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\begin{document}

\title{Fatal canine parvovirus type 2a and 2c infections in wild Chinese
pangolins ( Manis pentadactyla ) in southern China}



\author[1]{彦 华}%
\author[2]{Zhang Lina}%
\author[1]{Wang Kai}%
\author[1]{An Fuyu}%
\author[3]{Zhang Dongliang}%
\author[3]{Hailing Zhang}%
\author[1]{Xu Xuelin}%
\author[1]{Guo Ce}%
\author[1]{Yan Hongmei}%
\author[1]{Kuang Yingjie}%
\author[1]{Zhang Zhidong}%
\author[4]{Lu Rongguang}%
\affil[1]{Guangdong Academy of Forestry}%
\affil[2]{Guangdong Engineering Polytechic}%
\affil[3]{Chinese Academy of Agricultural Sciences Institute of Special Animal and Plant Sciences}%
\affil[4]{School of Medicine Chinese University of Hongkong Shenzhen No2001 Longxiang Avenue Shenzhen 518172 Guangdong Province China}%


\vspace{-1em}



  
  \date{May 21, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
The Chinese pangolin ( \emph{Manis pentadactyla}) is a critically
endangered scale-covered mammal belonging to the order Pholidota. Wild
pangolins are notably susceptible to pathogen infection and are
typically characterized by impoverished health. However, little is
currently known regarding the viruses prevalent among pangolins. In this
study, we report the detection of two subtypes of canine parvovirus type
2 (CPV-2), namely, CPV-2a and CPV-2c, both of which caused severe
diarrheal disease in two post-rescue pangolins with fatal consequences.
As in CPV-2-infected dogs, intensive lesion of the mucosal layer of the
small intestines is a prominent feature in infected pangolins.
Phylogenetic analysis revealed that the VP2 viral protein sequences
isolated from one pangolin were classified into the CPV-2c subclade,
with 99.8\% identities to a CPV-2c strain (MN832850) isolated from a
Taiwanese pangolin found in Taiwan Province. In contrast, VP2 sequences
obtained from the second pangolin were classified into the CPV-2a
subclade, with 99.8\% identities to a CPV-2a strain (KY386858) isolated
from southern China. In this study, we thus confirmed the infection of
pangolins with CPV-2c in mainland China and demonstrate that CPV-2a can
also infect pangolins. Based on these findings, we recommend that
further investigations should be conducted to establish the interspecies
transmission of these viruses among wild pangolins, wild carnivores, and
stray dogs.%
\end{abstract}%



\sloppy


\textbf{Fatal canine parvovirus type 2a and 2c infections in wild
Chinese pangolins (\emph{Manis pentadactyla}) insouthern China}

Zhang Lina \textsuperscript{1}, Wang Kai\textsuperscript{2}, An
Fuyu\textsuperscript{2}, Zhang Dongliang\textsuperscript{3}, Zhang
Hailing\textsuperscript{3}, Xu Xuelin\textsuperscript{2}, Guo
Ce\textsuperscript{2}, Yan Hongmei\textsuperscript{2}, Kuang
Yingjie\textsuperscript{2}, Zhang Zhidong\textsuperscript{2}, Lu
Rongguang\textsuperscript{4}*, Hua Yan\textsuperscript{2}\textbf{*}

1. Eco-Engineering Department, Guangdong Eco-Engineering Polytechnic,
Guangzhou 510520, China

2. Guangdong Provincial Key Laboratory of Silviculture, Protection and
Utilization, Guangdong Academy of Forestry, Guangzhou 510520, China

3. Key Laboratory of Special Animal Epidemic Disease of Ministry of
Agriculture and Rural Affairs, Institute of Special Animals and Plants,
Chinese Academy of Agricultural Sciences, No. 4899 Juye Street Changchun
130112, Jilin Province, China

4. School of Medicine, Chinese University of Hongkong Shenzhen, No.2001
Longxiang Avenue, Shenzhen 518172, Guangdong Province, China

*Correspondence1: wildlife530@hotmail.com

*Correspondence2: lurongguang@cuhk.edu.cn

Guangdong Provincial Key Laboratory of Silviculture, Protection and
Utilization, Guangdong Academy of Forestry, Guangzhou 510520, China.

\textbf{Abstract:} The Chinese pangolin (\emph{Manis pentadactyla} ) is
a critically endangered scale-covered mammal belonging to the order
Pholidota. Wild pangolins are notably susceptible to pathogen infection
and are typically characterized by impoverished health. However, little
is currently known regarding the viruses prevalent among pangolins. In
this study, we report the detection of two subtypes of canine parvovirus
type 2 (CPV-2), namely, CPV-2a and CPV-2c, both of which caused severe
diarrheal disease in two post-rescue pangolins with fatal consequences.
As in CPV-2-infected dogs, intensive lesion of the mucosal layer of the
small intestines is a prominent feature in infected pangolins.
Phylogenetic analysis revealed that the VP2 viral protein sequences
isolated from one pangolin were classified into the CPV-2c subclade,
with 99.8\% identities to a CPV-2c strain (MN832850) isolated from a
Taiwanese pangolin found in Taiwan Province. In contrast, VP2 sequences
obtained from the second pangolin were classified into the CPV-2a
subclade, with 99.8\% identities to a CPV-2a strain (KY386858) isolated
from southern China. In this study, we thus confirmed the infection of
pangolins with CPV-2c in mainland China and demonstrate that CPV-2a can
also infect pangolins. Based on these findings, we recommend that
further investigations should be conducted to establish the interspecies
transmission of these viruses among wild pangolins, wild carnivores, and
stray dogs.

\textbf{Introduction}

The Chinese pangolin (\emph{Manis pentadactyla} ) is a scale-covered
mammal belonging to the order Pholidota, which also contains the
congeneric Sunda or Malayan pangolin (\emph{M. javanica} ), the
Philippine pangolin (\emph{M. culioensis} ), and the Indian or
thick-tailed pangolin (\emph{M. crassicaudata} ) as co-occurring Asian
species (Gaudin, Emry, \& Wible, 2009). Pangolins have been established
to play pivotal ecological roles in the structure and function of
forests in southern China, and have accordingly been identified as key
indicator species, the status of which closely reflects that of
ecosystem health. However, as a consequence of widespread poaching and
trafficking, the number of pangolins has decreased substantially in
recent years, and across much of their distribution range, these mammals
are believed to be critically endangered.

The continued survival of wild pangolin populations is further
jeopardized by their generally poor state of health state, which is
believed to be attributable to pathogen infection. In this regard, it
has been reported that common etiological agents, such as canine
parvovirus (CPV), canine distemper virus (CDV), and parainfluenza virus
5 (PIV5), which are highly prevalent among mammals in the families in
Felidae and Canidae, can infect Pangolins (Chin et al., 2015; S. L. Wang
et al., 2020; X. Wang et al., 2019). Moreover, viral metagenomic
analyses have revealed the presence of coronavirus and Sendai virus
sequences in pangolins. (Liu, Chen, \& Chen, 2019).

CPV, a member of the family \emph{Parvoviridae} ,
genus\emph{Protoparvovirus,} and \emph{Carnivore protoparvovirus species
1}(Cotmore et al., 2014), has been characterized as an autonomously
replicable negative-sense single-stranded non-enveloped DNA virus, with
an icosahedral capsid protein of approximately 20 nm in diameter
(Organtini et al., 2015). The viral genome is approximately 5200 bases
in length and consists of two open reading frames (ORFs) with
palindromic structures at the 3' \selectlanguage{english}and 5' \selectlanguage{english}ends. ORF2 encodes the VP1 and
VP2 viral capsid proteins, the latter of which, consisting of 584 amino
acids, is the major capsid proteins that determines viral host range,
antigenicity, and hemagglutination properties (Sehata et al., 2017).
With respect to infectivity, it has been established that the residue at
position 300 of VP2 contributes to determining the host range (Allison
et al., 2016).

Given its rapid rate of nucleotide replacement, CPV is continually
evolving as a globally spreading virus (Shackelton et al., 2005), and at
present, four major CPV subtypes, namely, CPV-2, CPV-2a, CPV-2b, and
CPV-2c, are recognized worldwide (Behdenna et al., 2019, Mira et al.,
2019, Geng et al., 2015, Decaro et al., 2009, Li et al., 2019, De la
Torre et al., 2018).

Whereas transmission of subtype CPV-2 and its variants among wild
carnivores, particularly those in the families Felidae and Canidae, are
commonplace (Hoelzer \& Parrish, 2010; Voorhees et al., 2019), CPV-2
infections in animals belonging to the order Pholidota are rarely
reported. Recently, however, Wang et al. (2020) have reported that
CPV-2c subtype parvovirus can cause severe diarrhea-associated diseases
in Taiwanese pangolins, and in this study, we describe the discovery of
fatal CPV-2a and CPV-2c infection in two Chinese pangolins rescued from
forests in southern China.

\textbf{Materials and Methods}

\textbf{Sample collection}

Two sub-adult female Chinese pangolins (bodyweight: 1.1 kg), designated
P1 and P2, were found by local inhabitants in areas of natural forest
and taken to the Guangdong Wildlife Rescue Center (Guangzhou), although
both subsequently died within a week of rescue.

\textbf{Pathological observation andImmunohistochemistry}

Following post-mortem examination conducted at the Guangdong Wildlife
Rescue Center, entailing gross observations, tissues were collected from
different parts of the intestines, including the duodenum, jejunum,
ileum, colon, rectum and mesenteric lymph nodes, as well from other
major organs. These tissues were fixed in 10\% neutral formalin, then
sliced to give 5-\selectlanguage{greek}μ\selectlanguage{english}m sections, which were subsequently processed for
hematoxylin and eosin staining or immunochemical analyses using standard
protocols. Immunochemistry was performed using and in-house CPV
monoclonal antibody (mouse-origin; two-fold dilution) as the primary
antibody and horseradish peroxide (HRP)-conjugated anti-goat IgG (Sigma;
1:1000 dilution) as the secondary antibody. The histological features
and immunohistochemical results were observed and images captured using
an Olympus light microscope.

\textbf{Virus screening, isolation, and transmission electron microscopy
observations}

Viruses known to be associated with diarrheal diseases in pangolins,
including CPV, canine coronavirus (CCoV), canine distemper virus (CDV),
and dog circovirus (DogCV), were screened based on PCR analyses using
previously described primers (Deng et al., 2018; Hsu et al., 2016). For
the purposes of virus isolation, samples of pangolin tissues were
homogenized, diluted with phosphate-buffered saline, and purified by
passing through a 0.22-\selectlanguage{greek}µ\selectlanguage{english}m sterile filter. These filtered samples were
subsequently added to monolayers of F81 cells (ATCC-CL-0081) maintained
in minimum essential media supplemented with 10\% fetal bovine serum at
an amount equal to 10\% of the cell culture medium volume. The cells
were thereafter inspected daily for cytopathic effects. Cells showing
evidence of infection were collected, purified by ultracentrifugation,
and following negative staining with phosphotungstic acid, subjected to
transmission electron microscopy (Hitachi).

\textbf{ORF 2 sequence amplification and phylogeny}

Sequences of the VP2 protein were PCR amplified using previously
described primers (Lu et al., 2020), and sequences of the amplicons thus
obtained were verified using the Sanger method (Sangon). For
phylogenetic analysis, we constructed a tree based on the
maximum-likelihood (ML) tree method inferred using 144 \emph{Carnivore
protoparvovirus 1} VP2 gene sequences (Table S1). These sequences were
aligned using Clustal W (Larkin et al., 2007), and the ML tree was
generated using MEGA X software (Kumar, Stecher, \& Tamura, 2016) based
on 3-parameter (T92) model and gamma distribution with invariant sites
(G + I) (Tamura \& Nei, 1993). The reliability of the ML tree was
assessed based on 500 bootstrap replicates, and the original tree was
summarized and annotated using Figtree software.

\textbf{Results and Discussion}

\textbf{Clinical symptoms and pathological lesion} \textbf{s}

General lethargy, reduced food intake, and loose stools were among the
symptoms observed in the two pangolins examined in the present study.
Furthermore, whereas electrolyte disturbance was observed, there was no
evidence of leucopenia. The pangolins died naturally at 4-6 days
post-rescue, with post-mortem examinations revealing apparent congestion
and hemorrhage in the intestinal tract, thickened intestinal walls, and
necrosis of the mucosal tissue (Fig. 1A). Histopathological
observations, performed to determine lesions associated with CPV-induced
diarrhea, revealed intensive injury to the mucosal layer of the small
intestine to be a prominent feature, particularly the necrosis and
shedding of intestinal mucosal intraepithelial cells and glands, which
were observed in all parts of the small intestine (Fig. 1 B and C).
Moreover, the submucosal layers were found to be characterized by
inflammatory infiltrates, comprising primarily neutrophils and scattered
lymphocytes. In addition, we observed a reduction in the number of
lymphocytes in the spleen. The detection of bacteria in rectal tissues
provided evidence to indicate the probable occurrence of secondary
bacterial infection. In contrast, there was no indication of
histopathological changes in the heart, liver, lungs, or kidneys.

Immunohistochemical analyses were performed to determine the
localization of CPV antigen in the intestinal system of the CPV-infected
pangolins, representative high-magnification images of which are
presented in Fig 1. D and E. CPV antigens were detected in the duodenum,
jejunum, and ileum, although not in the mesenteric lymph nodes, with
strong HRP signals being observed in the crypt region and vicinity of
inflamed cells. These pathological changes in CPV-infected pangolins are
similar to those previously described in CPV-infected dogs, in which
hemorrhagic enteritis and crypt neurosis are prominent features
(Pollock, 1982).

Moreover, consistent with our observations of histopathological lesions,
the crypt was established to be the major CPV antigen-positive region
(Fig. 1). These pathological changes are consistent with those
previously reported by Wang et al. (2020), thereby indicating that that
CPV-2 targets and replicates within in the intestinal tract cells of
animals in the order Pholidota.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Fig. 1.} Pathological lesions and immunohistochemical analyses
of CPV-2 infected pangolins (sub-adult females) suffering from severe
diarrhea. Representative images of gross changes and histopathological
lesions are shown. Panel A presents a gross observation of the internal
organs, showing apparent congestion and hemorrhage. Panels B and C show
histopathological lesions in the jejunum. Pronounced injury in the
mucosa layer of small intestine is a prominent feature, notably the
necrosis and shedding of the intestinal mucosal intraepithelial cells
and glands. Panels D and E show the immunohistochemistry results for the
jejunum and ileum, respectively. The black arrows indicate the
significant pathological lesion or CPV antigen-positive area. Scale bars
are shown in the lower right-hand corners.

\textbf{Virus screening, isolation, and transmission electron microscopy
observations}

The findings of our PCR analyses revealed that samples obtained from the
two pangolins were CPV-positive, whereas there was no evidence to
indicate infection with CCoV, CDV, or DogCV. Moreover, typical
parvovirus-associated CPV were detected after four passages in F81
cells, and typical parvovirus-like particles, approximately 20 nm in
diameter, were observed in the cell supernatants (Fig. 2). Based on
these observations, we believe CPV-2s to be the etiological agents
causing pangolin diarrhea.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Fig 2.}A transmission electron micrograph showing a group of
parvo-like virus particles, indicated by the back arrows.

\textbf{Phylogeny and VP2 amino acid sequence substitutions}

The ML phylogenetic tree constructed in the present study was inferred
based on 144 reference VP2 sequences of Carnivore \emph{protoparvovirus}
1 and those of the parvoviruses obtained from the two pangolins examined
in this study. The original tree is shown in Fig. S1, and a summarized
version of the tree is shown in Fig. 3. The VP2 sequences in samples
obtained from pangolin P1 were classified into the CPV-2c subclade, with
99.8\% identity to a CPV-2c strain (MN832850) isolated from a Taiwanese
pangolin in China, whereas those from pangolin P2 were classified into
the CPV-2a subclade, with 99.8\% identity to a CPV-2a strain (KY386858).
Notably, CPV-2a and CPV-2c are the dominant strains prevalent among
domestic dogs in southern China (Chiang, Wu, Chiou, Chang, \& Lin, 2016;
Hao et al., 2020; Qi, Zhao, Guo, \& Sun, 2020) and in neighboring
tropical countries, respectively (Charoenkul et al., 2019; Hoang et al.,
2019; Inthong, Kaewmongkol, Meekhanon, Sirinarumitr, \& Sirinarumitr,
2020). In the present study, we established that the two Chinese
pangolins were respectively infected with CPV-2a and CPV-2c. The VP2
sequences obtained from pangolin P2 were classified into the CPV-2a
subtype group, closely related to CPV-2a strains, such as
CPV-2a/Dog/GY-9/China/16 (KY386858), CPV-2a/Dog/CN/JS1704/China/17
(MK518017), and CPV-2a/Dog/CN/HN1702/China/17 (MK517985) isolated from
dogs in Guiyang, Jiangsu, and Henan provinces in the central and
southern regions of China (Fig. 3). Moreover, the VP2 sequences obtained
from pangolin P1 were classified into the CPV-2c subtype group, closely
related to CPV-2c strains, such as CPV-2c/Dog/Vietnam/HN07/17,
CPV-2c/Pangolin/China/Taiwan/2018/18, and CPV-2c/Dog/South
Korea/K01708-1/17, which were isolated in the neighboring countries of
Vietnam and South Korea (Fig. 3). Accordingly, these findings provide
convincing evidence to indicate that the CPV-2 strains infecting the two
pangolins were of dog origin.

The amino acid sequence of the VP2 viral protein detected in pangolin P2
is characterized by an asparagine (N) residue at position 426 and refers
to other residues (such as residues 80, 103 and 297) in the VP2 protein,
on the basis of which, it should be identified as CPV-2a (new) subtype.
In contrast, the sequence in pangolin P1 has a glutamic acid (E) residue
at position 426, and accordingly should be recognized as the CPV-2c
subtype. Consistently, the P2 sequence has glutamine (Q) and alanine (A)
residues at positions 370 and 440, respectively, which are the same as
those of the CPV-2a subtype, whereas the P1 sequence is characterized by
arginine (R) and threonine (T) residues at positions370 and 440, as in
CPV-2c.

Most CPV-2 variants are characterized by a glycine residue at position
300 of the VP2 sequence, which has been demonstrated to be necessary for
the infection of canine and felid species (Allison et al., 2016;
Voorhees et al., 2019). The findings of numerous studies have indicated
that the CPV-2a variant can infect wild civet cats (\emph{Paradoxurus
musangus} ) (Mendenhall et al., 2016), red pandas (\emph{Ailurus
fulgens} )(Qin, Loeffler, Li, Tian, \& Wei, 2007), masked palm civets
(\emph{Paguma larvata} ), and Chinese ferret badgers (\emph{Melogale
moschata} ) (Chang \& Chen, 2021) in East Asian countries. The CPV-2c
variant has also been confirmed to infect masked palm civets and Chinese
ferret badgers (Chang \& Chen, 2021). Moreover, CPV-2 has been
established to be highly prevalent in farmed raccoon dogs and has
evolved in only 10 years (Lu et al., 2020).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Fig. 3.} Phylogenetic analysis of VP2 gene sequences from
pangolin and related parvoviruses. The ML tree was constructed based on
VP2 gene sequences of 142 parvoviruses belonging to the species
\emph{Carnivore protoparvovirus 1} (FPV {[}n = 3{]}; CPV-2 {[}n = 14{]}
or its variants CPV-2a {[}n = 68{]}, CPV-2b {[}n = 8{]}, or CPV-2c {[}n
= 52{]}). The VP2 gene sequences of FPV were used as an outgroup and
rooted by the oldest FPV sequence (FPV/Cat/US/FPV-d/64, U22189). The two
pangolin parvovirus isolates obtained in this study are indicated by red
triangle, and the reference virus is denoted by a black triangle.
Details of the several subclades grouped close to CPV-2b or CPV-2c are
presented in Fig S1. The scale bar shown at the bottom of the tree
denotes.

Pangolins remain critically endangered worldwide, owing to a range of
threats, not least of which is pathogen infection. To date, however,
there have been few studies that have examined the pathogens infecting
pangolins. In this study, however, we were able to confirm CPV-2c
infection among pangolins inhabiting mainland China and established that
these animals can also be infected with the CPV-2a variant. In this
regard, the findings of previous studies have provided evidence to
indicate that CPV frequently undergoes interspecies transmission among
domestic and wild animals, with asymptomatic individuals (e.g., stray
dogs and raccoons) inhabiting the peripheries of urban areas acting as
virus reservoirs that promote the spread of CPV-2 to wild animals
(Allison et al., 2012; Allison et al., 2013; Hirsch, Prange, Hauver, \&
Gehrt, 2013). Notably, the two strains detected in this study are
phylogenetically close to CPV strains derived from dogs. Moreover, stray
dogs have been observed attacking wild pangolins in areas in which urban
development has encroached on the habitats of these animals (S. L. Wang
et al., 2020), thereby indicating the potential for cross-species
transmission between stray dogs and wild pangolins.

In summary, in this study, we report the infection of Chinese pangolins
with two strains of canine parvovirus type 2 causing diarrheal diseases.
These two CPV-2 strains are closely related to CPV-2a and CPV-2c, which
are prevalent in China and neighboring countries, respectively, thereby
revealing that the CPV-2 strains infecting Chinese pangolins are related
to those known to be of dog origin. Based onour findings, we recommend
that further investigations should be conducted to assess the potential
interspecies transmission among wild Chinese pangolins and domestic or
feral dogs.

\textbf{ACKNOWLEDGEMENT}

This study was jointly supported by the Forestry Science and Technology
Innovation project in Guangdong Province (2022KJCX008). The authors
thank our colleagues,

\textbf{CONFLICT OF INTEREST}

The authors declare that they have no conflicts of interest.

\textbf{AUTHOR CONTRIBUTIONS}

Lu Rongguang conceived the study, designed the experiments, and analyzed
the data.

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