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\begin{document}

\title{Feline panleukopenia virus associated Hypogranular cerebellar hypoplasia
in young captive lions}



\author[1]{ningning Ma}%
\author[1]{Ziyi Guo}%
\author[1]{Mengmeng Deng}%
\author[1]{Zhan Liu}%
\author[2]{Shilin Xin}%
\author[1]{Tong Wang}%
\author[1]{Chenmeng Ding}%
\author[1]{Xiya Xu}%
\author[1]{Yongtao Li}%
\author[3]{Lu Chen}%
\author[2]{Yurong Yang}%
\affil[1]{Affiliation not available}%
\affil[2]{College of Animal Science and Veterinary Medicine ,Henan Agricultural University, Zhengzhou}%
\affil[3]{Henan Agricultural University}%


\vspace{-1em}



  
  \date{May 17, 2024}


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\selectlanguage{english}
\begin{abstract}
Here we described brain lesion in young captive lions after exhibiting
ataxia. The histopathological analyses of the brain showed typical
encephalitis with glial cell proliferation, neuronal atrophy and
necrosis, and encephalomalacia. We used a variety of identification
methods, including next-generation sequencing, polymerase chain reaction
(PCR), virus isolation, immunofluorescence staining (IFA), and electron
microscopy. Combined with clinical manifestations of the infected cub,
it determined that the dead cub have resulted from an FPV embryonic
infection. Through sequence analysis, it was found that the infected
virus may be a recombinant virus between FPV and CPV. this study not
only provides new gene sequences for the global study of FPV-infected
lions but also helps to further study the incidence and genetic
diversity of FPV.%
\end{abstract}%



\sloppy


\textbf{Feline panleukopenia virus associated Hypogranular cerebellar
hypoplasia in young captive lions}

Ningning Ma, Ziyi Guo, Mengmeng Deng, Zhan Liu, Shilin Xin, Tong Wang,
Chenmeng Ding, Xiya Xu, Yongtao Li, Lu Chen and Yurong Yang

College of Veterinary Medicine, Henan Agricultural University, Zhengzhou
,450002, PR People's Republic of China

\textbf{* Corresponding authors at:} College of Veterinary Medicine,
Henan Agricultural University,~Zhengzhou~,450002, PR People's Republic
of China

\textbf{E-mail addresses:}

E-mail: \emph{chenluhau@126.com} (Lu Chen);\emph{yangyu7712@sina.com}
(Yurong Yang)

\textbf{Abstract:} Here we described brain lesion in young captive lions
after exhibiting ataxia. The histopathological analyses of the brain
showed typical encephalitis with glial cell proliferation, neuronal
atrophy and necrosis, and encephalomalacia. We used a variety of
identification methods, including next-generation sequencing, polymerase
chain reaction (PCR), virus isolation, immunofluorescence staining
(IFA), and electron microscopy. Combined with clinical manifestations of
the infected cub, it determined that the dead cub have resulted from an
FPV embryonic infection. Through sequence analysis, it was found that
the infected virus may be a recombinant virus between FPV and CPV. this
study not only provides new gene sequences for the global study of
FPV-infected lions but also helps to further study the incidence and
genetic diversity of FPV.

\textbf{KEYWORDS:} Feline panleukopenia virus (FPV); young lions;
hypogranular cerebellar; brain lesion; embryonic infection

\textbf{INTRODUCTION}

Feline panleukopenia is caused by a feline panleukopenia virus (FPV),
which is a single-stranded DNA virus. Genetically, structurally, and
antigenically, FPV is closely related to the mink enteritis virus (MEV),
and canine parvovirus (CPV) (Truyen et al.,2013). FPV infections in
primarily domesticated cats and wild felids, including tigers (Panthera
Tigris) and lions (Panthera Leo) (Driciru et al., 2006; Duarte et al.,
2009; Goodrich et al.,2012), have been described based on clinical,
pathological, virologic, or molecular data. Previous studies show that
the symptoms associated with FPV infection in tigers and lions are
gastrointestinal enteritis and severe hemorrhagic enteritis,
respectively (Wang et al.,2019; Wang et al., 2017); Evidence of
neurological symptoms, such as ataxia, dysphagia, and hydrocephalus, has
not been described in lions infected with FPV previously We, therefore
describe previously undocumented neuropathologic changes and molecular
features associated with FPV infection in lions.

In this study, we report an outbreak of fatal FPV infection among
captive lions in the Zhengzhou Zoo in central China. The survey results
showed that a total of 39 lion cubs were born between April 2017 to July
2019, and the cubs were of different ages and varying degrees of
clinical characteristics.~Miscarriage in the early stage of pregnancy in
the lionesses resulted in 24 fetal deaths. Three cubs did not
breast-feed at birth and died within a week following birth. Another 12
normally born cubs were placed in similar or adjacent rooms. Signs of
limb weakness and walking instability began to appear at the age of 4-8
weeks. They were artificially fed with food and water, lost the ability
to act independently and had difficulty swallowing when they were about
a year old, and died of asphyxiation when eating meat.~Besides, there
were no other obvious clinical manifestations. The histopathologic
findings, together with the sequencing analysis, virus isolations, PCR,
IFA and Electron microscopy observation results, suggest that parvovirus
was the cause of the death in these animals. This is the first time a
case of recombination of CPV and FPV infection that caused lion brain
tissue lesions and neurological symptoms has been found.

\textbf{MATERIALS AND METHODS}

\textbf{2.1 Cell and Animal tissue samples}

Animal tissue samples were provided by Zhengzhou zoo; CrandellRees
feline kidney cell (CRFK) cells were purchased from Procell Life
Science\&Technology Co,.Ltd. In this study, CRFK were cultured in
Dulbecco's modified Eagle's medium (Gibco, USA) that had been
supplemented with 10\% fetal bovine serum (HyClone, USA) and penicillin
streptomycin (Life Technologies, USA). The CRFK were cultured to
approximately 80\% confluence and then infected with FPV HNZZ-2.

\textbf{2.2 Histopathologic examination}

Tissue samples were collected and set in 10\% buffered formalin
solution, processed and embedded in paraffin for histopathological
analysis. Threemillimetre-thick sections were stained with haematox-ylin
and eosin (H\&E) (McEndaffer et al., 2017).

\textbf{2.3 Metagenomic sequencing}

Illumina sequencing technology was used to complete the sequencing of
the pathogen genome, and the Illumina PE library was constructed. After
quality control of the obtained sequencing data, the metagenomic
sequencing of the virus was completed using Bioinformatics analysis
methods (Shanghai Tangpu Biotechnology Co., Ltd.).

\textbf{2.4 Viral PCR identification}

Total DNA was extracted from the culture supernatant using a DNA
Extraction Kit (TIANGEN BIOTECH (BEIJING)CO.,LTD.) according to the
manufacturer's pro-tocol. DNA extracts were used in conventional PCR
assays. The sequences of the primers are FPV-F (5'-
GGATGGGTGGAAATCACAGC-3') and FPV-R (5'-ATAACCAACCTCAGCTGGTC-3') (Qiao
et al., 2001).The amplification was generally achieved by means of 35
cycles of predenaturation at 95@\selectlanguage{english}C for 3min, denaturation at 95@\selectlanguage{english}C for
30s, annealing at 50@\selectlanguage{english}C-60 for 30 s and polymerization at 72@\selectlanguage{english}C for 10
min. After electrophoresis in a 1.0\% agarose gel and ethidium bromide
staining, the PCR products were excised from the gel and purified by a
commercial kit. For each amplified genome fragment, a pair of gene
specific primers are used for bidirectional sequencing (Sangon Biotech
Engineering (Shanghai) Co., Ltd.), and then the sequencing results are
spliced using DNAstar software to obtain a complete genome.

\textbf{2.5 Virus isolation}

Samples were collected from the spleen of dead lion cub from a park in
Zheng zhou Province, China. Samples were tested by a PCR assay as
described previously (Wu et al., 2015). FPV-positive samples were
dissolved in phosphate-buffered saline (PBS) and then homogenized with a
vortex. The samples were then centrifuged at 5000g for 5 min at 4 \selectlanguage{ngerman}°C.
Next, the supernatants were filtered using a 0.22 \selectlanguage{greek}μ\selectlanguage{english}m microporous
filtering film. The filtered supernatants were inoculated into CRFK.
Cell cultures were observed every 6 h for a cytopathic effect (CPE).

\textbf{2.6 Immunofluorescence assay}

CRFK were grown in 6-well microtitre plates and infected with FPV. After
48 h, the cells were fixed with 4\% paraformaldehyde for 15 min and then
washed three times with phosphate-buffered saline with Tween-20(PBST).
Cells were incubated with anti-FPV monoclonal antibody (Shenzhen Anti
Biotechnology Co., Ltd.) at 37 \selectlanguage{ngerman}°C for 1 h and then FITC-conjugated rat
anti-mouse secondary antibody (BIOMAN (Beijing) Technology Co., Ltd) for
1 h Finally, the labelled cells were observed viafluorescence microscopy
(Leal et al., 2007).

\textbf{2.7 Electron microscopy observation}

Took 20~\selectlanguage{greek}µ\selectlanguage{english}l of virus suspension~with a pipet-gun and dropped onto the
copper grid with~carbon film for 3-5min, and then use filter
paper~to~absorb~the excess liquid. Dropped 2\% phosphotungstic acid~on
the copper grid to stain for~1-2min, use filter paper to absorb excess
liquid, and dry at room temperature. The cuprum~grids are observed under
TEM and take images.

\textbf{2.8 Phylogenetic and Recombination analyses}

Phylogenetic analysis was performed using MEGA 7(Kumar et al., 2004).
Multiple alignments were carried out using CLUSTAL X version 1.83. A
phylogenetic tree was constructed using the neighbor-joining method and
distances were determined using the Kimura two-parameter method (Ohshima
et al., 2009; Whelan et al., 2001). The putative recombinant sequence
and its parents were identified using two different recombination
detection programs, namely RDP4 and Simplot software packages. RDP4 is
composed of the following 8 separate programs and enables automated
analyses of a nucleotide sequence alignment using all of the programs
concomitantly: RDP, Chimaera, BootScan, 3Seq, GENECONV, MaxChi, SIScan
and LARD (Martin et al., 2015 Ohshima et al., 2009). Simplot uses
conventional parameter settings.

\textbf{RESULTS}

\textbf{Histopathologic examination}

An 8-month-old female cub who was paralyzed was euthanized and sent for
pathological examination. The most significant lesion at autopsy was a
5cm round cavity in the center of the lion's brain, which was covered by
a connective tissue capsule (Figure 1). The histopathological analyses
of the brain showed typical encephalitis with glial cell proliferation,
neuronal atrophy and necrosis, and encephalomalacia. The cerebellum
showed nerve cell necrosis and capillary congestion. The spinal cord and
limb nerves showed vesicular degeneration and partial necrosis,
respectively (Figure 2).

\textbf{3.2 Metagenomic sequencing and PCR identification}

The result of the secondgeneration sequencing shows the detected virus
was FPV (Shanghai Tangpu Biotechnology Co., Ltd.). PCR results showed
that the spinal cord, blood, lymph, nerve, spleen, intestine, and liver
were all FPV positive.

\textbf{3.3 Virus isolation and identification}

One FPV strain, designated FPV HNZZ-2, was successfully isolated from a
sample from Zhengzhou Province, China. The results showed that
cytopathogenic effects observed in infected CRFK at 24h post-inoculation
included, swelling, netting, and shedding. However, the negative CRFK
control group showed uniform cell morphology with obvious borders and
good condition (Figure 3).

\textbf{3.4 IFA and Electron microscopy observation}

The presence of FPV in the cell culture fluids at the fifth passage
level was confirmed by PCR assay using the FPV speculative primer pair.
IFA results showed green fluorescence in infected CRFK cells probed with
an anti-FPV monoclonal antibody (Figure 4A), in contrast, there was no
fluorescence in normal CRFK cells (Figure 4B). The diameter of the
virion was approximately 20-25 nm which was observed under the electron
microscope and revealed no envelope, spherical shape, and the
icosahedral structure, and these were consistent with the structural
characteristics of panleukopenia virus (Figure 5).

\textbf{3.5 Comparison of the FPV HNZZ-2 gene sequence with other FPVs}

To analyze the molecular characteristics of the FPV HN-ZZ2 strain,
primers were designed based on the full gene sequence of the FPV
registered in GenBank. The genome sequence of the strain was amplified
by PCR (The primer sequence is shown in Table 1) (Qiao et al., 2001; Liu
et al., 2001) and the sequencing results were spliced using DNAstar
software to obtain a complete genome. Initially, the BLAST suite was
used to identify viral sequences based on their similarity to annotated
genomes in GenBank. Based on the best hits of blast search, 39 near
full-length genomes were selected (Information of reference strain,
Table 2). These genomic sequences and the complete genome sequence of
the FPV HN-ZZ2 strain were aligned and analyzed. Sequence analysis
showed that the nucleotide sequence was highly related to the reference
strain, with nucleotide sequence similarity ranging from 98.5\%
\textasciitilde{}99.6\%. The nucleotide sequence of FPV HN-ZZ2 strain
showed the highest homology with the FPV HH-1/86 strain, previously
isolated from Clouded Leopard (99.6\%), and the lowest homology with
MEV-L strain (98.5\%). The bioinformatic analysis showed that the
isolate encoded four proteins, including two nonstructural proteins of
NS1 (nt154-2160) and NS2 (nt154-423 and 1886-2123), and two structural
proteins of VP1 (nt 2160-2197 and 2270-4422) and VP2 (nt 2668-4422)
through alternative splicing. Subsequently, a phylogenetic tree for the
full nucleotide sequence of the FPV HN-ZZ2 strain and the 39 reference
strains was constructed using the Maximum Comprehensive Likelihood. All
phylogenetic analyses and tree editions were conducted using the MEGA 7
software. Phylogenetic analysis revealed that FPV HN-ZZ2 clustered
together with FPV but was also closely related to the CPV branch (Figure
6).

\textbf{3.6 Characterization of recombination detected in the genome of
FPV HNZZ-2}

Genetic recombination is generally considered as a key mechanism in
virus evolution. Several studies have reported multiple infections of
autonomous parvovirus, revealing that parvoviruses have a considerably
higher degree of variability (Jo\selectlanguage{ngerman}ão Vieira et al., 2008; Liu et al.,
2015). The putative recombinant sequence and its parents were identified
using two different recombination detection programs, namely RDP4 and
Simplot software packages. Interestingly, both Simplot and RDP4 programs
detected recombination signals in the nucleotide sequence of FPV
HN-ZZ2\selectlanguage{english}(Figure 7). RDP4 identified nucleotide position 2,841 as an
ending breakpoint and predicted a recombination region located at
nucleotide positions 1,956 and 2,841 of the alignment (Figure 7A and
7B). Among the nucleotide sequence data for all the 40 strains in the
RDP4 program, nucleotides 1,956-2,481of the FPV HN-ZZ2 possessed the
greatest similarity (99.7\%) to the corresponding strain CPV-L,
meanwhile, other parts have the highest homology with the FPV
Raccoon/RC9/BC2010 strain (99.3\%). Further to confirm whether
reorganization existed in FPV HN-ZZ2, Simplot was used, and nt position
2580 was identified as a putative breakpoint for recombination (Figure
7C). This indicated that the FPV HN-ZZ2 strain was likely to be a new
recombinant virus between CPV and FPV.

A phylogenetic tree based on the full-length NS1 and VP2 nucleotide
sequence was constructed using the isolated strain and 39 reference
strains (Figure 8). Phylogenetic analysis showed that the VP2 gene of
FPV HN-ZZ2 was in the same branch as CPV (Figure 8A), and the sequence
of the NS1 gene is closely related to FPV and MEV (Figure 7B). The
results are consistent with previous recombination analysis results,
further indicating that the isolate may be a recombinant virus.

In this study, the amino acids of NS1 at positions 23, 247, 443, 545,
595, and nucleotide at positions 309, 492, 621, 1543, 1959,1990 were
found to be identical to those of CPV reference strains. Interestingly,
the 60th amino acid of NS1 showed a mutation (I\selectlanguage{english}-V), which was different
from the three control strains (Table 3). Analysis of the VP2 protein
found that the amino acids at the host-specific sites at positions 80,
87, 93, 103, 300, 305, 323, 564, and 568 were identical to the amino
acid of the FPV reference strain. The 217th amino acid of VP2 showed a
mutation (T\selectlanguage{english}-R), which was different from the three control strains
(Table 4). The 300th amino acid was A (Ala) and the 562nd amino acid was
L (Leu), which also confirmed that the isolate FPV HN-ZZ2 belonged to
FPV rather than MEV (Shackelton et al., 2007). Whether new variants of
NS1 and VP2 causes the clinical symptoms to be different from the
classic strains remains unclear. Besides, compared with the FPV HN-ZZ1
strain, a unique substitution in NS1 and VP2 protein sequence was
observed (Table 5). Whether these amino acid differences are evidence of
host differences between FPV HN-ZZ2 and HN-ZZ1 remains to be studied.

\textbf{DISCUSSION}

Clinical disease associated with FPV in adult cats is usually
progressive and fatal, leading to fever, vomiting, and diarrhea,
associated with pancytopenia and necrotizing enteritis (Lamm et al.,
2008). Fetal infection by FPV most commonly leads to abortion or
stillbirths, whereas Late embryonic infection is more often accompanied
by brain lesions, including cerebellar hypoplasia, hydrocephalus and
hydranencephaly. In young wild and domestic carnivores, the infection
usually causes severe gastroenteritis, which is in most cases
hemorrhagic and there have been no previous reports of lions contracting
brain lesions (Stuetzer et al., 2014; Sharp et al., 1999). In this
study, the young lion mainly developed brain tissue changes. This is
similar to previous reports related to the pathological changes of FPV
infections in kittens such as lymphoplasma meningoencephalitis and
neuronal necrosis, the combined CNS malformations of hydranencephaly and
cerebellar hypoplasia (Stuetzer et al., 2014; Allison et al., 2013).
This study further conducted a comprehensive analysis of the full
sequence of FPV HN-ZZ2 and found that the NS1 gene had higher similarity
to CPV and VP2 gene has a high similarity with FPV.

In summary, combined with clinical manifestations and pathogen detection
results of the infected cub, it should be determined that the dead cub
have resulted from an FPV embryonic infection. This study speculated
that gene recombination forms a new virus. As a result of mutations, FPV
continues to expand its host range and change its tissue tropism to
become the main pathogen causing infections in many wild animals. FPV
has continuously evolved via the accumulation of point mutations in the
viral genome, resulting in two important implications. Following the
emergence of new antigen variants with different biological and
antigenic characteristics, the vaccine program may need to be adjusted
accordingly and the virus strains in commercial products are updated.
This study supports the need for continued epidemiological surveillance
to detect new FPV variants that may escape the host immune system and
detection methods. Besides, mixed breeding should be avoided for captive
animals to reduce the risk of disease transmission. Therefore, this
study not only provides new gene sequences for the global study of
FPV-infected lions but also helps to further study the incidence and
genetic diversity of FPV.

\textbf{ETHICAL STATEMENT}

Animal tissue samples used in the experiment were provided by relevant
personnel of Zhengzhou Zoo and the informed consent of the client has
been obtained.

\textbf{ACKNOWLEDGMENTS}

This work was supported by the National Natural Science Foundation of
China (grant number 30900151).

\textbf{CONFLICT OF INTEREST}

No potential conflict of interest was reported by the author(s).

\textbf{DATA AVAILABILITY STATEMENT}

The data that support the findings of this study are available from the
corresponding author upon reasonable request.

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\textbf{Figure legends}

\textbf{FIGURE 1} Brain lesions in the cub. Young lion mainly developed
brain tissue changes, such as hydrocephalus, a 5cm cavity in the center
of the brain

\textbf{FIGURE 2} FPV-related diseased tissue, lion (original
magnification x400) (A) The brain showed typical encephalitis with glial
cell proliferation, neuronal atrophy and necrosis, and encephalomalacia.
(B) The cerebellum showed nerve cell necrosis and capillary congestion.
(C) The spinal cord showed vesicular degeneration. (D) Limb nerves
showed partial necrosis

\textbf{FIGURE 3} Virus isolation and cytopathic effects of the FPV
HN-ZZ2 strain in CRFK cells (original magnification x200). The
FPV-positive samples were treated and inoculated into CRFK cells. The
cytopathogenic effects induced by the FPV HN-ZZ2 strain observed in CRFK
cells 24, 48, 60hours after inoculation were observed by light microsopy

\textbf{FIGURE 4} Identification in infected cells of FPV HN-ZZ2 through
IFA. (A) Cells infected with FPV are shown. Green fluorescence
represents the FPV antigen within infected cells. (B) Negative control

\textbf{FIGURE 5} Transmission electron microscopy technique of FPV
HN-ZZ2. The diameter of the virion was approximately 20-25 nm which was
observed under the electron microscope and revealed no envelope,
spherical shape, and the icosahedral structure, and these were
consistent with the structural characteristics of Feline FPV

\textbf{FIGURE 6} The phylogenetic tree constructed from the full
sequences of the HNZZ2 strain and the reference sequences. The branch
lengths are drawn to scale (number of substitutions per site). The trees
were rgenerated by the neighbor-joining method with bootstrap tests of
1000 replicates using the MEGA 7 software. The bootstrap support is
indicated above the respective branches with an asterisk. The FPV HN-ZZ2
strain described in the current study is indicated. The GenBank
accession numbers of the sequences under study are provided in Table 2

\textbf{FIGURE 7} The results of a recombination breakpoint analysis
performed using the RDP4 and Simplot program. (A and B) Recombination
detection by the RDP4 program shows that FPV HN-ZZ2 is generated from
recombination between CPV-L/Cania/China and canine
Raccoon/RC9/BC2010/Procyon lotor/Canada. Nucleotide position 2,841 is
identified as an ending breakpoint. A predicted recombination region is
found around positions 1,956 and 2,841 of the alignment. (C) The Simplot
program identifies nucleotide position 2,580 as a putative breakpoint
for recombination

\textbf{FIGURE} \textbf{8}Phylogenetic trees were inferred for the
nucleotides of the NS1(A) and VP2(B)genes. The trees were generated by
the neighbor-joining method with bootstrap tests of 1000 replicates
using the MEGA 7 software. The FPV HN-ZZ2 strain isolated in the current
study is indicated

\textbf{TABLE 1 Primers used for amplification and sequencing of FPV
HN-ZZ2}\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
Number & Primer names and location & Primer sequences (5'-3') &
Length(bp)\tabularnewline
\midrule
\endhead
1 & KP769859.1(120-940)-F & GAATGATAGGCGGTTTGTGTGTTTA &
820\tabularnewline
& KP769859.1(120-940)-R & GTCATGTGGACAATTTTTTTCTTTG &\tabularnewline
2 & KP769859.1(730-1400)-F & TGTATTGGAGTAGATGGTTGGTGAC &
670\tabularnewline
& KP769859.1(730-1400)-R & AATCCAATTCCATCCGTGCATTCTA &\tabularnewline
3 & KP769859.1(1290-1780)-F & ACAGCATTTGAATTAATACTTGAAA &
490\tabularnewline
& KP769859.1(1290-1780)-R & TGTTCAGGTCTTTCTTCACATCCAA &\tabularnewline
4 & KP769859.1 (1480-2100)-F & GTACAGGAAAATCTATTATTGCTCA &
620\tabularnewline
& KP769859.1 (1480-2100)-R & CCAGTGCAAGGTCCACTACGTCCGG &\tabularnewline
5 & W (1876-2495)-F & GTAAGCTTCCAGGAGACTTT & 620\tabularnewline
& W (1876-2495)-R & GTAAGCTTCGTCGTGTTCTT &\tabularnewline
6 & Q (2107-2747)-F & TAACTCCTCTGACTCCGGAC & 640\tabularnewline
& Q (2107-2747)-R & ACCACCGTCTGGTTGAACTG &\tabularnewline
7 & KP769859.1(2581-3160)-F & AAGATTGGGGGGGGAAAATAGGACA &
579\tabularnewline
& KP769859.1(2581-3160)-R & TCTCCTGGATTAAACCAAACTCCCC &\tabularnewline
8 & KP769859.1(2900-3640)-F & GGGGATTTCTACGGGTACTTTCAAT &
740\tabularnewline
& KP769859.1(2900-3640)-R & AATGCTCTATTTGTTTGCCATGTAT &\tabularnewline
9 & KP769859.1(3560-4120)-F & GATGAATTTGCTACAGGAACATTTT &
560\tabularnewline
& KP769859.1(3560-4120)-R & GTATAGTTAATTCCTGTTTTACCTC &\tabularnewline
10 & KP769859.1(3870-4720)-F & GGGGGAGCGCAAACAGATGAAAATC &
850\tabularnewline
& KP769859.1(3870-4720)-R & TTGTATACCATATAACAAACCTTCT &\tabularnewline
11 & KP769859.1(4510-4920)-F & AATCTCAACTAGCACCTAGAAAATT &
410\tabularnewline
& KP769859.1(4510-4920)-R & AAGGTTAGTTCACCTTATAGACAGT &\tabularnewline
\bottomrule
\end{longtable}

\textbf{The primer location refers to the position in the genome of
MG132167A strain. (GenBank: KP769859.1);The 5th and 6th primer
sequences are from references. (Liu et al.,2001; Qiao et al.,2001)}

\begin{CJK}{UTF8}{gbsn}\textbf{TABLE 2 The nucleotide sequence accession numbers of the
FPV、MEV and CPV strains analyzed in this study}\end{CJK}\selectlanguage{english}\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
& Strain & Accession number & Genetic type & Year submitted &
Origin\tabularnewline
\midrule
\endhead
& CPV/Raccoon/WI/37/10 & JN867618.1 & CPV & 2012 & Procyon
lotor/USA\tabularnewline
& CPV-L & MG763189.1 & CPV & 2018 & Canis
lupufamiliaris/China\tabularnewline
& SC02/2011 & JX660690.1 & CPV & 2012 & Dog/China\tabularnewline
& s5 & KF638400.1 & CPV & 2013 & Dog/China\tabularnewline
& Y1 & D26079.1 & CPV & 2008 & Canine parvovirus/Japan\tabularnewline
& CPV-447 & AY742934.1 & CPV & 2005 & Carnivore
parvoviruses/Germany\tabularnewline
& cpv/nj01/06 & EU310373.2 & CPV & 2008 & Dog/China\tabularnewline
& CPV-N & M19296.1 & CPV-2 & 1995 & USA\tabularnewline
& CPV-b & M38245.1 & CPV-2 & 1996 & USA\tabularnewline
& BJ/2010 & AEK69510.1 & CPV2a & 2011 & Canine/China\tabularnewline
& B-2004 & EF011664.1 & CPV2a & 2004 & Dog/China\tabularnewline
& 9985 & LC270891.1 & CPV2b & 2017 & Canis
lupusfamiliaris/Japan\tabularnewline
& 9985-46 & LC270892.1 & CPV2b & 2017 & Canis
lupusfamiliaris/Japan\tabularnewline
& CPV-LZ2 & JQ268284.1 & CPV2b & 2017 & Dog/China\tabularnewline
& UFMT & KY073269.1 & CPV2c & 2017 & Dog/Brazi\tabularnewline
& HUNCHUN-2 & AKF02396.1 & CPV2c & 2015 & Canis lupus
familiaris/China\tabularnewline
& UY120 & AIW67521.1 & CPV2c & 2014 & Dog/Uruguay\tabularnewline
& UY349 & KM457129.1 & CPV2c & 2014 & Dog/Uruguay\tabularnewline
& Raccoon/RC18/BC\_2016 & MF069447.1 & FPV & 2017 &
Procyonlotor/Canada\tabularnewline
& Raccoon/RC6/BC\_2015 & MF069445.1 & FPV & 2017 & Procyon
lotor/Canada\tabularnewline
& CU-4 & M38246.1 & FPV & 1996 & Feline panleukopenia
virus/USA\tabularnewline
& Raccoon/RC9/BC\_2010 & MF069446.1 & FPV & 2017 & Procyon
lotor/Canada\tabularnewline
& 193/70 & X55115.1 & FPV & 2005 & Australia\tabularnewline
& FPV-BJ04 & MH165481.1 & FPV & 2019 & Cat/China\tabularnewline
& FPV\_IZSSI\_42807\_15 & KX434462.1 & FPV & 2019 &
Cat/Italy\tabularnewline
& MHS2019 & MN908257.1 & FPV & 2020 & Tiger/China\tabularnewline
& MG132167A & KP769859.1 & FPV & 2016 & Cat/Belgium\tabularnewline
& FPV\_IZSSI\_3201\_1\_15 & KX434461.1 & FPV & 2019 &
Cat/Italy\tabularnewline
& GX01 & MG924893.1 & FPV & 2018 & Feline/China\tabularnewline
& HH-1/86 & KX900570.1 & FPV & 2017 & Jaguar/China\tabularnewline
& FPV-G & MG764510.1 & FPV & 2018 & Panthera tigris/China\tabularnewline
& HRB-CS1 & KP280068.1 & FPV & 2015 & Cat/China\tabularnewline
& HN-ZZ1 & KX685354.1 & FPV & 2017 & Tiger/China\tabularnewline
& FPV-L & MG764511.1 & FPV & 2018 & Panthera leo/China\tabularnewline
& Jun & MN400979.1 & FPV & 2020 & Feline/Korea\tabularnewline
& Rara & MN400980.1 & FPV & 2020 & Feline/ Korea\tabularnewline
& Gigucheon & MN400978.1 & FPV & 2020 & Feline/Korea\tabularnewline
& TN/FPV/2018 & MH559110.1 & FPV & 2018 & Felis
catus/India\tabularnewline
& XJ-1 & EF988660.1 & FPV & 2007 & Cat/China\tabularnewline
& GT-2 & AY955826.1 & FPV & 2005 & Tiger/China\tabularnewline
& MEVB & FJ592174.1 & MEV & 2009 & Mink/China\tabularnewline
& MEV-L & KT899746.1/ & MEV & 2015 & Mink/China\tabularnewline
& MEV-LHV & KT899745.1 & MEV & 2016 & Mink/China\tabularnewline
& MEVSD12/01 & KC713592.1 & MEV & 2013 & Mink/China\tabularnewline
& MEV/SMPV-11 & KP008112.1 & MEV & 2015 & Mink/China\tabularnewline
& MEV-LT18 & MK333230.1 & MEV & 2019 & Mink/China\tabularnewline
& MEV-HB18 & MN747143.1 & MEV & 2020 & Mink/ China\tabularnewline
& abashiri & D00765 & MEV & 2007 & Mink/Japan\tabularnewline
& MEV/LN-10 & HQ694567.2 & MEV & 2012 & Mink/China\tabularnewline
& MEV-SDNH & JX535284.1 & MEV & 2013 & Mink/China\tabularnewline
\bottomrule
\end{longtable}



\textbf{TABLE 3 Amino acid and nucleotide sequence variations in the NS1
of FPV HN-ZZ2 strain}\selectlanguage{english}
\begin{longtable}[]{@{}llllllllllll@{}}
\toprule
nt & 67 & 128 & 178 & 309 & 492 & 621 & 741 & 743 & 849 & 906 &
1048\tabularnewline
\midrule
\endhead
aa & 23 & 43 & 60 & * & * & * & 247 & 248 & * & * & 350\tabularnewline
FPVspecific & D & R & I & a & a & c & H & T & g & c & D\tabularnewline
FPV HN-ZZ2 & N & R & \textbf{V} & c & g & t & Q & T & t & t &
D\tabularnewline
CPVspecific & N & R & I & c & g & t & Q & I & g & c & D\tabularnewline
nt & 1224 & 1254 & 1327 & 1479 & 1509 & 1633 & 1720 & 1785 & 1926 & 1941
& 1959\tabularnewline
aa & * & * & 443 & * & * & 545 & 575 & 595 & * & * & *\tabularnewline
FPV specific & t & t & V & a & t & Q & I & H & a & g & t\tabularnewline
FPV HN-ZZ2 & t & g & I & a & c & E & I & Q & g & a & c\tabularnewline
CPV specific & c & t & I & g & t & E & I & Q & a & g & c\tabularnewline
\bottomrule
\end{longtable}

\textbf{In the comparison, the red mark shows the aa similarity between
FPV HN-ZZ2 strain and FPV and CPV ,while the green mark shows}
\textbf{differences in FPV, CPV, and MEV with unique mutations}

\textbf{An asterisk indicates the synonymous substitution of the coding
region.}

\textbf{TABLE 4 Amino acid and nucleotide sequence variations in the VP2
of FPV HNZZ2 strain}\selectlanguage{english}
\begin{longtable}[]{@{}lllllllllllll@{}}
\toprule
nt & 135 & 171 & 239 & 261 & 279 & 300 & 302 & 308 & 408 & 492 & 585 &
650\tabularnewline
\midrule
\endhead
aa & * & * & 80 & 87 & 93 & * & 101 & 103 & * & * & * &
217\tabularnewline
FPV specific & c & g & K & M & K & t & I & V & g & t & g &
T\tabularnewline
HN-ZZ2 & t & a & K & M & K & t & T & V & a & c & a & R\tabularnewline
CPV specific & c & a & R & L & N & c & I & A & g & t & g &
T\tabularnewline
nt & 694 & 756 & 810 & 871 & 900 & 915 & 967 & 1041 & 1092 & 1123 & 1185
& 1232\tabularnewline
aa & 232 & * & * & * & 300 & 305 & 323 & * & * & 375 & * &
411\tabularnewline
FPV specific & V & a & c & t & A & D & D & g & g & D & t &
E\tabularnewline
FPV HN-ZZ2 & V & g & t & c & A & D & D & a & a & D & c &
E\tabularnewline
CPV specific & I & a & t & c & G & Y & N & g & g & N & t &
E\tabularnewline
nt & 1290 & 1521 & 1566 & 1572 & 1691 & 1703 & & & & & &\tabularnewline
aa & * & * & * & * & 564 & 568 & & & & & &\tabularnewline
FPV specific & g & g & a & t & N & A & & & & & &\tabularnewline
FPV HN-ZZ2 & a & a & g & c & N & A & & & & & &\tabularnewline
CPV specific & g & a & a & c & S & G & & & & & &\tabularnewline
\bottomrule
\end{longtable}

\textbf{In the comparison, the red mark shows the aa similarity between
FPV FPV HN-ZZ2 strain and FPV and CPV,while the green mark shows}
\textbf{differences in FPV and CPV with unique mutations}

\textbf{An asterisk indicates the synonymous substitution of the coding
region.}

\par\null

\textbf{TABLE 5 Compared with the HN ZZ1, the amino acid sequence
variation in FPV HN-ZZ2 strain NS1 and VP2}\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
Strains & NS1 & NS1 & NS1 & NS1 & VP2 & VP2 & VP2\tabularnewline
\midrule
\endhead
& Aminoacid & Aminoacid & Aminoacid & Aminoacid & Aminoacid & Aminoacid
& Aminoacid\tabularnewline
& 33 & 46 & 47 & 545 & 21 & 37 & 217\tabularnewline
HN-ZZ1 & G & S & C & I & A & D & T\tabularnewline
FPV HN-ZZ2 & D & N & Y & V & T & G & R\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
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