Natural infection of parvovirus in wild fishing cats (Prionailurus viverrinus) reveals extant viral localization in kidneys

Chutchai Piewbang; Sabrina Wahyu Wardhani; Jira Chanseanroj; Jakarwan Yostawonkul; Suwimon Boonrungsiman; Nattika Saengkrit; Piyaporn Kongmakee; Wijit Banlunara; Yong Poovorawan; Tanit Kasantikul; Somporn Techangamsuwan

doi:10.1371/journal.pone.0247266

. 2021 Mar 2;16(3):e0247266. doi: 10.1371/journal.pone.0247266

Natural infection of parvovirus in wild fishing cats (Prionailurus viverrinus) reveals extant viral localization in kidneys

Chutchai Piewbang ^1,², Sabrina Wahyu Wardhani ^2,³, Jira Chanseanroj ⁴, Jakarwan Yostawonkul ^3,⁵, Suwimon Boonrungsiman ⁵, Nattika Saengkrit ⁵, Piyaporn Kongmakee ⁶, Wijit Banlunara ¹, Yong Poovorawan ⁴, Tanit Kasantikul ⁷, Somporn Techangamsuwan ^1,^2,^*

Editor: Simon Clegg⁸

PMCID: PMC7924760 PMID: 33651823

Abstract

Carnivore protoparvovirus-1 (CPPV-1), a viral species containing feline panleukopenia virus (FPV) and canine parvovirus (CPV) variants, are widely spread among domestic and wild carnivores causing systemic fatal diseases. Wild fishing cats (Prionailurus viverrinus), a globally vulnerable species, have been found dead. Postmortem examination of the carcasses revealed lesions in intestine, spleen and kidney. CPPV-1 antigen identification in these tissues, using polymerase chain reaction (PCR) and immunohistochemistry (IHC), supported the infection by the virus. PCR- and IHC-positivity in kidney tissues revealed atypical localization of the virus while in situ hybridization (ISH) and transmission electron microscopy (TEM) with the pop-off technique confirmed the first description of viral localization in kidneys. Complete genome characterization and deduced amino acid analysis of the obtained CPPV-1 from the fishing cats revealed FPV as a causative agent. The detected FPV sequences showed amino acid mutations at I566M and M569R in the capsid protein. Phylogenetic and evolutionary analyses of complete coding genome sequences revealed that the fishing cat CPPV-1 genomes are genetically clustered to the FPV genomes isolated from domestic cats in Thailand. Since the 1970s, these genomes have also been shown to share a genetic evolution with Chinese FPV strains. This study is the first evidence of CPPV-1 infection in fishing cats and it is the first to show its localization in the kidneys. These findings support the multi-host range of this parvovirus and suggest fatal CPPV-1 infections may result in other vulnerable wild carnivores.

Introduction

The genus Prionailurus, family Felidae, is categorized as a group of spotted, small-sized, wild cats that are native to Asia [1]. The Prionailurus genus comprises four formally recognized wild cat species including the leopard cat (P. bengalensis), the flat-head cat (P. planiceps), the rusty-spotted cat (P. rubiginosus) and the fishing cat (P. viverrinus) [2]. The fishing cat lives mainly in the vicinity of swamps and mangroves. It lives exclusively in South and Southeast Asia and is more prevalent in Thailand [3, 4]. At present, fishing cat populations are threatened as the wetlands where they live have been destroyed over the last decade. As a result, this species is currently considered vulnerable and is on the Red List of the International Union for Conservation of Nature (IUNC) [3]. Due to the loss of their habitat, fishing cat colonies have been migrating and occasionally living in areas where domesticated animals and humans live [5]. This phenomenon of shared environment may lead to increased pathogen spillover between susceptible wild and domestic animals and vice versa. Reports of fatal outbreaks in either wild or domestic animals that may have resulted from pathogen spillover have been documented over the past few decades [6–9]. Thus, the transmission dynamics of pathogens from those animals should be examined and needs further investigation.

Carnivore protoparvovirus-1 (CPPV-1), a viral species including mink enteritis virus (MEV), raccoon parvovirus (RPV), feline panleukopenia virus (FPV) and variants of canine parvovirus (CPV), has been recognized as an important pathogen associated with fatal diseases in both wild and domestic Canidae and Felidae families [10, 11]. FPV, a common pathogen of the Felidae species, has been hypothesized to be a common ancestor of CPV [12–14]. CPV frequently infects animals in the Canidae family and there is increasing evidence of infection in the Felidae family counterpart [9, 15, 16]. Previously, FPV and CPV have been hypothesized to be a host-restricted virus. However, several findings subsequently indicated that some animals in the family Canidae might play a role as a reservoir for FPV or vice versa [11, 17, 18]. Moreover, several reports have indicated that the FPV and CPV variants (CPV-2a, -2b, and -2c) share susceptible hosts. These hosts are not only domestic animals but also a variety of carnivorous species [15, 19, 20], suggesting a multi-host range of CPPV-1 [11].

From 1996 to 1997, CPV variants were first detected in leopard cats and were initially designated as leopard cat parvovirus (LCPV), suggesting the first description of CPPV-1 in the genus Prionailurus [21, 22]. Some of these LCPV strains were revealed to be genetically divergent from the previously described CPV-2a and -2b due to a mutation of the G300A amino acid in the capsid (VP) protein. This resulted in a change in its antigenic properties that is now used as a unique mutation point to differentiate CPV-2c from other CPV variants [22]. Discovery of the new CPV variant (currently known as CPV-2c) in leopard cats supported the idea of co-evolution of CPPV-1 among animal species. Later, infections of FPV and CPV variants were reported in leopard cats [9]. This suggested that other species in the same genus as the leopard cat (genus Prionailurus) may be susceptible to CPPV-1 infection. Until now, susceptibility to CPPV-1 infection in other species in the genus Prionailurus is still largely unknown. Deciphering how CPPV-1 variants are evolving and how newly emerging variants behave may help elucidate how to prevent a possible outbreak of the new variant. Even if the new virus gains wider host ranges, fatal outbreaks or atypical lesions associated with the infection might be observed in a new, susceptible host. This study describes a fatal CPPV-1 infection in wild fishing cats (Prionailurus viverrinus). Genetic characterization of the obtained CPPV-1 reveals FPV as a causative agent. This finding provides the first evidence of FPV infection in this species, and it unveils the unique cellular tropism of this virus. Further elucidation of FPV infection and its variants in this species is necessary for its account for global infection in numerous vulnerable species.

Materials and methods

Animals and routine postmortem examination

In June 2019, two wild fishing cats (P. viverrinus), designated case nos. 1 & 2 were found dead in the suburban area in Nakhon Ratchasima Province, Thailand. Later in August 2019, another fishing cat, designated case no. 3, was being rescued from the area where the first two fishing cats were found. Due to the severity of dehydration, case no. 3 died during referral to the animal hospital. These three fishing cats were submitted for postmortem investigation as a routine process. Unfortunately, samples of free-roaming domesticated animals or wild species harbored in the same area were not available; thus, they were not included in this study. Routine postmortem examination was performed on all fishing cats. Selective vital organs including lung, liver, heart, spleen, and kidney were sampled from all fishing cats, while the intestine and mesenteric lymph node were additionally collected from fishing cat no.3. All selected tissues were submitted for histological examination and individually sampled for further molecular assays. For histology, the tissues were immersed in 10% (v/v) neutral buffered formalin for at least 24 hours and routinely processed. Then they were stained with hematoxylin and eosin (H&E) using standard procedures prior to investigation by an ACVP board-certified veterinary pathologist (TK). Fresh tissue samples obtained from the three fishing cats were stored at –80°C for molecular studies. All procedures were performed in accordance with the guidelines and regulations following the approval of the Chulalongkorn University Animal Care and Use Committee (No. 1931036).

General virological molecular assays

The fresh tissue samples including heart, lung, liver, spleen, and kidney of all fishing cats plus additional intestinal and mesenteric lymph node tissues of fishing cat no. 3, were subjected to viral nucleic acid extraction by individually homogenizing them with 1% (v/v) phosphate-buffered saline (PBS). Subsequently, the total viral nucleic acid was extracted using a commercial viral nucleic acid extraction II kit (Geneaid, Taipei, Taiwan) following the manufacturer’s suggestion. The extracted nucleic acids were then qualified and quantified by their A₂₆₀/A₂₈₀ absorbance ratio using the spectrophotometry method (NanoDrop, Thermo Scientific™, Waltham, MA, USA). All extracted nucleic acids were further subjected to viral molecular investigation using polymerase chain reaction (PCR) analysis with several pan-virologic-family PCR testing panels including the detection of the herpesvirus [23], paramyxovirus [24], pneumovirus [25], calicivirus [26], influenza virus [27], bocavirus [26, 28], parvovirus [29] and coronavirus [30, 31]. The PCR protocols, reagents, cycling conditions and positive/negative controls used in the reactions are described in the S1 File. PCR detection specific of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of feline was used as an internal control as described previously [32]. Subsequently, the positive PCR amplicons were visualized using a QIAxcel capillary electrophoresis platform (Qiagen, Hilden, Germany) as previously described [33]. The positive amplicons of each PCR assay were purified and subjected to bidirectional Sanger sequencing (Macrogen Inc, Seoul, South Korea). Due to moderate autolysis of the tissues, it was not possible to perform bacteriological investigation on these fishing cats.

In situ CPPV-1 detection using immunohistochemistry (IHC), in situ hybridization (ISH) and Transmission Electron Microscopy (TEM)

Initial virologic molecular screening revealed positive amplicons of pan-parvovirus PCR and the sequencing results also showed potential DNA sequences of CPPV-1. These findings prompted us to further confirm the presence of CPPV-1 and to localize the distribution of CPPV-1 in the tissues of fishing cats. Formalin-fixed paraffin-embedded (FFPE) tissues including lung, liver, heart, spleen, kidney and intestine were subjected to CPPV-1 IHC detection using a mouse monoclonal anti-canine parvovirus antibody (ab59832, Abcam, Cambridge, UK) with the horseradish peroxidase (HRP) detection system (EnVision polymer, Dako, Glostrup, Denmark). Briefly, the FFPE sections were cut to 4-μm thickness and further deparaffinized and rehydrated. The slides were then pretreated, endogenous peroxidase blocked and processed as previously described [11]. Sections of the intestinal tissue of a FPV-infected cat and a CPV-infected dog were used as positive controls, while identical sections incubated with purified goat-mouse and rabbit IgGs (IHC universal negative control reagent, code ADI-950-231-0025, Enzo Life Sciences, Farmingdale, NY, USA), were used as negative controls. Regarding the renal tubular necrosis present in the areas where CPPV-1 IHC positive signals were observed, the kidney sections of all fishing cats were subjected to Periodic Acid-Schiff (PAS) special staining, in order to demonstrate the cellular architecture of kidney tissues.

Furthermore, the FFPE kidney sections of fishing cat nos. 1 & 3 were subjected to ISH and TEM analysis in order to support the result of positive CPPV-1 IHC staining and to ultra-structurally demonstrate the viral particles in renal tissues, respectively.

For the ISH, the CPPV-1 DNA probe covering 393 bp [34] of the capsid gene of CPPV-1 was constructed using a PCR DIG Probe Synthesis Kit (Roche Diagnostics, Basel, Switzerland), according to the manufacturer’s suggestions. The thermal cycling reaction and condition were performed as previously described [34] with the exception by using the digoxigenin (DIG)-labeled oligonucleotides. The constructed hybridization probe was visualized and confirmed by size resolution on 1.5% (w/v) agarose gel electrophoresis. The ISH with chromogenic DNA was done as previously described [26] with minor modifications. Briefly, after deparaffinization, rehydration, and subsequent rinsing in PBS, the 4-μm-thick FFPE slides were subjected to proteolytic digestion, post-fixation, and pre-hybridization. The slides were then hybridized with the 25 ng/μL ISH probe at 42°C overnight. The hybridization signals were visualized by coupling with 100 μL of anti-DIG-AP Fab fragments (Roche, Basel, Switzerland) (1:200 in 1X Blocking solution) in combination with Liquid Permanent Red (LPR) (Dako, Glostrup, Denmark). Slides were then counterstained with hematoxylin. Red precipitates in the correlation of cellular morphology were thought to be positive for ISH. For negative controls, the tilapia lake virus (TiLV) probes [35] were employed, instead of the CPPV-1 probe.

The TEM samples were prepared in accordance with pop-off techniques with modifications [36–38] and were stained with heavy metals as previously described [39, 40]. The ultrastructure was investigated using TEM (HT7800; Hitachi, Tokyo, Japan) operated at 80 kV.

Full-length genetic characterization of fishing cat CPPV-1

As the results of virologic PCR and IHC screening were in agreement and the results of partial VP-2 genome sequences obtained from pan-parvoviral PCR suggested genetic similarity between case nos. 1 & 2, but not for case no.3, we further characterized the full-length CPPV-1 sequence from these fishing cats (case nos. 1 & 3) and classified the CPPV-1 origin using a set of degenerated primer pairs specific for FPV and CPV retrieved from a previous publication [26]. Briefly, the extracted nucleic acids obtained from spleen and kidneys of two fishing cats plus additional intestinal tissue of fishing cat no. 3, were individually amplified using a GoTaq^® Hot Start Green Master Mix (Promega, Madison, WI, U.S.A.) and specific primers (S1 File). The PCR conditions have been described previously [26]. The target amplicons were visualized using 2% (w/v) agarose gel electrophoresis and further purified using a Monarch DNA Gel extraction kit (New England Biolab, Frankfurt, Germany) prior to commercial Sanger sequencing. The complete genome sequences of the CPPV-1 isolates obtained from the two fishing cats were deposited in GenBank (accession numbers MW145540-MW145541).

Phylogenetic, recombination and evolutionary analyses of fishing cat CPPV-1

The full-length genome sequences of the fishing cat CPPV-1 strains were aligned with the published complete CPPV-1 sequences obtained from various host species, available in GenBank using MAFFT V. 7 (http://mafft.cbrc.jp/alignment/server/) and MEGA7 software (http://www.megasoftware.net). The sequence alignments were then subjected to phylogenetic tree construction. The phylogenetic tree was created using the maximum likelihood (ML) method with the Hasegawa–Kishino–Yano with gamma distribution (HKY+G) model, which was selected using the find-best-fit model algorithm in MEGA7 according to the Bayesian information criterion. The tree was bootstrapped with 1,000 replicates. Sequence pairwise similarity among CPPV-1 genomes was calculated using the maximum composite likelihood model and their evolutionary distance was analyzed in MEGA7. The output pairwise nucleotide identities were generated and visualized in Microsoft Excel format (S2 File). The output alignment sequence of CPPV-1 was then used as a template for genetic recombination analysis using two statistically independent software packages. Briefly, the alignment identified the potential recombination strains using integrated Recombination Detection Program 4 (RDP4) Package V. Beta 4.94 software and this was crosschecked with a similarity plot and bootscanning analysis in the SimPlot software package V. 3.5.1. The settings of the programs and interpretation of the results were conducted as previously described [26, 41].

For evolutionary analysis of the fishing cat CPPV-1, a dataset of 224 complete genome sequences containing 26 FPV-, 6 MEV- and 192 CPV sequences, all originating from 18 countries between 1979 and 2019, was retrieved from the GenBank database. Evolutionary analysis was performed with the 2 potential CPPV-1 sequences obtained from fishing cats using the Bayesian Markov chain Monte Carlo (BMCMC) model implemented in BEAST V. 2.4.8 [42]. The jModelTest [43] was performed to identify the best fitting nucleotide substitution model for multiple alignment sequences. The best-fit substitution models under lognormal relaxed and strict clock models at constant population sizes as priors were implemented to account for varied evolutionary rates among lineages. The coalescent Bayesian skyline tree prior and empirical base frequencies were obtained under the best-fit clock model and run for 100 million chains, sampling every 10,000^th generation, with the first 10% discarded as burn-in. The convergence of parameters was confirmed by calculating the effective sample size using the TRACER program V. 1.7.0 [44]. The maximum clade credibility trees were annotated using TreeAnnotator V. 1.8.3 [42]. The phylogenetic tree with estimated divergence, variable timeline, posterior probability and 95% highest posterior density (HPD) was generated and displayed using FigTree V. 1.4.3 [45].

Retrospective study of CPPV-1 antigen in wildlife carnivores

In order to explore the role of CPPV-1 associated with morbidity and mortality with unknown causes in wild carnivores in the distant and recent past, 305 selective fresh samples were included for genomic extraction and identification targeting of the CPPV-1 capsid gene as described above. These samples have been obtained since 2013 as either fecal swabs or intestinal contents. They were obtained from 136 zoo animals from 27 different carnivorous species (S1 Table).

Results

Postmortem findings and tissue localization of fishing cat CPPV-1

All necropsied fishing cats were moderately emaciated with varying degrees of dehydration while 2 of the 3 fishing cats (case nos. 1 & 2) showed moderate autolysis. Prominently, the spleen and kidneys were congested in all fishing cats. Fishing cat no.3 had macroscopic lesions of catarrhal enteritis that contained a watery brown-yellowish content in their lumens and congestion of the mesenteric lymph node. Histologically, the lung of fishing cat no. 3 revealed severe congestion with infiltration of mononuclear cells in alveoli. The liver showed mild periportal hepatitis, characterized by mononuclear cell infiltration at liver portal triads.

Similar degrees of splenic congestion with few numbers of splenic lymphoid follicles were observed in all fishing cats. The lymphoid follicles were depleted and the remaining lymphoid follicles amid collapsed splenic architecture with increased numbers of prominent splenic trabeculae (Fig 1A). There were scattered karyorrhectic debris of lymphocytes with accumulations of eosinophilic fibrillar materials in the center of such follicle. Few numbers of these lymphocytes contain 5–7 μm basophilic intranuclear inclusion bodies that marginate the nuclear chromatin.

Shortening of intestinal villi with collapse of mucosal architecture was present in the intestinal section of fishing cat no. 3 (Fig 1B). Some crypts were dilated, and they contained eosinophilic proteinaceous material and were lined by markedly attenuated or necrotic crypt epithelial cells. Many cryptal epithelial cells were pyknotic and karyorrhectic and rare cells contained similar basophilic intranuclear inclusion bodies. CPPV-1 IHC was used to demonstrate the presence of the virus in tissues and to describe the pathological role of CPPV-1 in the lesions observed in routine postmortem examination. The residing mononuclear cells in the spleen revealed positive intense immunoreactivity of CPPV-1 in all fishing cats (Fig 1C). The immunoreactivity was also observed in most of the cytoplasm of cryptal epithelial cells and was compatible with the intestinal lesions of fishing cat no. 3 (Fig 1D).

Notably, various degrees of renal tubular vacuolation and multifocal renal hemorrhage were evident in all fishing cats (Fig 2A). Some renal tubular epithelial cells were hypereosinophilic with nuclear pyknosis, while the rare vacuolated tubular epithelial cells revealed vesiculated nuclei with marginating nuclear chromatin (Fig 2B). Regarding the kidney lesions, we further performed PAS staining to demonstrate the cellular architecture of kidney tissue and to exclude systemic hypoxia as being the possible cause of this lesion. PAS staining demonstrated the renal tubular basement membranes were intact (Fig 2B, inset).

Within the kidney sections, CPPV-1 immunolabelling was strong and frequently seen in the cytoplasm of renal tubular epithelial cells (Fig 2C) and urothelial cells at the renal pelvis in all cases. No evidence of immunogenic reaction was observed in the negative controls (S1 Fig). The atypical CPPV-1 localization in renal tissues revealed by IHC suggested that we should conduct additional investigations to confirm the presence and localization of the CPPV-1 in kidney using the ISH and TEM with pop-off technique. The CPPV-1 ISH-immunoreactivity was positive in all fishing cats and diffusely localized in the nucleus of renal tubular epithelial cells, where the tubular lesions were observed (Fig 2D). No reaction was observed in the negative controls (S1 Fig.)

In accordance with results of positive immunological signals of ISH that localized in the nucleus of renal epithelial cells, numerous small electron-dense viral particles, estimated at about 19–22 nm in diameter, were observed. These were clustered in the nuclei of these renal tubular and urothelial cells (Fig 3).

Fig 3 — Transmission electron microscopy (TEM) using the pop-off technique. Ultrastructural demonstration of clustering of electron-dense particles (arrows). The icosahedral particle size was 19–22 nm diameter located in the nucleus of renal tubular epithelial cells (inset). Scale bars as shown in the figure.

Detection of fishing cat CPPV-1 and genomic analysis

Primarily, the pan-family PCR panels specific for various viruses including herpesvirus, paramyxovirus, pneumovirus, calicivirus, influenza virus, bocavirus, coronavirus and parvovirus were tested on the extracted viral nucleic acid samples. This revealed positive results only with the pan-parvovirus PCR in intestine, lymph node and kidney samples of the three fishing cats. Conversely, other pan-PCRs for the other virus detections were negative in all tested tissue samples.

From previously obtained results of pan-parvovirus PCR positivity in all fishing cats, we further characterized the coding genome sequences in two fishing cats using several primer pairs in the extracted nucleic acid samples obtained from intestine (case no.1) and kidney (case no.3). The 4,462 and 4,430 base pairs of complete coding genomes of two fishing cats CPPV-1 strains were detected and were designated strain 19ZP004-TH/2019 (case no.1, accession no. MW145540) and 19ZP005-TH/2019 (case no.3, accession no. MW145541), respectively. After genetic analysis, the complete coding genomes revealed genetically similar results among CPPV-1 strains obtained from fishing cats by showing that these fishing cat CPPV-1 were FPV. Deduced amino acid comparison between the CPPV-1 variants and the fishing cats FPV are described in Table 1. Of note, the fishing cat FPV revealed unique amino acid mutations of I566M and M569R in the structural (VP1 & 2) protein, that were not detected in previous CPPV-1 variants.

Table 1. Alignment of capsid amino acid sequence of CPPV-1 isolated from fishing cats with previously described CPPV-1 variants retrieved from the NCBI database.

The accession numbers, the CPPV-1 variant, infected host, country and year of sample collection are presented.

Isolations	Amino acid position of VP2 protein
Isolations	80	87	93	101	103	232	297	300	305	323	370	426	564	566	568	569
MN908257 FPV Tiger China 2019	K	M	K	T	V	V	S	A	D	D	Q	N	N	I	A	M
MG764511 FPV Lion China 2015	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
MN862743 FPV Mink Canada 2018	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
MF069447 FPV Raccoon Canada 2016	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
MN862748 FPV River Otter Canada 2019	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
MN862744 FPV Pine Marten Canada 2016	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
MN451652 FPV Arctic Fox Finland 1983	.	.	.	.	.	.	.	V	.	.	.	.	.	.	.	.
MH127910 FPV Leopard Cat Taiwan 2017	n/a	n/a	n/a	n/a	n/a	n/a	n/a	.	.	.	.	.	.	.	.	.
MH127911 FPV Leopard Cat Taiwan 2017	n/a	n/a	n/a	n/a	n/a	n/a	n/a	.	.	.	.	.	.	.	.	.
Fishing cat CPPV-1/ZP004	.	.	.	.	.	.	.	.	.	.	.	.	.	M^*	.	R^*
Fishing cat CPPV-1/ZP005	.	.	.	.	.	.	.	.	.	.	.	.	.	M^*	.	R^*
MG924893 FPV Cat China 2016	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
MN127779 FPV Cat Thailand 2018	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
MN127781 FPV Cat Thailand 2019	.	.	.	.	.	I	.	.	.	.	.	.	.	.	.	.
KP019621 Civet CPPV-1 Thailand 2013	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
KR002804.1 CPV 2a Dog China 2014	R	L	N		A	I	A	G	Y	N	.		S	.	G	.
KR002799.1 CPV 2b Dog China 2013	R	L	N		A	I	A	G	Y	N	.	D	S	.	G	.
MF177228.1 CPV 2c Dog Italy 2009	R	L	N		A	I	A	G	Y	N	.	E	S	.	G	.
MN747143.1 MEV Mink China 2018	R	.	N	I	A	I	.	D	.	N	.		S	.	G	.

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CPPV-1: carnivore protoparvovirus-1; CPV: canine parvovirus; FPV: feline parvovirus; MEV: mink enteritis virus.

*Unique amino acid mutations observed in fishing cat CPPV-1; n/a: not available data.

Phylogenetic and evolutionary analysis

For comparing the genetic relationship between detected fishing cat CPPV-1 and other previously published CPPV-1 genomes, phylogenetic analysis based on complete coding genome sequence was conducted. The ML phylogenetic tree showed that most FPV, MEV and CPV formed a very distinct clade among groups (Fig 4). The two CPPV-1 genomes from the studied fishing cats (indicated by red triangles) were clustered together with the same lineage of previously published FPV sequences obtained from various hosts, confirming that the obtained fishing cat CPPV-1s were FPV. The detected genomes were clustered together and most closely related to the FPV genome detected in domestic cats in Thailand (accession no. MN127780). To further elucidate the evolutionary process of detected fishing cat CPPV-1, several recombination detections and evolutionary analyses on a dataset of 224 CPPV-1 complete genome sequences were conducted. The recombination analysis revealed that no recombination events were found in fishing cat CPPV-1 sequences.

Fig 4 — The phylogenetic tree showed that the fishing cat CPPV-1 sequences (indicated by red triangles) were clustered together with the FPV genomes with the genome most related to the FPV genome detected in the Thai cat (MN127780). The GenBank accession numbers of previously described CPPV-1 sequences used in this analysis were indicated.

The evolutionary tree of these data showed two main distinct separated clades (Fig 5). One clade included FPV and MEV and another clade included all CPV sequences. The overall evolutionary rate was estimated at 1.08 × 10⁻⁴ substitutions/site/year (95% HPD: 1.95–2.87 × 10⁻⁴). The evolutionary tree showed that fishing cat FPV sequences were clustered in the same lineage of FPV as that isolated from Thailand. They have shared genetic evolution with FPV genomes detected in domestic cats in China and may have the same origin since around 1970.

Retrospective study of CPPV-1 in other wild samples

A retrospective study of 136 zoo-wild animals derived from 27 different carnivores did not reveal the presence of CPPV-1 genomic antigen as detected by PCR.

Discussion

Circulation of CPPV-1 variants that includes CPV and FPV have been identified in both domestic and wild carnivores. While fatal infection of CPPV-1 in domestic dogs and cats is common, this infection is sporadically seen in wild carnivores [46]. Several studies have indicated that many wild carnivores are susceptible to CPPV-1 infection including wild felids, minks, otters, skunks, raccoons, foxes, wolfs, coyotes, civets and martens [11, 19]. Particular to the genus Prionailurus, only leopard cats have been reported to be infected with CPPV-1 variants [9]. In this study, we describe the natural infection of FPV, a member of CPPV-1, in wild fishing cats (Prionailurus viverrinus). This is the first report with a description of distinct viral distribution and tropism in kidney tissues as confirmed via antigenic detection using PCR, IHC, ISH and TEM analysis of the ultra-structure.

Cross-species transmission of CPPV-1 variants between domestic and wild carnivores has been observed, and some fatal CPPV-1 outbreaks have been speculated to be associated with pathogen spillover among habitats [9, 11]. Due to the current loss of wildlife habitat, wild colonies have been migrating and sharing habitats with domestic animals, which has led to increased pathogen spillover between susceptible animals. In this study, we found that the fishing cat CPPV-1 genomes have shared an evolutionary pattern with the FPV strains detected in domestic cats in Thailand. These results show a similar evolutionary pattern to the FPV strains from common ancestors isolated from domestic cats in China since the 1970s. This result may imply either possible evidence of cross-species transmission between domestic cats and fishing cats or co-evolution of the CPPV-1 variants. However, owing to the limitation of samples obtained from free-roaming animals in the same habitats and the small numbers of investigated fishing cats, the primary reservoir of the CPPV-1 in this study could not be identified and further investigation is needed.

Although several studies have indicated that various amino acid residues in the capsid gene are associated with both the ability to infect new hosts and the antigenicity of CPPV-1 [18, 47], the single amino acid mutation at position 300 of the capsid protein (VP2) has been reported as a key determinant for multiple host ranges [48]. In this study, the CPPV-1 variant obtained from the fishing cats revealed the same amino acid patterns as found as in FPV sequences, with the presence of alanine (A) as a key amino acid determinant at capsid position 300. While the fishing cat capsid protein of CPPV-1 revealed asparagine (N) and alanine (A) amino acids located at positions 564 and 568, respectively (which are considered to be critical for FPV replication in the feline host), they had 2 unique amino acid mutations at positions I566M and M569R. These mutations can differentiate this virus from previously described CPPV-1 variants. Previous analysis of the ancestral reconstruction found that FPV can infect distinct carnivores with few changes in the VP2 gene [49]. Thus, the FPV-like parvovirus was tentatively named as a potentially novel virus detected in the fishing cats and this may support the first identification of this virus in this species. However, a small number of investigated fishing cats compared to a small number of other sequences may not give a definitive interpretation. This needs further study for more definite conclusions to be drawn.

Fishing cat CPPV-1 localizations were observed in various tissues including intestinal epithelial cells and lymphoid cells, where the most common tissue localization of the CPPV-1 infection is found in other species [20, 26, 50, 51]. Interestingly, since initial investigation of viral genomic detection using PCR and viral localization using IHC revealed distinct viral localization in kidney tissues, we performed PAS staining to demonstrate the cellular architecture of renal tissue and to exclude systemic hypoxia as being the possible cause of tubular necrosis. PAS staining revealed the most tubular basement membranes were intact, suggesting that this lesion may not result from systemic hypoxia [52]. However, other causes such as toxin-induced tubular necrosis can cause this lesion. Thus, the role of FPV-like parvovirus infection associated with kidney lesions still needs further investigation. Moreover, we attempted to further elucidate extant localization of the virus in kidney tissue using ISH and TEM with the pop-off technique [37] in the areas where CPPV-1 IHC-immunoreactivity was detected. The electron-dense particles were observed in the nucleus of renal tubular epithelial cells and urothelial cells, supporting the positive in situ immunoreactivity of CPPV-1 in kidney tissue. FPV-infected cats shed the virus predominantly in feces, but the presence of active virus in urine has been described [53, 54]. Recent studies have indicated that parvovirus infection has a role in renal failure [55]. However, the localization of FPV and the CPV counterpart in the kidney tissue has not previously been investigated. Furthermore, a mouse kidney parvovirus (MKPV) has been proven to be a nephrotropic virus by showing an affinity to infect and localize in renal tubular epithelial cells, resulting in kidney diseases [56, 57].

Based on our knowledge, CPPV-1 localization in the kidneys has not been previously described. This suggests the novel description of a unique viral localization in this species or even if it can be found in FPV-infected cats. This finding may support previous studies which found FPV in the urine. Unique pathological findings may be subjective and may be observed as infection in individuals. Thus, a large-scale investigation of fishing cat CPPV-1 will facilitate a definitive interpretation. In addition, specimens that were submitted mostly from dead animals and had already undergone postmortem autolysis are not suitable for further organ collection from these animals. Thus we were not able to determine the viral localization in other organs. Elucidation of atypical viral localization of fishing cat CPPV-1 would be a useful investigation.

CPPV-1 has been identified in various wild carnivores and it is associated with fatal outbreaks. The negative evidence of retrospective CPPV-1 detection in zoo-wild samples in this study may result from either no close-contact among susceptible animals or, on the other hand, prolonged sample storage that affects the stability of genomic materials. The novel identification of CPPV-1 associated with mortal infection of fishing cats in this study raises concerns about a novel, potentially virulent disease in this vulnerable animal. Therefore, further study of the transmission of CPPV-1 in fishing cats is essential to prevent a loss of other susceptible animal species. Accordingly, prevention strategies should be emphasized in order to manage CPPV-1 infections, not only in this species but also in free-roaming domestic animals that may serve as potential hosts for this virus.

Supporting information

S1 Fig

Photomicrograph of negative controls for CPPV-1 IHC (A-C) and CPPV-1 ISH (D). No CPPV-1 IHC reaction is present within the negative control section of (A) intestine, (B) spleen, and (C) kidney. (D) No immunoreactivity is present within a kidney section incubating with the TiLV probe (non-related probe).

(TIF)

Click here for additional data file.^{(34.6MB, tif)}

S1 Table. List of wildlife carnivore species enrolled in the retrospective study of CPPV-1 detection.

(DOCX)

Click here for additional data file.^{(14.8KB, docx)}

S1 File. Amplification procedures used for routine pan-virologic-family detection and full-length genetic characterization of fishing cat CPPV-1.

(DOCX)

Click here for additional data file.^{(37.5KB, docx)}

S2 File. Pairwise nucleotide distances of fishing cat CPPV-1 and previously published CPPV-1 genomes.

The nucleotide similarity of complete coding sequences of various CPPV-1 variants is compared with the nucleotide sequences of CPPV-1 obtained from fishing cats in this study. Accession nos. are indicated.

(XLS)

Click here for additional data file.^{(495KB, xls)}

Acknowledgments

We would like to thank Mr. Poowadon Chai-in, for development of sample preparation techniques for TEM, and Dr. Robert D. Gaffin and Dr. Ariyaporn T. Gaffin for proofreading.

Data Availability

Obtained CPPV-1 sequences of fishing cat nos 1 & 3 have been deposited in NCBI GenBank under accession numbers MW145540-MW145541, respectively. All other relevant data are within the manuscript and its Supporting Information files.

Funding Statement

C.P. was supported by the Ratchadapisek Somphot Fund for Postdoctoral Fellowship, Chulalongkorn University. S.W. is financially afforded by scholarship program for ASEAN countries, Chulalongkorn University. This research was funded by The Thailand Research Fund (RSA6180034), Grant for Joint Funding of External Research Project, Ratchadaphisek Somphot Endowment Fund and Veterinary Science Research Fund (RES_61_364_31_037), Chulalongkorn University, and Veterinary Pathogen Bank, Faculty of Veterinary Science, Chulalongkorn University.

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PLoS One. doi: 10.1371/journal.pone.0247266.r001

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PONE-D-20-33818

Natural infection of parvovirus in wild fishing cats (Prionailurus viverrinus) reveals extant viral localization in kidneys

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Reviewer #1: The authors investigated three cases of natural canine ürptpüarvovirus-1 (CPPV-1) infection in wild fishing cats. In their article they describe a new tropism of parvovirus to renal tissue by using PCR, immunohistochemistry and transmission electron microscopy.

Major comments:

1. In the materials and methods (Line 116ff) the authors describe that they isolate nucleic acids from fresh samples. Since the authors also claim a high degree of autolysis in cases 1&2 they have to specify: 1.) nucleic acids were isolated from which organs in which cats, and 2.) PCRs for virus familys were performed on which organs from which cats.

2. The authors describe that they perform pan-virologic-family PCRs - they should specify for which families they used PCR and for which RT-PCR.

3. In addition, the used PCR/RT-PCR kits have to be specified including PCR conditions and applied positive controls for PCR/RT-PCR.

4. Where process controls included during nucleic acid extraction since autolysis may have a significant impact on the performance of the PCR reaction.

In the following sentence the authors state that histologically all fishing cats showed desquamative enteritis, lymphoid depletion in lymph nodes and spleen. What does this mean in the context of the sentence before? Please include a picture detailing lymphoid depletion in spleen and lymph nodes. Were these findings associated with a positive signal in PCR and/or IHC for CPPV-1?

6. Line 230f: What do you mean with severely collapsed intestinal mucosa?

7. Regarding the tubular necrosis: Do the authors perform PAS-stain to exclude hypoxia as being the cause of the tubular necrosis? Were the necrotic cells positive for CPPV-1?

8. The IHC-picture of the kidney looks like as if almost all renal tubular cells were positive for CPPV-1 antigen. To confirm their finding the authors should perform in situ hybridizytion to show a correlation/specificity of the IHC for CPPV-1.

9. Regarding the retrospective study (lines 315ff): What do the authors mean by "A retrospective study of 136 zoo-wild animals in 27 carnivores revealed the presence of CPPV-1 genomic antigen."? Does this mean 27 cases were positive? The authors should explain which organs were investigated in which animal and what methods including nucleic acid extraction and PCR method were used. Do the authors also perform IHC? If all samples were negative, how can the authors show that this result is not related to tissue storage / fixation?

Minor comments:

Line 90 "... CPPV-1 reveals FPV is a ...": please change is to as

Line 327 "... and tropism and in kidney...": please delete the second "and".

Reviewer #2: Carnivore protoparvovirus-1 (CPPV-1), include feline panleukopenia virus (FPV) and canine parvovirus (CPV), which are widespred among domestic and wild carnivores, causing systemic fatal diseases. Wild fishing cats (Prionailurus viverrinus), is a vulnerable species. Virological (PCR and TEM) and gross and microscopic investigations, identified the opresence of an FPV-like parvovirus in fishing cats found dead. Postmortem examination revealed severe enteritis, lymphadenopathy and nephritis. On whole genome sequencing, the virus closely resembled FPV sequences with two peculiar amino acid mutations I566M and M569R in the capsid protein.

The manuscript is of relevant scientific interest. The introduction is well written. The materials and methods seem adequate. The discussion is correct and rather balanced. I have only minor comments.

i) there is a confusion with the terms CPPV-1 and FPV, that are nearly the same thing. I would suggest to use consistently the term FPV-like parvovirus

ii) there are some parts of the manuscript that could be deleted, reworded or re-phrased.

Abstract: check English. Shorten the final part, very generic.

Line 81-83 it is not clear

Lines 81-88: rephrase

Line 90

Line 316

Line 322: please delete/replace the semicoma (;) after infection

Line 325: FPV is not a variant of CPPV-1

Line 346-347: I would delete the comments (Suggesting… Therefore….)

Line 360-361: rephrase

Line 363: odd sentence (Supporting viral genomic detection…). Rephrase

iii) A table with the list (and details) of primers used (consensus and specific), even as supplemental file, should be added.

iv) General comment: the English is good but should be further refined.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2021 Mar 2;16(3):e0247266. doi: 10.1371/journal.pone.0247266.r002

Author response to Decision Letter 0

8 Dec 2020

Rebuttal response on PONE-D-20-33818

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Response: We have revised the manuscript following PLOS ONE's style requirements.

2. To comply with PLOS ONE submissions requirements, please provide methods of sacrifice in the Methods section of your manuscript.

Response: The three fishing cats that enrolled in this study were naturally died by first 2 fishing cats were found dead and another fishing cats died during referral. Thus, we have no information regarding the method of sacrifice. Details of animals in this study were described in Materials and Methods section.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: N/A

________________________________________

3. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

5. Review Comments to the Author

Reviewer #1: The authors investigated three cases of natural canine carnivore protoparvovirus-1 (CPPV-1) infection in wild fishing cats. In their article they describe a new tropism of parvovirus to renal tissue by using PCR, immunohistochemistry and transmission electron microscopy.

Major comments:

Response: With the request from reviewer, we have provided additional information of the sample collection by describing details as “The fresh tissue samples including heart, lung, liver, spleen, and kidney of all fishing cats plus additional intestinal and mesenteric lymph node tissues of fishing cat no. 3, were subjected to viral nucleic acid extraction.” Page 6, lines 113-115. For additional information of PCRs for virus families, we have added more details regarding the PCR protocols in S1_File.

2. The authors describe that they perform pan-virologic-family PCRs - they should specify for which families they used PCR and for which RT-PCR.

Response: We have added more details in S1_file.

3. In addition, the used PCR/RT-PCR kits have to be specified including PCR conditions and applied positive controls for PCR/RT-PCR.

Response: We have provided more details as description in S1_file.

4. Where process controls included during nucleic acid extraction since autolysis may have a significant impact on the performance of the PCR reaction.

Response: Thank you for your concern. At this point, we do respect that the autolysis tissues may significantly impact on the performance of the PCRs, thus the tissue samples which revealed moderate to marked autolysis (presented on necropsy desk) would be not included for the molecular study. Furthermore, we have validated all extracted nucleic acids by further performing the PCR to detect the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as an internal housekeeping gene in order to validate the performance of samples and extraction process. Thus, we have provided the information as “The PCR protocols, reagents, cycling conditions and positive/ negative controls used in the reactions are described in the S1 File. PCR detection specific of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal control as described previously (31).” In page 6, lines 124-127.

5. Regarding the postmortem findings (lines 214ff) the authors stated that "Other organs showed advanced autolysis and so they could not be investigated." Do the authors mean by histology, immunohistochemistry, PCR, TEM? Please specify. In the following sentence the authors state that histologically all fishing cats showed desquamative enteritis, lymphoid depletion in lymph nodes and spleen. What does this mean in the context of the sentence before? Please include a picture detailing lymphoid depletion in spleen and lymph nodes. Were these findings associated with a positive signal in PCR and/or IHC for CPPV-1?

Response: Thank you for question regarding the postmortem findings that the presented statements may lead confusion. Due to the fact that fishing cats no. 1&2 have been found dead and the most of organs revealed autolysis, which is presenting on necropsy desk, resulting in other organs could not be investigated. Thus, we do delete “Other organs showed advanced autolysis and so they could not be investigated” and revise the statement regarding the post-mortem findings as “All necropsied fishing cats were moderately emaciated with varying degrees of dehydration while 2 of the 3 fishing cats (case nos. 1 and 2) showed moderate autolysis. Prominently, the spleen and kidneys were congested in all fishing cats. Note that fishing cat no.3 had macroscopic lesions of catarrhal enteritis that contained a watery brown-yellowish content in their lumens and congestion of the mesenteric lymph node.” in page 11, lines 236-240. Furthermore, we have provided the H&E and CPPV-1 IHC pictures of lymph node as your requested in Fig 1 and the IHC-positive signals were identified in the area of lymphoid necrosis as described in Fig 1 legend.

6. Line 230f: What do you mean with severely collapsed intestinal mucosa?

Response: We have revised the sentence as “(B) Fishing cat no. 3. The intestinal mucosa is diffusely and severely desquamated, the intestinal villi are mostly short, many crypts are lost or contain karyorrhectic debris (inset) and there are decreased numbers of goblet cells.”. Fig1 legend. (Page 12, lines 254-256.)

7. Regarding the tubular necrosis: Do the authors perform PAS-stain to exclude hypoxia as being the cause of the tubular necrosis? Were the necrotic cells positive for CPPV-1?

Response: We have performed the PAS staining in kidney section of all fishing cats. The results were shown in Fig 2B. and described as “Regarding the severe renal tubular necrosis, we further performed PAS staining in kidney sections to demonstrate the cellular architecture of kidney tissue and to exclude systemic hypoxia as being the possible cause of this lesion. PAS staining demonstrated the disruption of renal tubular epitheliums while the architectures of most renal tubular basement membranes were intact (Fig 2B).” in Result section (pages 12-13, lines 264-268) and “we performed PAS staining to demonstrate the cellular architecture of renal tissue and to exclude systemic hypoxia as being the possible cause of tubular necrosis. PAS staining revealed disruption of tubular epithelium while most tubular basement membranes were intact, suggesting that this lesion may not result from systemic hypoxia (51).” in Discussion section (Page 21, lines 406-409). Of note. The CPPV-1 IHC signals were frequently seen in the area of tubular necrosis as we have revised as “The CPPV-1 ISH-immunoreactivity was positive in all fishing cats and diffusely localized in the nucleus of renal tubular epithelial cells, where the tubular necrosis lesions were observed (Fig 2D).” in Results section (page 15, lines 275-277).

8. The IHC-picture of the kidney looks like as if almost all renal tubular cells were positive for CPPV-1 antigen. To confirm their finding the authors should perform in situ hybridization to show a correlation/specificity of the IHC for CPPV-1.

Response: We performed the ISH in kidney sections, derived from all fishing cats and the result picture was shown in Fig 2D. Details regarding the protocols and results were described in Materials and Methods section (pages 7-8, lines 153-171) and Results section (page 13, lines 275-278, respectively.

Response: The statement regarding the result of retrospective study is confused so we revised the sentence as “A retrospective study of 136 zoo-wild animals derived from 27 different carnivores did not reveal the presence of CPPV-1 genomic antigen as detected by PCR” in page 19, lines 360-361. For the methods and samples used for retrospective study, we have provided the information in the Materials and Methods section as “305 selective fresh samples were included for genomic extraction and identification targeting of the CPPV-1 capsid gene as described above.” in page 11, lines 229-231. Furthermore, we have revised the discussion as the prolonged sample storage may affect the PCR result in as “The negative evidence of retrospective CPPV-1 detection in zoo-wild samples in this study may result from either no close-contact among susceptible animals or, on the other hand, prolonged sample storage may affect the stability of genomic materials..” in page 22, lines 432-434.

Minor comments:

Line 90 "... CPPV-1 reveals FPV is a ...": please change is to as

Line 327 "... and tropism and in kidney...": please delete the second "and".

Response: We have revised them as reviewer suggestions.

Reviewer #2: Carnivore protoparvovirus-1 (CPPV-1), include feline panleukopenia virus (FPV) and canine parvovirus (CPV), which are widespred among domestic and wild carnivores, causing systemic fatal diseases. Wild fishing cats (Prionailurus viverrinus), is a vulnerable species. Virological (PCR and TEM) and gross and microscopic investigations, identified the presence of an FPV-like parvovirus in fishing cats found dead. Postmortem examination revealed severe enteritis, lymphadenopathy and nephritis. On whole genome sequencing, the virus closely resembled FPV sequences with two peculiar amino acid mutations I566M and M569R in the capsid protein.

Response: Thank you for your feeling positive with our manuscripts.

i) there is a confusion with the terms CPPV-1 and FPV, that are nearly the same thing. I would suggest to use consistently the term FPV-like parvovirus

Response: We have revised as reviewer suggestion.

ii) there are some parts of the manuscript that could be deleted, reworded or re-phrased.

Abstract: check English. Shorten the final part, very generic.

Line 81-83 it is not clear

Lines 81-88: rephrase

Line 90

Line 316

Line 322: please delete/replace the semicoma (;) after infection

Line 325: FPV is not a variant of CPPV-1

Line 346-347: I would delete the comments (Suggesting… Therefore….)

Line 360-361: rephrase

Line 363: odd sentence (Supporting viral genomic detection…). Rephrase

Response: We have revised as reviewer suggestions.

iii) A table with the list (and details) of primers used (consensus and specific), even as supplemental file, should be added.

Response: Details of primers used in this study were described in S1_File.

iv) General comment: the English is good but should be further refined.

Response: The English have been reviewed and revised by native speakers.

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Response: On behalf of corresponding author, I do agree to make public revision.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Attachment

Submitted filename: Rebuttal response PONE-D-20-33818.docx

Click here for additional data file.^{(31.3KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0247266.r003

Decision Letter 1

Simon Clegg

5 Jan 2021

PONE-D-20-33818R1

Natural infection of parvovirus in wild fishing cats (Prionailurus viverrinus) reveals extant viral localization in kidneys

PLOS ONE

Dear Dr. Piewbang,

==============================

Many thanks for submitting your manuscript to PLOS One

It was reviewed by two experts in the field, and they have recommended some modifications be made prior to acceptance

In particular, please examine the comments regarding the images and the text within the manuscript (which may require inclusion of a pathologist)

I therefore invite you to make these changes and to write a response to reviewers which will expedite revision upon resubmission

I wish you the best of luck with your modifications

Hope you are keeping safe and well in these difficult times

Thanks

Simon

==============================

Please submit your revised manuscript by Feb 19 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Simon Clegg, PhD

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: No

Reviewer #3: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #3: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #1: The manuscript has been improved, however there are still major inconsistencies:

The majority of my comments below deal with the discrepancy between the manuscript text and the displayed photomicrographs and I was wondering, if just these pictures are not representative and the cases show in general, what the authors describe, or if it would be useful to ask a board certified veterinary pathologist reviewing the described findings. This is especially important as the inconsistencies include several organs, e.g. intestine, kidney and spleen. Of particular importance: the picture of the intestine shows a degree of autolysis and I am not convinced that the pictures support the written morphological lesions.

Before publication the authors have to clarify the inconsistency between their morphological description and the pictures included in the manuscript despite the manuscript has been significantly improved compared to the initial submission.

1.) In the abstract the authors state: “Postmortem examination of the carcasses revealed severe inflammation of intestine, lymph node and kidney.”

In their description the authors neither detected inflammation in the intestine nor in the lymph nodes. This description does not fit their morphological description in the results and in the figure legends.

2.) The authors state in the abstract: “CPPV-1 antigen identification in these tissues, using polymerase chain reaction (PCR) and immunohistochemistry (IHC), supported the natural infection of the virus.” How can these techniques support a natural infection?

3.) In the result section the authors state: “Similar degrees of severe lymphoid depletion in spleen were observed in all fishing cats (Fig. 1A).” In the figure description the authors state: “(A) Fishing cat no. 1. Lymphoid depletion of splenic white pulps (asterisks) that indicate by hypocellularity (inset).” These findings are neither visible in the picture nor in the insert. In figure 1A lymphoid follicles are visible and a lymphoid depletion cannot be detected. Furthermore, a severe congestion of the spleen as state in the result section cannot be seen.

4.) With respect to Fig 1B: The findings described by the authors are not visible in the picture: ”Severe diffuse desquamative enteritis, evidenced by shortening villi and necrosis of cryptal epithelium (Fig.1A) was presented in intestinal section of fishing cat no.3 (Fig.1B).”

5.) With respect to Fig 1C: How do the authors know that the immunopositive cells are circulating mononuclear cells or histiocytes as stated in the results/description of figure 1?

6.) The tubular necrosis described by the authors as shown in the pictures is not convincing.

7.) In addition, the authors state: “The PAS staining demostrates the disruption of renal tubular epitheliums while the most architecture of renal tubular basement menbranes were intact (Fig. 2B). This sentence is confusing and the picture especially the insert does not show tubular epithelial cell necrosis. Furthermore, “The tubular lining epithelium are swollen, and the tubular basement membranes are intact (inset).” as described by the authors can not be seen in the pictures.

Reviewer #3: This is an interesting manuscript and one which could be of concern to the fishing cats. I have made a few comments below, but my biggest one is around the pathology images and the text, which do not appear to match the text, so would be grateful if you could check this. But overall it reads well and the comments are only minor, with the above mentioned exception

Line 63-65- there is increasing evidence of CPV in cats (both diseased and asymptomatic), but it is less than in dogs, maybe reword this sentence?

Line 78-79- this reads a little strange- please reword

Line 79- suggested may sound better than warranted

Line 102- comma after cats

Line 112- I think general virological assays isn’t correct- its more molecular or immunological assays

Line 113- comma after cats

Line 118- instructions may be better than suggestions

Line 126- the GAPDH gene of what? Cats or a pathogen/ commensal bacteria?

Line 138- further spelt incorrectly

Line 146-149- this appears unclear and would benefit from some clarification in the text

Line 158-159- repetitive

Line 200- a bootstrap value of 10 000 is often better than 1000

Line 232- it would be nice to know what these species were

Line 238- remove note

Line 248- in the spleen

Figures- I have a few concerns regarding the photos as they do not look representative of what the authors describe. Some show autolysis which makes it difficult to interpret. Do you have any more clear photos. Some of the findings which you mention in the results are not visible in these images

Line 277- in the negative controls

Line 282- you define PAS here despite using it previously, please define at first use

Line 379- co-evolution is suggested for CPV global emergence- what is to say that isn’t the case here rather than pathogen spill over?

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: No

PLoS One. 2021 Mar 2;16(3):e0247266. doi: 10.1371/journal.pone.0247266.r004

Author response to Decision Letter 1

8 Jan 2021

Rebuttal response on PONE-D-20-33818R1

Reviewer #1: The manuscript has been improved, however there are still major inconsistencies:

The majority of my comments below deal with the discrepancy between the manuscript text and the displayed photomicrographs and I was wondering, if just these pictures are not representative and the cases show in general, what the authors describe, or if it would be useful to ask a board-certified veterinary pathologist reviewing the described findings. This is especially important as the inconsistencies include several organs, e.g. intestine, kidney and spleen. Of particular importance: the picture of the intestine shows a degree of autolysis and I am not convinced that the pictures support the written morphological lesions.

Response: Thank you for your kind review. Regarding the morphological descriptions, we kindly requested Dr. Tanit Kasantikul, an American board-certified veterinary pathologist for reviewing and describing the histology of the infected fishing cats. The newly obtained pathological descriptions and some figures were revised according reviewer and the pathologist suggestion.

1.) In the abstract the authors state: “Postmortem examination of the carcasses revealed severe inflammation of intestine, lymph node and kidney.”

Response: We have revised as “Postmortem examination of the carcasses revealed lesions of intestine, spleen and kidney.” Page 2, lines 28-29.

Response: We have revised the sentence as “CPPV-1 antigen identification in these tissues, using polymerase chain reaction (PCR) and immunohistochemistry (IHC), supported the infection of the virus.” Page 2, lines 29-30.

Response: We revised the Fig. 1A by providing the lower magnification picture to present the spare lymphoid follicles in spleen. Of noted, we have revised the pathological description both in text and figure legend that was addressed by the pathologist as described as “Similar degrees of splenic congestion with few numbers of splenic lymphoid follicle were observed in all fishing cats. The lymphoid follicles were depleted and the remaining lymphoid follicles amid collapsed splenic architecture with increased numbers of prominent splenic trabeculae (Fig 1A). There were scattered karyorrhectic debris of lymphocytes with accumulations of eosinophilic fibrillar materials in the center of such follicle. Few numbers of these lymphocytes contain 5-7 um basophilic intranuclear inclusion bodies that marginate the nuclear chromatin.” in main text (pages 11-12, lines 245-251) and “(A) Fishing cat no. 1. Diffuse congested spleen with sparse numbers of lymphoid follicles. Center of one of the remaining lymphoid follicles contained eosinophilic fibrillar material (fibrin) intermixed with scattered karyorrhectic debris of lymphocytes (lymphocytolysis) (inset). Few numbers of these lymphocytes contained 5-7 um basophilic intranuclear inclusion bodies that marginated the nuclear chromatin (arrow).” in the Fig.1 legend (pages 12, lines 264-268).

Response: We have revised the description as “(B) Fishing cat no. 3. Shortening of villi with occasional dilated crypts that contained eosinophilic proteinaceous substances and were lined by markedly attenuated or necrotic crypt epithelial cells (inset). Many crypt epithelial cells were pyknotic and karyorrhectic and rare cells contained similar basophilic intranuclear inclusion bodies (arrows)”. Page 12, lines 268-272.

5.) With respect to Fig 1C: How do the authors know that the immunopositive cells are circulating mononuclear cells or histiocytes as stated in the results/description of figure 1?

Response: We have revised the description as “(C) Fishing cat no. 1. The CPPV-1 immunoreactivity is frequently observed in the cytoplasm of mononuclear cells, where in the area of splenic lymphoid follicle. Page 12, lines 272-274.

6.) The tubular necrosis described by the authors as shown in the pictures is not convincing.

Response: We have changed the Figs. 2A-2B and their legend regarding the pathologist suggestions.

7.) In addition, the authors state: “The PAS staining demostrates the disruption of renal tubular epitheliums while the most architecture of renal tubular basement membranes were intact (Fig. 2B). This sentence is confusing and the picture especially the insert does not show tubular epithelial cell necrosis. Furthermore, “The tubular lining epithelium are swollen, and the tubular basement membranes are intact (inset).” as described by the authors can not be seen in the pictures.

Response: We have revised the figure and text as “PAS staining demonstrated the renal tubular basement membranes were intact (Fig 2B, inset) (page 13, lines 284-285).

Response: Thank you for you review. We have provided the newly obtained pathological description throughout the pathology pictures, which are examined and reviewed by board-certified veterinary pathologist.

But overall it reads well and the comments are only minor, with the above mentioned exception

Line 63-65- there is increasing evidence of CPV in cats (both diseased and asymptomatic), but it is less than in dogs, maybe reword this sentence?

Response: We have revised the sentence as “CPV frequently infects animals in the Canidae family and there is increasing evidence of infection in the Felidae family counterpart.” Page 3, lines 265-267.

Line 78-79- this reads a little strange- please reword

Response: We have revised the sentence as “Later, infections of both FPV and CPV variants were reported in leopard cats (9).” Page 4, lines 80-81.

Line 79- suggested may sound better than warranted

Line 102- comma after cats

Response: We have revised as reviewer suggestion.

Line 112- I think general virological assays isn’t correct- its more molecular or immunological assays

Response: We have revised it as “General virological molecular assays” Page 6, line 115.

Line 113- comma after cats

Line 118- instructions may be better than suggestions

Response: We have revised as reviewer suggestion.

Line 126- the GAPDH gene of what? Cats or a pathogen/ commensal bacteria?

Response: We have added the details of GAPDH as “PCR detection specific of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of feline was used as an internal control as described previously (31). Page 6, lines 128-130.

Line 138- further spelt incorrectly

Response: We corrected it.

Line 146-149- this appears unclear and would benefit from some clarification in the text

Response: We have revised the text as “Sections of the intestinal tissue of a FPV-infected cat and a CPV-infected dog were used as positive controls, while identical sections incubated with….” Page 7, lines 148-149.

Line 158-159- repetitive

Response: We have corrected the sentence as “The thermal cycling reaction and condition were performed as previously described (33)”. Page 8, lines 160-161.

Line 200- a bootstrap value of 10 000 is often better than 1000

Response: Thank you for your suggestion, we do agree with your suggestion about higher bootsrapping parameter will be better. However, since genetic diversity among CPPV-1 is relatively low, 1000 repetitive bootstrapping parameter is enough for analysis.

Line 232- it would be nice to know what these species were

Response: We have provided the list of wildlife carnivores that enrolled in this study in S1 Table.

Line 238- remove note

Line 248- in the spleen

Response: We have corrected as reviewer suggestion.

Response: Thanks for your concerns, we have revised the figures and their legends following pathologist suggestion. Since the intestinal histological picture seems to be more difficult to interpret due to the fact of tissue autolysis; however, we regret to say that we have only this despite all sections were reviewed by pathologist.

Line 277- in the negative controls

Line 282- you define PAS here despite using it previously, please define at first use

Response: We have revised as reviewer suggestions.

Line 379- co-evolution is suggested for CPV global emergence- what is to say that isn’t the case here rather than pathogen spill over?

Response: We have added more details about it in the discussion section as “This result may imply either possible evidence of cross-species transmission between domestic cats and fishing cats or co-evolution of the CPPV-1 variants.” Page 20, lines 397-399.

PLoS One. doi: 10.1371/journal.pone.0247266.r005

Decision Letter 2

Simon Clegg

2 Feb 2021

PONE-D-20-33818R2

Natural infection of parvovirus in wild fishing cats (Prionailurus viverrinus) reveals extant viral localization in kidneys

PLOS ONE

Dear Dr. Piewbang,

==============================

Many thanks for submitting your manuscript to PLOS One

It was reviewed by two experts in the field, and they have recommended some further minor modifications be made prior to acceptance

I therefore invite you to make these changes and to write a response to reviewers which will expedite revision upon resubmission

I wish you the best of luck with your modifications

Hope you are keeping safe and well in these difficult times

Thanks

Simon

==============================

Please submit your revised manuscript by Mar 19 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Simon Clegg, PhD

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors did a great job and significantly improved the manuscript. However, there are still some issues:

In the materials and methods (Animals and routine postmortem examination section) the authors state: "Selective vital organs including lung, liver, heart, spleen, and kidney were sampled from all fishing cats, while the intestine and mesenteric lymph node were additionally collected from fishing cat no.3." while in the (full-length genetic characterization of fishing cat CPPV-1) section the authors write: "Briefly, the extracted nucleic acids obtained from intestine, spleen and kidneys of two fishing cats were individually amplified using a GoTaq® Hot Start Green Master Mix (Promega, Madison, WI, U.S.A.) and specific primers (S1 File)."

--> How can you isolate nucleic acids from the intestine of two cats when samples are only available from cat 3? Please clarify.

In the description of figure 1 the size of the scale bars is missing.

In the abstract in line 29 I suggest to change ... revealed lesions of intestine ... to revealed lesions in intestine ...

Reviewer #3: The manuscript is much improved from a previous version, and the pathology images are much better. A few minor issues are detailed below, but I fully expect that this can be accepted when these minor issues are addressed

Line 26- are widely spread (add in word)

Line 27- a globally vulnerable …. (reword)

Line 30- infection by the virus (reword)

Line 67- this needs a reference, and some good references here for cats infected with CPV- both symptomatic and asymptomatic

Line 81- replace of, with as the

Within your materials and methods you take some samples from some animals, but not from others, yet the samples not taken appear later in the study for the PCR analysis. Can you please check this just for clarity?

Line 408- determinant may sound better than determination?

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: No

PLoS One. 2021 Mar 2;16(3):e0247266. doi: 10.1371/journal.pone.0247266.r006

Author response to Decision Letter 2

2 Feb 2021

Rebuttal response on PONE-D-20-33818R2

Reviewer #1: The authors did a great job and significantly improved the manuscript. However, there are still some issues:

--> How can you isolate nucleic acids from the intestine of two cats when samples are only available from cat 3? Please clarify.

Response: Thank you for your positive review on our manuscript. There is a missing information regarding the samples used for genome sequencing and we do revise them as “the extracted nucleic acids obtained from spleen and kidneys of two fishing cats plus additional intestinal tissue of fishing cat no. 3, were…” in page 9, lines 183-184.

In the description of figure 1 the size of the scale bars is missing.

Response: the size of the scale bars in Fig. 1 is already described as found in page 13, line 276.

In the abstract in line 29 I suggest to change ... revealed lesions of intestine ... to revealed lesions in intestine ...

Response: We revised it as reviewer suggestion.

Line 26- are widely spread (add in word)

Line 27- a globally vulnerable …. (reword)

Line 30- infection by the virus (reword)

Response: Thank you for your recommendation, we have revised them as reviewer suggestions.

Line 67- this needs a reference, and some good references here for cats infected with CPV- both symptomatic and asymptomatic

Response: We have provided the potential references as your suggestion as found in page 3, line 67.

Line 81- replace of, with as the

Response: There is some missing information regarding the samples used for genome sequencing of the CPPV-1, so we have revised the sentence as “the extracted nucleic acids obtained from spleen and kidneys of two fishing cats plus additional intestinal tissue of fishing cat no. 3, were…” in page 9, lines 183-184.

Line 408- determinant may sound better than determination?

Response: We revised it as reviewer suggestion.

Attachment

Submitted filename: Rebuttal response on PONE-D-20-3318R2.docx

Click here for additional data file.^{(14.6KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0247266.r007

Decision Letter 3

Simon Clegg

4 Feb 2021

Natural infection of parvovirus in wild fishing cats (Prionailurus viverrinus) reveals extant viral localization in kidneys

PONE-D-20-33818R3

Dear Dr. Piewbang,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

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Kind regards,

Simon Clegg, PhD

Academic Editor

PLOS ONE

Additional Editor Comments:

Many thanks for resubmitting your manuscript to PLOS One

As you have addressed all the comments and the manuscript reads well, I have recommended it for publication

You should hear from the Editorial Office shortly.

It was a pleasure working with you and I wish you the best of luck for your future research

Hope you are keeping safe and well in these difficult times

Thanks

Simon

PLoS One. doi: 10.1371/journal.pone.0247266.r008

Acceptance letter

Simon Clegg

22 Feb 2021

PONE-D-20-33818R3

Natural infection of parvovirus in wild fishing cats (Prionailurus viverrinus) reveals extant viral localization in kidneys

Dear Dr. Piewbang:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Simon Clegg

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig

(TIF)

Click here for additional data file.^{(34.6MB, tif)}

S1 Table. List of wildlife carnivore species enrolled in the retrospective study of CPPV-1 detection.

(DOCX)

Click here for additional data file.^{(14.8KB, docx)}

S1 File. Amplification procedures used for routine pan-virologic-family detection and full-length genetic characterization of fishing cat CPPV-1.

(DOCX)

Click here for additional data file.^{(37.5KB, docx)}

S2 File. Pairwise nucleotide distances of fishing cat CPPV-1 and previously published CPPV-1 genomes.

(XLS)

Click here for additional data file.^{(495KB, xls)}

Attachment

Submitted filename: Rebuttal response PONE-D-20-33818.docx

Click here for additional data file.^{(31.3KB, docx)}

Attachment

Submitted filename: Rebuttal response on PONE-D-20-3318R2.docx

Click here for additional data file.^{(14.6KB, docx)}

Data Availability Statement

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PERMALINK

Natural infection of parvovirus in wild fishing cats (Prionailurus viverrinus) reveals extant viral localization in kidneys

Chutchai Piewbang

Sabrina Wahyu Wardhani

Jira Chanseanroj

Jakarwan Yostawonkul

Suwimon Boonrungsiman

Nattika Saengkrit

Piyaporn Kongmakee

Wijit Banlunara

Yong Poovorawan

Tanit Kasantikul

Somporn Techangamsuwan

Roles

Abstract

Introduction

Materials and methods

Animals and routine postmortem examination

General virological molecular assays

In situ CPPV-1 detection using immunohistochemistry (IHC), in situ hybridization (ISH) and Transmission Electron Microscopy (TEM)

Full-length genetic characterization of fishing cat CPPV-1

Phylogenetic, recombination and evolutionary analyses of fishing cat CPPV-1

Retrospective study of CPPV-1 antigen in wildlife carnivores

Results

Postmortem findings and tissue localization of fishing cat CPPV-1

Fig 1. CPPV-1 infection in fishing cats.

Fig 2. CPPV-1 infection in fishing cats.

Fig 3. CPPV-1 viral particles in the nucleus of renal tubular epithelial cells.

Detection of fishing cat CPPV-1 and genomic analysis

Table 1. Alignment of capsid amino acid sequence of CPPV-1 isolated from fishing cats with previously described CPPV-1 variants retrieved from the NCBI database.

Phylogenetic and evolutionary analysis

Fig 4. Phylogenetic topology revealing the genetic relationship of the full-length coding sequences between the fishing cat CPPV-1 and other CPPV-1 variants.

Fig 5. Evolutionary analysis of the fishing cat CPPV-1 with other CPPV-1 variants.

Retrospective study of CPPV-1 in other wild samples

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

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