\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[ngerman,greek,english]{babel}



\usepackage{float}








\begin{document}

\title{First report of variant pseudorabies virus infection in goats in China:
a neglected infectious disease to ruminants}



\author[1]{lei Tan}%
\author[1]{Kaiwen Xu}%
\author[2]{Shibiao Yang}%
\author[1]{Wen Wang}%
\author[3]{Gao Lin}%
\author[1]{Lincheng Yang}%
\author[3]{Jun Yao}%
\author[1]{Aibng Wang}%
\affil[1]{Hunan Agricultural University}%
\affil[2]{Affiliation not available}%
\affil[3]{Yunnan Animal Science and Veterinary Institute}%


\vspace{-1em}



  
  \date{May 29, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Pseudorabies virus (PRV), the cause agent of Aujeszky's disease, is an
infectious pathogen which greatly affects the heathy development of pig
industry worldwide. The low specific host tropism of PRV allows this
virus to infect a variety of animals, such as pigs, cattle, minks, dogs,
and even possible humans. However, the occurrence of PRV natural
infection in goats has never been documented. Herein we provided robust
evidences demonstrating the first case of a variant PRV infection
leading to the acute goat death in Yunnan Province, China, which might
be resulted from mixed feeding with PRV-infected fattening pigs.
Therefore, this report not only highlights the potential threat of newly
emerging variant PRV strain(s) to goat industry, but also appeals the
development of effective and safe vaccines against PRV variants for
goats/ruminants in future.%
\end{abstract}%



\sloppy


\textbf{First report of variant pseudorabies virus infection in goats in
China: a neglected infectious disease to ruminants}

\textbf{Running title: Pseudorabies virus infection in goats}

Lei Tan\textsuperscript{1}, Kaiwen Xu\textsuperscript{1}, Shibiao
Yang\textsuperscript{2}, Wen Wang\textsuperscript{1}, Lin
Gao\textsuperscript{2}, Lingchen Yang\textsuperscript{1}, Jun
Yao\textsuperscript{2}, Aibing Wang\textsuperscript{1,3}

\textsuperscript{1}Lab of Animal Disease Prevention \& Control and
Animal model, Hunan Provincial Key Laboratory of Protein Engineering in
Animal Vaccines, College of Veterinary Medicine, Hunan Agricultural
University (HUNAU), Changsha, Hunan, 410128 China.

\textsuperscript{2}Yunnan Tropical and Subtropical Animal Virus Diseases
Laboratory, Yunnan Animal Science and Veterinary Institute, Kunming,
Yunnan, 650224 China.

\textsuperscript{3}PCB Biotechnology LLC, Rockville, Maryland, USA
20852.

\textbf{Correspondence} : Aibing Wang, College of Veterinary Medicine,
Hunan Agricultural University, Changsha, China.

\textbf{Email} : \emph{bingaiwang@hunau.edu.cn}

And

Jun Yao, Yunnan Tropical and Subtropical Animal Virus Diseases
Laboratory, Yunnan Animal Science and Veterinary Institute, Yunnan,
China.

\textbf{Email} : \emph{yaojun\_joshua@hotmail.com}

\textbf{Summary}

Pseudorabies virus (PRV), the cause agent of Aujeszky's disease, is an
infectious pathogen which greatly affects the heathy development of pig
industry worldwide. The low specific host tropism of PRV allows this
virus to infect a variety of animals, such as pigs, cattle, minks, dogs,
and even possible humans. However, the occurrence of PRV natural
infection in goats has never been documented. Herein we provided robust
evidences demonstrating the first case of a variant PRV infection
leading to the acute goat death in Yunnan Province, China, which might
be resulted from mixed feeding with PRV-infected fattening pigs.
Therefore, this report not only highlights the potential threat of newly
emerging variant PRV strain(s) to goat industry, but also appeals the
development of effective and safe vaccines against PRV variants for
goats/ruminants in future.

\textbf{Keywords:} pseudorabies virus variant, goat, natural infection,
China

\textbf{Introduction}

Pseudorabies virus (PRV), also called as Suid alphaherpesvirus 1 that
belongs to the genus \emph{Varicellovirus} of the
sufamily\emph{Alphaherpesviridae} , within the family
\emph{Herpesviridae} , is an enveloped virus with a length of nearly 143
kb linear double-stranded DNA genome encoding more than 72 genes (Sun et
al., 2016; Szpara et al., 2011). The unique natural and reservoir host
for PRV are pigs (including wild boars), however the first suspected
infection of this pathogen was recorded in American cattle with clinical
signs characterized by intense itching in 1813 (Laval \& Enquist, 2020).
Since the initial isolation of PRV in 1902, the presence of this
pathogen was widely documented almost in all pig raising countries,
indicating that PRV has quickly swept across the world and posed a huge
threat to various animal species, especially to the development of pig
industry (Lee \& Wilson, 1979).

Pseudorabies (PR) is a highly contagious disease that causes severe
clinical symptoms in the infected pigs, thereby leading high morbidity
and mortality especially to the sucking piglets. The infected piglets
usually present clinical signs characterized by diarrhea, vomiting,
nervous system disorders (such as tremor, dyskinesia, and lethargy) (Sun
et al., 2016). Vaccination is regarded as the most effective approach
for the eradication of PR. Though the infectious disease has
successfully been eradicated in some developed countries via the wide
application of virulent gene-deleted live vaccines, PRV remains a fatal
pathogen circulating in Chinese pig population (Freuling, M\selectlanguage{ngerman}üller, \&
Mettenleiter, 2017; Sun et al., 2018). In this regard, at least two
great PR outbreaks have been witnessed in China, mainly resulting from
PRV classical strains prevalent in the 1990s and variant strains
circulating after 2011, respectively.

PRV has a broad species tropism, which facilitates it to infect a
variety of mammals, including carnivores (Jin, Gao, Liu, Zhang, \& Hu,
2016; H. Liu et al., 2017; Serena et al., 2018), ruminants (Cheng et
al., 2020), and rodents. Even a case of human encephalitis caused by a
PRV variant strain has been reported recently (Q. Liu et al., 2020).
However, naturally occurring PRV infection in goats has not been
reported yet. In current study, we provided the first case of a variant
PRV infection causing the acute goat death in Yunnan province, China.
Meanwhile, the genetic characteristics of this PRV strain YNG were
characterized in detail.

\textbf{Materials and methods}

\textbf{2.1 Clinical symptoms and specimen collection}

A total of 136 black goats showing clinical signs of possible PRV
infection were included in this study. The clinical symptoms in these
goats were carefully observed and characterized. Splanchnic (including
lymphonodus, lung, kidney, and liver) and brain tissues from the dead
goats were collected for pathogenic diagnosis by polymerase chain
reaction (PCR) or reverse transcriptase-coupled polymerase chain
reaction (RT-PCR), and virus isolation. Meanwhile, 10 serum and 16 fecal
samples from fattening pigs in this farm were collected for the
detection of antibodies and pathogens, respectively. Additionally,
related information including feeding and housing of these goats was
surveyed and documented.

\textbf{2.2 Identification of PRV in collected specimens}

The homogenates of each tissue specimen from goats or fecal samples from
pigs living nearby were mixed with sterile phosphate-buffered saline
(PBS) and undergone repeated freezing and thawing cycles, then
centrifuged at 12000×g for 10 min. The viral nucleic acids in the
supernatants were extracted using a commercial DNA/RNA extraction kit
(Takara, Dalian, China), and stored at -80 \selectlanguage{english}.

Prepared DNA samples were used for detecting the presence of PRV by PCR
method with a pair of primers listed in \textbf{Tab. 1} , PCR reaction
(25 \selectlanguage{greek}µ\selectlanguage{english}L) mixture included 12.5 \selectlanguage{greek}µ\selectlanguage{english}L of 2\selectlanguage{ngerman}×Taq Plus PCR Master mix (Takara,
Dalian, China), 1 \selectlanguage{greek}µ\selectlanguage{english}L of each primer (10 pmol), 4 \selectlanguage{greek}µ\selectlanguage{english}L of DNA template, and
6.5 \selectlanguage{greek}µ\selectlanguage{english}L of DPEC treated water, the cycling parameters were performed as
described previously (Tan et al., 2020). Prepared RNA samples were
exploited to detect Rabies virus (RV) and Caprine arthritis encephalitis
virus (GAEV), respectively, as previously established methods (Li et
al., 2013). PCR products were visualized in 1\% agarose gel
electrophoresis.

\textbf{2.3 Virus isolation}

Cerebrum specimens from the dead goat were handled as describe above,
the supernatants were filtrated through a 0.22 um filter (Millipore,
USA) and incubated with monolayer PK15 cells. The cells were cultured in
Dulbecco modified eagle medium (DMEM) supplied with 5\% newborn bovine
serum (NBCS) and 1\% streptomycin/penicillin. After one-hour incubation,
the supernatants were removed and replaced with maintenance medium (DMEM
with 2\% NBCS and 1\% streptomycin/penicillin). When obvious cytopathic
effects (CPEs) were observed, the cells and supernatants were harvested.
After three cycles of thawing and refreezing, PCR was performed to
detect the presence of PRV nucleic acid in the supernatants. Isolated
and identified PRV virus was designated as YNG strain.

\textbf{2.4 Indirect immunofluorescence assay (IFA)}

PK15 cells infected with YNG strain were fixed with 4\% paraformaldehyde
for 20 min and permeabilized with 0.5\% Triton X-100 for 10 min at room
temperature, respectively. Then the cells were blocked with 3\% bovine
serum albumin (BSA) for 1h, and probed with mouse monoclonal antibodies
against the \emph{gB} and \emph{gE} proteins of PRV (1:1000) for 4 h.
Following the removal of the supernatants, the cells were washed with
PBS three times, the second antibodies (goat anti-mouse-FITC) (1:2500)
were applied and incubated for 45 min in dark environment. The images
were taken using a fluorescent microscope (Olympus, Tokyo, Japan).

\textbf{2.5 One-step growth curve experiment}

To investigate the growth characteristics of YNG strain in vitro,
monolayer Vero and PK15 cells seeded in 24-well plates were incubated
with different strains (YNG and YNP, an isolation from pig farms where
was close to this PRV-outbreaking goat farm) at multiplicity of
infection (MOI) of 0.01 for 1 h. Both the cell debris and supernatant
were collected after repeated freezing and thawing cycles, the viral
copies and titers were determined via real-time PCR after the DNA
extraction and TCID\textsubscript{50} assay, respectively.

\textbf{2.6 Sequence alignment and phylogenetic analysis}

To analyze the genetic characteristics of PRV strain isolated from goat
in the present study, the major viral immune (\emph{gB} , \emph{gC} ,
and\emph{gD} ) and virulence genes (\emph{gE} and \emph{TK} ) of YNG
strain were amplified by PCR with specific primers (\textbf{Tab. 1} ).
Positive PCR products were purified, sequenced by Tsingke company
(Changsha, China), these sequences were submitted to the Genbank
database. These newly sequences, together with their corresponding
reference sequences downloaded from GenBank database, were aligned using
DNAStar version 7.10 (Lasergene) software for comparison. Phylogenetic
trees were also re-constructed using the neighbor-joining (NJ) method in
MEGA 7.0 software with 1000 bootstrap replicates based on the \emph{gC}
and\emph{gE} gene, respectively.

\textbf{2.7 Enzyme-linked immunosorbent assay (ELISA)}

16 serum specimens from pigs were collected to examine the presence
of\emph{gE} antibodies using a commercial ELISA kit (IDEXX Laboratories,
USA).

\textbf{2.8 Experimental infection of mice}

Twenty-four 5-week-old female Kunming mice purchased from Hunan SJA
laboratory animal CO., LTD were randomly divided into four groups, six
mice in each group were challenged with
10\textsuperscript{4}TCID\textsubscript{50} of various PRV strains (YNG,
YNP, and another classical PRV strain genetically close to Ea strain,
HNLY) via hind footpad injection, respectively, while the mice in the
control group were inoculated with equal volume of DMEM. These mice in
distinct groups were maintained in the same environment, and their
health conditions were monitored three times per day. The survival curve
of mice in each group was produced by using the GraphPad Prism 8.0
software (GraphPad software, La Jolla, CA, USA).

\textbf{2.9 Statistical analysis}

All experiments received more than three replicates, the statistical
significances of data obtained from different groups were analyzed by
GraphPad Prism version 8.0 (GraphPad software, La Jolla, CA, USA), and
ns represents p\textgreater{}0.05, *p\textless{}0.05,
**p\textless{}0.01, ***p\textless{}0.001, respectively.

\textbf{Results}

\textbf{3.1 Detection of the causative agent(s) causing the death of
goats}

In November 2015, most of black goats showing clinical signs of possible
PRV infection were reported in a farm in Yunnan, China. Subsequently, a
detailed survey was conducted, the symptoms in these goats included high
fever, appetite loss, skin itch and fidgety with no ages and sexes
limitation (\textbf{Fig. 1A-B} ). Additionally, some of the ill goats
displayed severe diarrhea (\textbf{Fig. 1C} ), and all of these goats
died eventually. The pathological examination showed the occurrence of
pneumonia hemorrhage, lymphadenopathy syndrome with hemorrhage, brain
hyperemia and hemorrhage in dead goats (\textbf{Fig. 1D} ).

Further investigation revealed that these goats were fed together with
fatted pigs only by the separation of a wall, while all pigs in this
farm had been immunized with the PRV attenuated live vaccine (Bartha-K61
strain) at the development stage of piglets. Furthermore, all animals
were fed by the same breeder.

To explore the possible causative agent(s) leading to the death of goats
in this case, the viral nucleic acids were extracted from brain tissues
of the dead goats, PCR and RT-PCR methods were performed to detect the
presence of pathogens such as PRV, RV, and GEAV. Interestingly, the
specimens presented PRV positive while RV and GEAV negative.
Furthermore, the treatment of these diseased goats with antibacterial
drugs, such as Cefatriaxone and Sulfadiazine, had poor therapeutic
effect. Meanwhile, 10 serum specimens from the fattening pigs raised
near the goats were positive for PRV \emph{gE} antibody, while only 1 of
16 fecal samples from these pigs were tested positive for PRV by PCR
method (data not shown). Thus, we concluded that PRV infection was the
leading cause resulting in the death of goats.

\textbf{3.2 Isolation and identification of PRV strain from the dead
goat}

To characterize the features of PRV strain from goat, the supernatants
of cerebrum specimen from the dead goat were incubated into monolayer
PK15 cells for 1 h, and the cells were then harvested upon observing the
appearance of over 80\% CPEs. Obvious CPEs could be observed less than
16 h post-infection (\textbf{Fig. 2A} ), furthermore, the presence of
PRV nucleic acids (namely, PRV-positive) was confirmed by PCR in the
supernatants of the first, second, third, fifth, and seventh passages of
cells incubated with YNG strain (\textbf{Fig. 2B} ). Additionally, the
infectious virions of YNG and YNP strains in Vero cells were specially
recognized by mouse monoclonal antibodies against the \emph{gB}
and\emph{gE} proteins of PRV via IFA (\textbf{Fig. 2C} ).

\textbf{3.3 Growth characteristics of YNG strain comparing with other
variant strains}

One-step growth curve experiment, CPE observation, and TCID50 assay were
conducted to analyze the growth characteristics of these PRV strains
aforementioned in PK15 and Vero cells, with YNG strain as the control.
The viral copies were determined according to the standard curve: lg
{[}virus copies{]} = -0.2293x+9.832, r\textsuperscript{2}=0.9963. As
shown in \textbf{Fig. 3} , YNG strain exhibited a similar growth curve
as that of YNP strain both in PK15 and Vero cells. The virus titers of
YNG and YNP strains in PK15 and Vero cells at the 36 hpi were
10\textsuperscript{7.14}, 10\textsuperscript{6.88} and
10\textsuperscript{6.80},
10\textsuperscript{6.75}TCID\textsubscript{50}/mL, respectively
(\textbf{Fig. 3} ). Likewise, there was no obvious difference of CPEs in
PK15 and Vero cells triggered by distinct PRV strains, suggesting that
YNG strain should be a PRV variant.

\textbf{3.4 Analysis of sequence variation in the newly goat PRV strain}

To further address the genetic characteristics of YNG strain, the major
viral immune (\emph{gB} , \emph{gC} , and \emph{gD} ) and virulence gene
(\emph{gE} and \emph{TK} ) sequences were successfully amplified and
sequenced. The findings revealed that the lengths of \emph{gB}
,\emph{gC} , \emph{gD} , \emph{gE} and \emph{TK} genes of YNG strain
were 2745 bp, 1464 bp, 1209 bp, 1737 bp, and 1023 bp, respectively
(GenBank accession numbers: MW458931, MW392295, MW458932, MW458933,
MW458934). Sequence analysis results also showed that there existed
continuous nucleotides insertion
encoding\textsuperscript{65}AASTPAA\textsuperscript{71} in the \emph{gC}
gene in comparison to referring PRV strains in genotype I, such as
Bartha and Becker strains (\textbf{Fig. 4A} ). Similarly, a 6-nucleotide
deletion (encoding \textsuperscript{278}SP\textsuperscript{279}) in the
\emph{gD}gene was identified in YNG strain relative to classical PRV
strains (eg. Fa and Ea) prevalent in Chinese pig population before 2011,
indicating that YNG strain is a PRV variant (\textbf{Fig. 4B} ).
Compared with other referring variants, only few amino acid variations
in \emph{gB} gene of YNG strain were identified, while no amino acid
variations of \emph{TK} ,\emph{gC} , and \emph{gD} genes were found.
Notably, compared to other PRV strains, a 3-nt deletion (encoding D at
the position of 490) was found in the \emph{gE} gene of YNG strain and
another variant PRV strain (JX/CH/2016, Genbank number accession:
MK806387) isolated in Guangdong province, China (\textbf{Fig. 4C} ).

To define the evolutionary position of YNG strain among numerous
referring strains from different regions or hosts, \emph{gC}
and\emph{gE} gene based phylogenetic trees were constructed,
respectively (\textbf{Fig. 4D\&E} ). The results demonstrated that all
PRV strains prevalent in the world could be divided into two genotypes
(genotype I and II), and the genotype II was composed of classical PRV
strains prevalent before 2011 and variant PRV strains circulated after
2011 in China. Notably, most of PRV strains isolated from different
hosts (pigs, dogs, wolves, sheep, foxes, human, etc.) prevalent in
China, including YNG strain, belonging to the genotype II, presented a
random distribution. Additionally, the phylogenetic position of YNG
strain was closer to these of variant PRV strains than classical PRV
strains, and far away from the traditional vaccine strains (such as
Bartha) belonging to the genotype I.

\textbf{3.5 The pathogenicity of YNG strain in vivo}

In order to evaluate the pathogenicity of YNG strain in vivo, Kunming
mice (six-week-old) were chosen as animal models and inoculated with 0.1
mL of 10\textsuperscript{5} TCID\textsubscript{50}/mL of YNG, YNP, or
HNLY strain via intramuscular injection at the hind leg, respectively.
The clinical signs and death time of the mice in different groups were
carefully observed. The results revealed that all of the mice challenged
with YNG or YNP strain displayed severe itching before 42 hpi, and died
before 72 hpi, while the mice inoculated with HNLY strain only exhibited
slightly clinical signs before 72 hpi (\textbf{Fig. 5A} ). Additionally,
the brains of mice infected with YNG and YNP strains showed similar
pathological changes, such as severe hemorrhage, while those challenged
with HNLY strain only presented slight hemorrhage lesion when compared
with the control group (\textbf{Fig. 5B} ). Accordingly, higher viral
copies in the cerebrum, kidney, and spleen of YNG or YNP strain-infected
mice were found, relative to those in HNLY strain-infected mice
(\textbf{Fig. 5C} ), indicating a similar pathogenicity of YNG strain as
PRV variant to mice.

\textbf{Discussion}Though extensive efforts have been devoted to the
eradication of the disease caused by this pathogen, PRV remains pandemic
in some countries/regions, mainly in Asia and Europe (Q. Liu et al.,
2020). For instance, the latest researches revealed that the average
positive rate of PRV nucleic acid among 16256 tissue specimens collected
from the mainland China between 2012 and 2017 was 8.3\% (Sun et al.,
2018), while the sero and molecular prevalence rates of wild PRV in pigs
in Shandong province of China from 2015 to 2018 even reached to 52.7\%
(8667/16457), 15.7\% (257/1638), respectively (Ma et al., 2020),
indicating the still severe status of wild PRV in Chinese pig
populations. The current study reported the first case of natural PRV
infection in goats, thereby further expanding the list of non-natural
hosts challenged by this pathogen. Thus, our new finding, together with
previously confirmed species including humans and its lethality to these
susceptible animals (Ai et al., 2018; Cheng et al., 2020; Jin et al.,
2016; H. Liu et al., 2017) and multiple infection ways (Q. Liu et al.,
2020), collectively suggest that PRV may be a zoonotic pathogen (Ai et
al., 2018; Yang et al., 2019) and more attention should be paid to it in
the future.

Large numbers of ruminants raised in China, severe prevalence of PRV in
Chinese pig population and frequent transportation of various animals
(pigs, goats/sheep, cattle, etc.,) among different regions greatly
enhance the possibility of the contact between pigs and these PRV
susceptible animals, thereby leading to the transmission of PRV from
host pigs to non-host animals (Tan et al., 2021). Consistent with this,
our study for the first time confirmed the PRV transmission from pigs to
goats, mainly due to the close contact of raising places for those two
species. This conclusion was stemmed from a series of evidences: a)
serum or fecal specimens from pigs raised nearby presented PRV antibody
or nucleic acid positive; b) PRV viruses were confirmed and isolated
from infected or died goats; c) isolated YNG strain exhibited highly
genetic sequence homology with PRV variant strain YNP, moreover, its
pathogenicity was successfully reproduced in mice as its counterpart
YNP. These highlight the really threat of PRV to goats or even
ruminants, as partially supported by a previous report (Kong et al.,
2013). As a precaution measure, far physical isolation of raising places
for different animal species is therefore suggested.

Genetically, PRV strains prevalent in the world are divided into two
genotypes, most of PRV strains in China belong to the genotype II (He et
al., 2019), while the latter one can be further classified into two
sub-genotypes (PRV classical and variant strains). Furthermore, PRV
variant strains emerged since 2011 (e.g. JS-2012, HN1201, hSD-1/2019) in
China showed higher pathogenicity to animal models than classical
strains (i.e. Ea and Fa) (Luo et al., 2014; Tong et al., 2015). More
direct evidences substantiating the conclusion were that vaccination
with PRV Bartha-K61 strain could prevent the occurrence of classical PRV
infection but fail to provide complete protection against the newly
emerging PRV variants (8, 9). Importantly, the attenuated live vaccine
(Bartha-K61 strain) instead led to high pathogenicity to sheep (Kong et
al., 2013). Likewise, immunization with Bartha-K61 strain or HB98 strain
(another PRV attenuated live vaccine designed based on Ea strain) caused
histopathological lesions in dogs (Lin et al., 2019; Willemse, Rondhuis,
Goedegebuure, \& Maas, 1977). It is worth noting that the recombinant
PRV variant strain with US7\&US8\&UL23 gene deletion is highly lethal to
dogs, while the US7\&US8\&UL23\&US3 gene-deleted recombinant PRV strain
is safe to this species. Of course, whether this genetically modified
PRV strain can be developed for vaccine candidates against variant PRV
infection in dogs needs further investigations (Yin et al., 2020). Taken
together, these facts described above suggest that currently commercial
vaccines might be not suitable for the eradication of PR in ruminants
(eg. sheep/goats and cattle), accordingly, safer vaccines with efficient
protection should be developed for this purpose in the future.

In summary, we reported the first case of the occurrence of PR in goats
and confirmed that the goat-originated PRV strain was a Chinese variant
strain by genetic sequence analysis. These suggest that variant PRV
strains should receive sufficient attention in terms of the healthy
development of goat industry in China. Considering the confirmed PRV
infection to humans (Q. Liu et al., 2020), the potential transmission of
PRV from goats to humans cannot be ruled out.

\textbf{Acknowledgement}

This work was supported by the General Program of National Natural
Science Foundation of China (Grants No. 31571432/31802252), the National
Key Research and Development Program of China (No. 2017YFD0501800), the
Hunan Provincial Natural Science Foundation of China (2020JJ4041), the
Postgraduate Scientific Research Innovation Project of Hunan Province
(CX20200659), and the Finance Project of Yunnan Province (No.
20161105560334).

\textbf{Conflict of interests}

The authors declare that they have no competing interests.

\textbf{Ethical approval}

All animal experiments were conducted according to the rules of Animal
Ethics Committee of the Hunan Agricultural University, Changsha, China
(43321503), and the mice were housing in the animal facility of Hunan
Agricultural University (Changsha, Hunan, China).

\textbf{References}

Ai, J. W., Weng, S. S., Cheng, Q., Cui, P., Li, Y. J., Wu, H. L., . . .
Zhang, W. H. (2018). Human Endophthalmitis Caused By Pseudorabies Virus
Infection, China, 2017.\emph{Emerg Infect Dis, 24} (6), 1087-1090.
doi:10.3201/eid2406.171612

Cheng, Z., Kong, Z., Liu, P., Fu, Z., Zhang, J., Liu, M., \& Shang, Y.
(2020). Natural infection of a variant pseudorabies virus leads to
bovine death in China. \emph{Transbound Emerg Dis, 67} (2), 518-522.
doi:10.1111/tbed.13427

Freuling, C. M., M\selectlanguage{ngerman}üller, T. F., \& Mettenleiter, T. C. (2017). Vaccines
against pseudorabies virus (PrV).\emph{Vet Microbiol, 206} , 3-9.
doi:10.1016/j.vetmic.2016.11.019

He, W., Auclert, L. Z., Zhai, X., Wong, G., Zhang, C., Zhu, H., . . .
Su, S. (2019). Interspecies Transmission, Genetic Diversity, and
Evolutionary Dynamics of Pseudorabies Virus. \emph{J Infect Dis, 219}
(11), 1705-1715. doi:10.1093/infdis/jiy731

Jin, H. L., Gao, S. M., Liu, Y., Zhang, S. F., \& Hu, R. L. (2016).
Pseudorabies in farmed foxes fed pig offal in Shandong province, China.
\emph{Arch Virol, 161} (2), 445-448. doi:10.1007/s00705-015-2659-9

Kong, H., Zhang, K., Liu, Y., Shang, Y., Wu, B., \& Liu, X. (2013).
Attenuated live vaccine (Bartha-K16) caused pseudorabies (Aujeszky's
disease) in sheep. \emph{Vet Res Commun, 37} (4), 329-332.
doi:10.1007/s11259-013-9568-8

Laval, K., \& Enquist, L. W. (2020). The Neuropathic Itch Caused by
Pseudorabies Virus. \emph{Pathogens, 9} (4).
doi:10.3390/pathogens9040254

Lee, J. Y., \& Wilson, M. R. (1979). A review of pseudorabies
(Aujeszky's disease) in pigs. \emph{Can Vet J, 20} (3), 65-69.

Li, Y., Zhou, F., Li, X., Wang, J., Zhao, X., \& Huang, J. (2013).
Development of TaqMan-based qPCR method for detection of caprine
arthritis-encephalitis virus (CAEV) infection.\emph{Arch Virol, 158}
(10), 2135-2141. doi:10.1007/s00705-013-1728-1

Lin, W., Shao, Y., Tan, C., Shen, Y., Zhang, X., Xiao, J., . . . Xie, Q.
(2019). Commercial vaccine against pseudorabies virus: A hidden health
risk for dogs. \emph{Vet Microbiol, 233} , 102-112.
doi:10.1016/j.vetmic.2019.04.031

Liu, H., Li, X. T., Hu, B., Deng, X. Y., Zhang, L., Lian, S. Z., . . .
Yan, X. J. (2017). Outbreak of severe pseudorabies virus infection in
pig-offal-fed farmed mink in Liaoning Province, China. \emph{Arch Virol,
162} (3), 863-866. doi:10.1007/s00705-016-3170-7

Liu, Q., Wang, X., Xie, C., Ding, S., Yang, H., Guo, S., . . . Li, W.
(2020). A novel human acute encephalitis caused by pseudorabies virus
variant strain. \emph{Clin Infect Dis} . doi:10.1093/cid/ciaa987

Luo, Y., Li, N., Cong, X., Wang, C. H., Du, M., Li, L., . . . Qiu, H. J.
(2014). Pathogenicity and genomic characterization of a pseudorabies
virus variant isolated from Bartha-K61-vaccinated swine population in
China. \emph{Vet Microbiol, 174} (1-2), 107-115.
doi:10.1016/j.vetmic.2014.09.003

Ma, Z., Han, Z., Liu, Z., Meng, F., Wang, H., Cao, L., . . . Liu, M.
(2020). Epidemiological investigation of porcine pseudorabies virus and
its coinfection rate in Shandong Province in China from 2015 to 2018.
\emph{J Vet Sci, 21} (3), e36. doi:10.4142/jvs.2020.21.e36

Serena, M. S., Metz, G. E., Lozada, M. I., Aspitia, C. G., Nicolino, E.
H., Pidone, C. L., . . . Echeverria, M. G. (2018). First isolation and
molecular characterization of Suid herpesvirus type 1 from a domestic
dog in Argentina. \emph{Open Vet J, 8} (2), 131-139.
doi:10.4314/ovj.v8i2.3

Sun, Y., Liang, W., Liu, Q., Zhao, T., Zhu, H., Hua, L., . . . Wu, B.
(2018). Epidemiological and genetic characteristics of swine
pseudorabies virus in mainland China between 2012 and 2017. \emph{PeerJ,
6} , e5785. doi:10.7717/peerj.5785

Sun, Y., Luo, Y., Wang, C. H., Yuan, J., Li, N., Song, K., \& Qiu, H. J.
(2016). Control of swine pseudorabies in China: Opportunities and
limitations. \emph{Vet Microbiol, 183} , 119-124.
doi:10.1016/j.vetmic.2015.12.008

Szpara, M. L., Tafuri, Y. R., Parsons, L., Shamim, S. R., Verstrepen, K.
J., Legendre, M., \& Enquist, L. W. (2011). A wide extent of
inter-strain diversity in virulent and vaccine strains of
alphaherpesviruses. \emph{PLoS Pathog, 7} (10), e1002282.
doi:10.1371/journal.ppat.1002282

Tan, L., Li, Y., He, J., Hu, Y., Cai, X., Liu, W., . . . Wang, A.
(2020). Epidemic and genetic characterization of porcine epidemic
diarrhea virus strains circulating in the regions around Hunan, China,
during 2017-2018. \emph{165} (4), 877-889.
doi:10.1007/s00705-020-04532-7

Tan, L., Yao, J., Yang, Y., Luo, W., Yuan, X., Yang, L., \& Wang, A.
(2021). Current Status and Challenge of Pseudorabies Virus Infection in
China. \emph{Virol Sin} , 1-20. doi:10.1007/s12250-020-00340-0

Tong, W., Liu, F., Zheng, H., Liang, C., Zhou, Y. J., Jiang, Y. F., . .
. Tong, G. Z. (2015). Emergence of a Pseudorabies virus variant with
increased virulence to piglets.\emph{Vet Microbiol, 181} (3-4), 236-240.
doi:10.1016/j.vetmic.2015.09.021

Willemse, A., Rondhuis, P. R., Goedegebuure, S. A., \& Maas, J. H.
(1977). {[}An atypical form of Aujezky's disease after vaccination
(author's transl{]}. \emph{Tijdschr Diergeneeskd, 102} (6), 386-388.

Yang, X., Guan, H., Li, C., Li, Y., Wang, S., Zhao, X., . . . Liu, Y.
(2019). Characteristics of human encephalitis caused by pseudorabies
virus: A case series study.\emph{Int J Infect Dis, 87} , 92-99.
doi:10.1016/j.ijid.2019.08.007

Yin, H., Li, Z., Zhang, J., Huang, J., Kang, H., Tian, J., \& Qu, L.
(2020). Construction of a US7/US8/UL23/US3-deleted recombinant
pseudorabies virus and evaluation of its pathogenicity in dogs.
\emph{Vet Microbiol, 240} , 108543. doi:10.1016/j.vetmic.2019.108543

\textbf{Figure captions}

\textbf{Figure 1} \selectlanguage{english}:Typical clinical signs and pathological changes were
observed in PRV-infected goats, such as skin itch and fidgety
(\textbf{A\&B} ), diarrhea (\textbf{C} ). Also, severe hyperemia and
hemorrhage were shown on the cerebrum from the infected goat (\textbf{D}
).

\textbf{Figure 2} : Isolation and identification of YNG strain from
goat. (\textbf{A} ) Obvious the cytopathic effects (CPE) of PK15 cells
induced by the infection of the third passages of YNG strain, the CPE of
cells in the left were characterized by being rounded and floated. The
right cells were set as negative group, Scale bar = 200 um. (\textbf{B}
) The existence of infectious PRV virions in the cell cultures of the
first, second, third, fifth, and seventh passages were confirmed via
PCR, which could yielded the expected DNA bands (\textasciitilde{}450
bp) of PRV gE in 1\% agarose gel electrophoresis. DNA+ and DNA-- were
regarded as positive and negative controls during DNA extraction,
respectively. And PCR- was the negative control during PCR
amplification. (\textbf{C} ) Indirect immunofluorescent assay (IFA) for
the detection of PRV in Vero cells using anti-PRV gE and gB primary
antibodies. Scale bar = 100 um

\textbf{Figure 3} : The proliferation characteristics of YNG strains in
vitro. (\textbf{A} ) One-step growth curves of YNG and YNP strains on
PK15 and Vero cells at MOI of 0.01, the total PRV copies from the
supernatant and cells in triplicates were measured via real-time PCR.
(\textbf{B} ) The viral titers of YNG and YNP at 36 hpi were determined
in PK15 cells by TCID\textsubscript{50}.

\textbf{Figure 4:} Analysis of sequence variation in YNG strain.
(\textbf{A} ) A 7-aa insertion
(\textsuperscript{64}AASTPAA\textsuperscript{70}) in the gC gene of PRV
strains in genotype II compared with that in genotype I. (\textbf{B} ) A
2-aa deletion (\textsuperscript{278}SP\textsuperscript{279}) in the gD
gene of PRV variants in genotype II and Becker strain compared with that
of classical PRV strains in genotype II. (\textbf{C} ) A 3-nt deletion
(\textsuperscript{1469}ACG\textsuperscript{1471}) encoding D at the
position of 490 in the \emph{gE} gene of YNG strain and another variant
PRV strain prevalent in China (GenBank no. MK806387 (JX/CH/2016)
compared with that of the other strains. (\textbf{D\&E} ) Phylogenetic
trees based on \emph{gC} and \emph{gE} gene sequences of YNG strain and
other reference strains generated by the neighbor-joining method in MEGA
7.0 software. The red and black prismatic represented YNG strain
obtained in this study and PRV strain isolated from human, respectively.

\textbf{Figure 5} : The pathogenicity of YNG strain in mouse model.
(\textbf{A} ) Survival curve (n=6/group) in Kunming mice after
intramuscular injection with different PRV strains
(10\textsuperscript{4} TCID\textsubscript{50}/mouse). (\textbf{B} )
Pathological changes of cerebrum from mouse in different groups.
(\textbf{C} ) The viral copies in different organs from mice infected
with different PRV strains were determined according to the standard
curve constructed by the threshold cycle (CQ) values of the serial
10-fold dilution of standard plasmid with known copies via real-time PCR
method. Values are means+-standard deviation (SD) = 3, significant
differences were determined when P\textgreater{}0.05 (ns),
P\textless{}0.05(*), P\textless{}0.01(**), and P\textless{}0.001(***).

\textbf{Hosted file}

\verb`Table 1.doc`  available at \url{https://authorea.com/users/332067/articles/708989-first-report-of-variant-pseudorabies-virus-infection-in-goats-in-china-a-neglected-infectious-disease-to-ruminants}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-1A/Fig-1A}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-1B/Fig-1B}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-1C/Fig-1C}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-1D/Fig-1D}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-2A/Fig-2A}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-2B/Fig-2B}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-2C/Fig-2C}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-3/Fig-3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-4A/Fig-4A}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-4B/Fig-4B}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-4C/Fig-4C}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-4D/Fig-4D}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-4E/Fig-4E}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-5A/Fig-5A}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-5B/Fig-5B}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-5C/Fig-5C}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

