS vaccine, swine fever vaccine, live pseudorabies vaccine,
foot-and-mouth disease O+A vaccine, Japanese encephalitis vaccine and
parvovirus vaccine. Sows were immunized with a live PED vaccine and an
inactivated vaccine at 30 days before delivery and 14 days before
delivery, respectively.

Clinical presentation and interventions

On October 28, 2020, at first, 80\% of the sows in a farrowing unit
developed depression, fatigue, and anorexia. Three days later, they
developed mushy persistent diarrhea. The body temperature was normal or
slightly higher, they showed anorexia and had fully recovered about 7
days after onset. Piglets presented with epidemic diseases with watery
diarrhea and vomiting as the main clinical symptoms. Vomiting usually
occurred after eating or feeding on breast milk. The severity of
symptoms varied with age. The younger the age, the more severe the
symptoms. In a period of 1 to 3 days after newborn piglets within 7 days
of age developed diarrhea, they were severely dehydrated and died, with
a mortality rate of 90\% to 100\%. Diarrhea occurred successively in
sows in other units 5 days later. After on-site anatomy and laboratory
testing of feces and tissues, PEDV was determined to be the pathogen
underlying this epidemic. Therefore, on October 31, the staff closed the
pigsty where diarrhea occurred, treated the sows and piglets with
amoxicillin- or lincomycin-containing drinking water for health care,
and disinfected the pigsty with Denaishuang dry powder and kept it dry;
on November 7, intramuscular injection of autogenous vaccine was
administered; on November 11, the farrowing house was disinfected, the
pigsty was dried and decontaminated with hot water at 60, the sick pigs
were given salt solution for replenishment, and the farrowing house was
cleaned, disinfected, and dried; on November 13, weaned piglets with
secondary infection were treated with antibiotics. On November 16th, the
sow swinery of the mating house was restored, the personnel were
released from lockdown, and the empty swinery units were cleaned and
disinfected. After adopting a series of comprehensive prevention and
control measures such as emergency vaccination with autogenous vaccine,
the mortality rate of newborn piglets on the farrowing room of this farm
dropped significantly at half a month after vaccination, and production
gradually returned to stable (Figure 1).

In general, four farrowing units showed clinical symptoms of PED, and
the mortality rate of suckling piglets was 40\%. A total of 85 sows
died, and 187 litters had abortion, with an incidence rate of 30\%. A
total of 6,455 piglets died, with an incidence rate of 70\%. At the
current price, the net profit per pig was 300 US dollars, and the direct
loss was nearly 2 million US dollars. Some weaned piglets also
experienced diarrhea, weight loss, and growth retardation. The total
loss from piglet feeding was 12\%. In the fattening house, there was no
sign of PED.

Epidemiological investigations revealed that at half a month before the
outbreak of PED, PEDV broke out in another pig farm 800 meters away from
the farm.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Laboratory findings

After preliminary confirmation of the PEDV pathogen by the pig farm
laboratory, fecal samples, milk and serum of 20 sows, as well as the
feces and intestines of three infected piglets were sent to South China
Agriculture University for sequence analysis and serological testing. In
order to reveal the molecular characteristics of the virus and determine
the relationship between the current epidemic strains in China and other
subtype strains, the Bootstrap method of MEGA6.0 software was used to
analyze the evolutionary tree of domestic isolates and reference strains
from different sources at different times, which indicated that the
strain formed a new and unique branch in the PEDV genotype II (Figure
2), which had the closest genetic distance to the genotype I strain. The
S1 gene homology comparative analysis of this strain showed that it had
98.1\%-100\% homology with 59 epidemic strains uploaded by NCBI in the
past year, 91.8\% homology with CV777 strain, 97.8 homology with the
AJ1102 vaccine strain, and 97.3\%-98.1\% homology with the 89 strains
prevalent in 2014-2015 measured by our laboratory (Table 1), indicating
that the PEDV of the current outbreak had mutated. Please see
Supplementary Figure 1 for specific similarities. The similarity with
the 2020 strain was also poorer by 1-2\% compared to the 2014-2015
strain, indicating that PEDV had continuously mutated.

To evaluate the immunizing effect of the PED vaccine in this farm, 20
diseased sows in farrowing season were randomly selected before the
immunization with autogenous inactivated vaccines and numbered 1-20.
Colostrum, feces, and serum were collected respectively to test the
levels of PED antigen and IgA antibody in colostrum, as well as the
levels of PED antigen in feces and specific IgG antibody in serum. The
results showed that the pigs had a good immune response after
immunization with the PED vaccine, with high levels of IgA and IgG
antibodies. However, for the three sows numbered 8, 11, and 20, despite
the high levels of IgA in milk and serum IgG antibodies, there were
still detectable PED antigens (Table 2). PED antigens were detected in
feces of more sows, indicating that the antibody levels produced by the
existing commercial vaccines cannot completely neutralize the new
epidemic strain of PEDV.

To evaluate the fecal virus shedding of sows during the disease course,
we conducted a two-week monitoring of virus shedding on 7 diseased sows.
The results showed that the affected sows had intermittent virus
shedding within two weeks after delivery. Some of the sows shed a great
load of virus due to individual differences (Table 3).

\textbf{Discussion and conclusions}

In recent years, PEDV has repeatedly broken out in China and has become
one of the deadliest pathogens infecting pig herds. Existing measures
such as immunization, biosafety, and feeding with tissues from diseased
animals cannot prevent its spread (James Lowe, 2014, Olanratmanee, E.O.,
2010). Although vaccines prepared with G1a subtype strains have been
developed and promoted for decades, it is precisely because of long-term
use that its shortcomings are exposed. The amino acid mutation rate of
the immunogen S gene of epidemic strains and vaccine strains has reached
as high as 10\% (Seong-Hee Kim, 2015). The rapid development and
promotion of new vaccines based on G2b subtype antigens have effectively
prevented the spread of PEDV in China in the past 10 years. At present,
all the genotypes of PEDV exist in China.

Owing to the pressure of herd immunity, the S gene of the coronavirus
has frequently mutated, and changes in some amino acids have caused
antigenic drift and escape from the protection of existing vaccines
(Chun-Ming Lin, 2016). Therefore, periodic update of coronavirus vaccine
strains becomes urgent. However, even the latest PED variant vaccine
strain was isolated in 2011. For example, the antigenic difference has
been confirmed in the virus neutralization experiment between the
epidemic strain and vaccine strain of bovine coronavirus (Toru Kanno,
2013). Neutralization of the virus with hyperimmune serum showed that
there is only one serotype of PEDV. However, an increasing number of
field surveys show that a mismatch between virus sequence analysis and
serum neutralization test indicates a big difference between the
antigenicity of PEDV epidemic strain and the vaccine strain. To date,
the attenuated vaccine of the mutant strain continues to play a partial
protective role (Dongbo SUN, 2016). However, an increasing number of pig
farms, which have undergone normal immunization procedures continue to
have outbreaks of PEDV, and the isolated strains have confirmed it to be
highly pathogenic epidemic strain.

Following the occurrence of PED in the pig farm, the outbreak has spread
fast and has incurred huge losses; thus, a vaccine strain that matches
the that of the epidemic in the pig farm must be selected for the
immunization program. At present, there are only few PED vaccine strains
available in China, and pig farms will most probably use autogenous
inactivated vaccines for emergency immunization. Inactivated vaccines
mainly act through humoral immunity and can produce a large amount of
IgA. IgA antibodies are considered the most important protective
antibodies in pig colostrum and provide a protective effect for piglets
(En Zhang, 2020). Our research results show that although the vaccine
could produce high titers of IgA antibody, some pigs still shed virus in
the milk. This shows that the existing vaccine may not completely
neutralize the PEDV epidemic strain. PEDV is mainly transmitted through
feces and mouth, and the viral load carried in feces is very high. Data
show that 1 g of feces dissolved in 100 L of water is highly infectious
to pigs. Our continuous monitoring of the fecal virus shedding of
diseased sows found that they could intermittently shed virus for more
than two weeks; thus, it is particularly important to strengthen
disinfection and adopt biosafety measures. From the perspective of the
hazards of PEDV, before 2015, PEDV caused fulminant epidemics
nationwide. Since 2016, as China has gradually gained a clear
understanding of the prevention and control of PED, acute fulminant
epidemics have decreased significantly. It is worth noting that the
pathogenicity of highly pathogenic strains has not decreased.

To summarize, PEDV outbreaks on large-scale pig farms occurred from
regularly in the second half of 2020. The protective effects of
inactivated and attenuated live PEDV vaccines available on the market
are not satisfactory. The disease may develop despite elevated antibody
titers following immunization with vaccines, which indicates that the
existing vaccines may not provide adequate immune protection against
epidemic strains. This may be related to the overall health of the pig
herd and weak cross-protection between strains. The farm described in
this report, where the infection occurred, could finally control the
epidemic through immunization with autogenous inactivated vaccines,
biosafety measures, and strict cleaning and disinfection. Therefore, the
prevention and control of porcine epidemic diarrhea warrant
comprehensive prevention and control measures. By appropriate selection
of a suitable vaccine and effective management measures ideal prevention
and control may be achieved.

\textbf{Declarations}

Acknowledgments

Not applicable

Funding

This research was funded by the Industry Technology System of Modern
Agriculture Construction Fund (CARS-35).

Availability of data and materials

Please contact the authors with data requests.

Authors' contributions

LG analyzed the data and drafted the manuscript. QG and ZZ carried out
most of the experiments. HW participated in the study design. GZ
conceived the study. All authors read and approved the final manuscript.

Consent for publication

All authors consent for publication.

Competing interests

The authors declare that they have no conflict of interest.

\textbf{Figure Legends}

Fig 1. Time-mortality dynamic curve. The X axis shows time, and the Y
axis shows the number of died pigs. The arrow is the time for the farm
to inject its own inactivated vaccine.

Fig 2. Phylogenetic trees of isolates and a representative virus based
on the S gene sequence. the tree was performed by neighbor-joining
method using MEGA with 1,000 bootstrap replicates. The names of GenBank
accession numbers, the strains, years, and places of isolation are show
on the right in order, and the genogroups of virus are shown on the
right of strains.

Supplementary Fig 1. Percentages of nucleotide identity among 26 PEDV
isolates based on S gene sequence.

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