Isolation and genetic characterization of parvoviruses from domestic cats reveals emergence of CPV-2c in India: A first report

Abstract

The objective of the present study was to isolate and characterize the VP2 gene of parvoviruses from domestic cats in India. For that, 38 fecal samples were screened by PCR with 36.84% positivity. Sequence analysis of those isolates showed canine parvovirus type-2c (CPV-2c) as the predominant variant, followed by feline panleukopenia virus (FPV) and 2a. Phylogenetic analysis of the CPV-2c sequences revealed clustering with Singaporean, South Korean, Mongolian and Bangladeshi dog 2c sequences. Phylogenetic analysis of the 2a isolate (MZC 2) was found to be clustered with Indian, Thai and Singaporean dog 2a isolates. Similarly, all the four FPV sequences were ancestrally related to Indian dog and cat FPV sequences hinting towards interspecies transmission between dogs and cats. Both synonymous and non-synonymous mutations were evident in CPV-2c, 2a and FPV sequences indicative of active evolution. In cell culture medium, CPV-2 showed cytopathogenic effects at the third passage level. In conclusion, the study provided the first report of CPV-2c in cats from India, which demands for extensive epidemiological surveillance to monitor interspecies spread and to shed more light on viral phylogenomics, their distribution in the country and in the Southeast Asian region and usage of current vaccines.

Introduction

Canine parvovirus-2 (CPV-2) and Feline panleukopenia virus (FPV) are members of the genus Protoparvovirus, variants of the species Protoparvovirus carnivoran 1 and family Parvoviridae, and are major pathogens of domestic and wild carnivores causing fatal haemorrhagic gastroenteritis (Milićević et al. 2023). CPV-2 after its emergence in the late 1970s as a host variant of FPV evolved into newer antigenic variants such as CPV-2a, 2b and 2c; New CPV-2a, 2b and 2c; Asp-300 (2a/2b), which then completely replaced the original antigenic type and are now distributed worldwide (Decaro and Buonavoglia 2012). In the 1980s, CPV-2a had emerged from CPV-2 as the first antigenic variant, which differs from the original type in 5-6 amino acid (aa) positions of the VP2 protein (Decaro et al. 2011). A second antigenic variant, CPV-2b, had a further mutation (asparagine to aspartic acid at aa residue 426) in the VP2 protein (Parrish et al. 1991). In 2000, a third antigenic variant, CPV-2c, was detected which showed aa change asparagine/aspartic acid to glutamic acid at residue 426 of the VP2 protein (Buonavoglia et al. 2001). Further, the CPV-2a and CPV-2b variants showing amino acid change 297 Ser→Ala have been designated as the new CPV-2a and new CPV-2b, respectively (Decaro et al. 2009). The original CPV-2 was unable to infect cats but new variants of CPV-2 have the capability to infect both cats and dogs (Clegg et al. 2012).

Among the CPV-2s, CPV-2a was first detected in cats in the late 1980s from non-symptomatic cats in Japan (Mochizuki et al. 1993). Later on, CPV-2b was detected in cats from USA (Truyen et al. 1996). Subsequently, CPV-2 variants were found to spread across all continents and retrieved from cats with gastroenteritis in Italy, Thailand, Germany, USA, India, Portugal, and Spain (Truyen et al. 1996, Gamoh et al. 2003, Decaro et al. 2010, Decaro et al. 2011, Mukhopadhyay et al. 2016, Balboni et al. 2018, Charoenkul et al. 2019, Calatayud et al. 2020). CPV-2 variants have also been isolated from the feces of clinically healthy domestic and wild felines, suggesting that the virus was either shed long after infection or that it causes subclinical or very mild disease in this species (Markovich et al. 2012). In addition, there has been a report of coinfection by multiple CPV variants in a cat with CPV-2a and the 426 Glu variant (Battilani et al. 2007). Mixed infections by FPV and CPV-2 or FPV and CPV-2a have also been reported (Url et al. 2003; Battilani et al. 2013).

Underscoring the importance of cats as a potential source of genetic heterogeneity and recombination for parvoviruses, and as very few reports are available with respect to molecular characterization of parvoviruses in domestic cats from India, the current study was conceptualized for isolation, VP2 sequence analysis and the phylogenomics of CPV-2/FPV virus strains which were detected in suspected cats. Further, to the best of the author's knowledge this is the first report of CPV-2/FPV in cats from North eastern states of India like Mizoram and Manipur.

Materials and methods

Study area, population, sample collection and epidemiological data

A total of 38 rectal swabs were collected from suspected (cats with hemorrhagic gastroenteritis, anorexia and depression) cases irrespective of age, sex and breed from four different states (Assam, Maharashtra, Manipur, and Mizoram) of India between 2019 and 2022 (Table 1). The samples were initially screened for CPV-2 infection by rapid antigen detection kit (Genbody, South Korea) followed by confirmation by polymerase chain reaction (PCR). Epidemiological data with respect to age, sex, breed, vaccination status and clinical signs of the affected cats were collected. Clinical signs were scored as per Van Nguyen et al. (2006) with minor modifications (Table 2).

Table. I. Summary of descriptive data of the cat (n=14) and dog FPV samples (OP778053) used for the present study. * Dog FPV sample.

Table. II. Clinical score card used for the cats with CPV-2/FPV gastroenteritis.

PCR screening and amplification of VP2 gene fragment

Viral DNA was extracted from stool samples using commercially available kit (QIAamp Fast DNA Stool Mini Kit, Qiagen). The quantity and the purity of DNA were checked in a microvolume spectrophotometer (Eppendorf BioSpectrometer basic). The DNA thus extracted was kept at −20ºC until further use. The extracted template DNA was screened for the presence of CPV-2 using the Hfor/Hrev primer pair listed in Table 3, which amplifies a 630 base pair (bp) fragment of the VP2 gene encoding capsid protein (Buonavoglia et al. 2001). The DNA prepared from CPV-2 vaccine strain (Canigen DHPPiL, Virbac) was used as positive control in the PCR assay. PCR amplification was carried out in 25 μL reaction mixture containing 50-100 ng template DNA, 12.5 μL of 2x PCR master mix [Thermo Fisher Scientific containing Taq DNA polymerase (0.05 U/µL), reaction buffer, 4 mM MgCl2, and 0.4 mM of each dNTP], 1 µL each of forward and reverse primer (10 pmol/μl) and nuclease free water to adjust the volume. PCR amplification consisted of initial denaturation at 95ºC for 5 mins followed by 35 cycles of denaturation at 95ºC for 30 s, annealing at 55ºC for 30 s and extension at 70ºC for 1 min and final extension at 70ºC for 10 mins. The PCR-amplified products were electrophoresed on 1.5% agarose gel in Tris acetate EDTA buffer and visualized under UV light and documented by gel documentation system (Vilber Bioprint, France).

Table. III. Primers for the amplification of partial VP2 gene of CPV-2/FPV.

Cloning of PCR product

Positive PCR products were purified from agarose gel by GeneJet Gel Extraction Kit (Thermo Scientific, K0702) followed by cloning with CloneJET PCR Cloning Kit (Thermo Scientific, K1232) as per the instructions given by the manufacturer.

Genotyping of CPV-2 isolates

The recombinant plasmid carrying correct insert, isolated from the representative clone was sent to Central Instrumentation Facility, Biotech Center, University of Delhi South Campus, India for sequencing. The sequence chromatogram was visualized and aligned in BioEdit v 7.2.5 analysis software (Isis Therapeutics, Carlsbad, CA, USA) to get a clean sequence of 630 nucleotides followed by BLAST analysis (https://blast.ncbi.nlm.nih.gov/Blast) to confirm the presence of CPV-2/FPV. The deduced aa sequences obtained were aligned with corresponding sequences available in GenBank using Clustal W of Mega 11 (Tamura et al. 2011) with default parameters. The aligned sequences (both nucleotide and aa) of VP2 genes of all the isolates were then submitted to GenBank for allotment of accession numbers.

Phylogenetic analysis

All the sequences obtained from this study along with the reference and vaccine sequences retrieved from GenBank were aligned using ClustalW and the phylogenetic analysis was performed employing the neighbor joining approach with Tamura 3-parameter model using MEGA 11 programme and deduced aa sequences of VP2, percentage homology and differences were analysed using DNA Star sequence analysis software (Tamura et al. 2011).

Virus isolation

Ten CPV-2 PCR positive fecal samples were filtered and used for virus isolation in the Madin darby canine kidney (MDCK) cell line. The infected monolayers were harvested on day 3 post inoculation (with or without a cytopathic effect [CPE]) by three cycles of alternative freezing, thawing and centrifuged at 6000 g for 15 mins in a refrigerated centrifuge. The supernatants were collected in a microcentrifuge tube and stored at −80ºC until further use. Finally, the virus in the cell culture fluids was confirmed at the third passage level by PCR assay using the Hfor/Hrev primer pair.

Statistical analysis

Variant-wise clinical score comparison was done using Independent-Samples Kruskal-Wallis Test. The results were considered significant when P was < 0.05. Statistical analyses were carried out using statistical software (SPSS version 27.0).

Results

Polymerase chain reaction and epidemiological data

Out of total 38 fecal samples screened for CPV-2, 14 (36.84%) samples were found positive by PCR. Signalment data and descriptive statistics of the CPV-2/FPV positive cats are summarized in table 1. The median age of the cats was found to be between 3-6 months (range 1 month – 02 years). None of the cats were found to be vaccinated against FPV. Breed-wise occurrence was found to be highest among mixed breed (71.42%) followed by domestic short-haired (14.28%) and non-descript cats (14.28%). Major clinical signs observed (n=14) were anorexia (85.71%), mild to moderate depression (85.71%), moderate vomiting (71.42%) and hemorrhagic watery diarrhea (14.28%). Variant-wise clinical score comparisons (Table 4) revealed significant (P<0.05) difference in the degree of severity when CPV-2a, 2c and FPV affected cats were compared against healthy control. Similarly, mean clinical score comparison of FPV (9.5±2.61) affected cats were significantly (P<0.05) altered compared to CPV-2a (11.5±3.32) and 2c (12.14±3.51) affected cats. However, non-significant difference was observed when mean clinical score of CPV 2a was compared against CPV 2c.

Table. IV. Variant-wise clinical score (Mean±S.E.) comparison against healthy control on the day of presentation. S- significant at P>0.05 NS- Non-significant.

Sequencing and amino acid mutation

In order to characterize the detected CPV-2 virus, the coding VP2 gene sequence of 630 nucleotides was obtained from all the isolates. Based on the 426 amino acid (aa) residues of the deduced VP2 protein, 08/14 (57.14%) CPV-2s were classified as 2c, 04/14 (28.57%) was found to be FPV and 02/14 (14.28%) viruses were characterized as the 2a variant. The original CPV type 2 was not found. Several synonymous and non-synonymous mutations were noticed in all the types of CPV-2 variants (2a and 2c) and FPV under study when compared against database and the amino acid substitutions are summarized in Table 5. Non-synonymous mutations observed by CPV-2c isolates were Ser292Asn, Val294Leu, His309Gln, His318Gln, Gln370Arg, His404Gln, Leu404Gln, Glu424Val, Gln426Glu, Tyr427Asp, Val436Ile and Leu447Ile. Non-synonymous mutations observed by FPV isolates were Val401Ile and Ile466Asn. Similarly, non-synonymous mutations observed by CPV-2a isolates were Ser292Asn, Pro293Ser, Ile336Val, His404Gln, His426Asn and Ala440Thr.

Table. V. Amino acid variations in the CPV-2a, 2b and 2c VP2 capsid protein of feline isolates from the present study against reference, dog FPV sequence (Mizoram 17; OP778053) and vaccine strains. * Dog FPV sequence for comparison. Superscript a, b, c, d and e are indicative of reference strains for CPV-2, CPV-2b, FPV, CPV-2a and 2c, respectively

Phylogenetic analysis

The phylogenetic tree based on aa sequences of the present isolates against reference, field and the vaccine strains is depicted in Figure 1. The 2c isolates from the present study formed 3 separate clusters (viz. OP961983 and OP729184; OQ024198 and OQ024199; OQ024197, OQ024195, OQ024196, OQ024194 and OP729183). Besides, the 2c isolates were grouped together along with Indian (OP125772), Singaporean (ARR75645), Mongolian (QBB89700), South Korean (UID86069) and Bangladeshi (WCD68703) 2c isolates from the database (aa homology 98.9% - 100%) following the same evolutionary pattern, albeit, forming separate lineages. The aa homology within the 2c isolates was between 95.1% to 100%. Further, all the 2c sequences were closely grouped with dog 2c sequence (Figure 2) from our previous study (unpublished) with 99.5% to 100% aa homology indicating existence of possible transmission of CPVs between dogs and cats.

In the present study, isolate MHC 20 (CPV-2a) was found to be clustered with 2c isolate, MNC 27. Another 2a isolate (MZC 2) was found to be clustered with Indian, Thai and Singaporean 2a isolates with aa homology of 98.4%-98.9%. Both the 2a isolates (MHC 20 and MZC 2) had aa homology of 97.4% with each other. In addition, 2a isolates were also found to be clustered with dog 2a sequence from our previous study (unpublished) (Figure 2) with aa homology of 98.4%-98.9% which was indicative of possible transmission of CPVs between dogs and cats.

Similarly, all the four FPV sequences formed two separate clusters (MZC 37 and MZC 38; MZC 16 and MZC 36) and had amino acid homology of 99.5%-100% with each other and were ancestrally related to Indian dog (OP778053) and cat FPV sequences (aa homology of 98.9% to 100%) from the database hinting towards transmission of FPV between dogs and cats.

Figure. 1. Phylogenetic analysis of partial VP2 sequences of CPV-2 variants and FPV isolates from the cats against the reference and vaccine strains using the Neighbour-joining method with Bootstrap consensus tree (1000 replications). The Tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The isolates obtained in this study are red coloured with accession number, followed by sample number, strain identity, host and year of isolation and. This analysis involved 55 amino acid sequences. Drawn using MEGA version 11.0.

Figure. 2. Phylogenetic analysis of partial VP2 sequences of CPV-2 variants from the cats against the reference and dog CPV-2 strains from our previous study using the Neighbour-joining method with Bootstrap consensus tree (1000 replications). The Tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The isolates obtained in this study are red coloured for 2c isolates and blue coloured for 2a isolates. This analysis involved 27 amino acid sequences. Drawn using MEGA version 11.0.

Virus isolation

In MDCK cell culture, typical cytopathogenic effects (Figure 3) (rounding and detached cells) were observed for seven out of 10 CPV-2 positive cat samples at the third passage level (72 hours of incubation) and confirmed with PCR. No effort was made to revive FPV because of lack of feline specific cell lines.

Figure. 3. Citopathic effect of CPV-2c on MDCK cell culture. Left: Uninfected cells, Right: MDCK infected with CPV-2c (MHC 18) showing rounding and sloughing of cells (200X magnification).

Discussion

The present study revealed the presence of both CPV-2 and FPV infections among the cat population from four states of India. Of the 38 suspected cats screened by conventional PCR assay, 14 (36.84%) were found positive for CPV-2 variants/FPV. The PCR positivity of 36.84% was found to be in agreement with previous reports (Parthiban et al. 2014, Jing et al. 2015, Mukhopadhyay et al. 2016, Karanam et al. 2022). As such there are only few reports of CPV-2/FPV infection among cats in India and to the best of the author's knowledge, this is the first report of CVP-2c in cats from India and first report of CPV-2/FPV infection in cats from states like Mizoram and Manipur. So, the study provided in depth molecular insights into the distribution and evolution dynamics of the CPV-2/FPV from different geographical regions of India.

The median age of the cats was found to be between 3-6 months (range of 1month to 2 years) and was in accordance with earlier report (Stuetzer and Hartmann 2014). This might be attributed to viral replication in mitotically active tissues such as intestinal mucosa, lymphoid tissue and bone marrow (Parker et al. 2001).

The most prominent clinical signs among the affected cats were anorexia (85.71%), depression (71.42%), vomition (71.42%) and diarrhea (50%) which was in agreement with earlier report (Miranda et al. 2014; Nelson and Couto 2014). To assess the severity of clinical signs across variants, mean clinical score of FPV, 2a and 2c affected cats were compared against healthy control as well as against each other on the day of presentation. There was significant increase in severity of clinical signs in all the affected cats compared to healthy control. Severity of clinical signs was also significantly more pronounced in 2a and 2c affected cats compared to FPV affected cats. 2c has been reported to cause severe disease in adult dogs and also in dogs that have completed the vaccination protocols (Decaro and Buonavoglia 2012). However, non-significant alteration of clinical severity among 2a and 2c affected cats might be due to small sample size and need to be repeated in larger sample size. As such many studies noted a striking variety in the clinical course of CPV-2 infection in dogs and cats ranging from subclinical infection to acute fatal illness (Miranda et al. 2014). This variation was largely attributed to age of infection, lack of protective immunity, stress level and having a higher number of dividing cells and concurrent infections (Schoeman et al. 2013).

One striking finding in the current investigation was lack of vaccination of 100% of the cats, which raised a major concern about the screening population's lack of disease-protective immunity and was in agreement with previous report (Mukhopadhyay et al. 2016).This further complicates the scenario as cats act as carriers and can easily transmit the infection to unprotected dogs.

CPV-2c was the predominant variant (57.14 %) in the study population which was indicative of shifting trend of CPV-2 antigenic variant in this part of the country as earlier reports from different parts of India reported FPV or CPV-2a or new CPV-2a as the predominant variant (Parthiban et al. 2014, Mukhopadhyay et al. 2014, Karanam et al. 2022) among feline population. Further, our recent large-scale study to determine the prevalence of CPV-2 variants in canine population (unpublished) also revealed CPV-2c as the predominant variant (61.26%). At present, CPV-2c represents only a small percentage of parvovirus in canine and feline populations in India; however, looking into the current trend from the present study, authors presumed a major antigenic shift in favor of CPV-2c both in canine and feline populations. This presumption will be confirmed in future studies from the country as similar to what has happened in the past in other countries, the new variant may likely become the predominant variant in the field (Pereira et al. 2007). To make the matter worse, CPV-2c have already revealing itself as the predominant feline variant in some of the Asian countries along with FPV, CPV-2a, new CPV-2a and new CPV-2b (Pan et al. 2023, Tang et al. 2022).

All the CPV-2c isolates from the present study showed a synonymous mutation with aa Glu (type 2c) at residue 426 of the VP2 protein confirming them as CPV-2c variant (Decaro and Buonavoglia 2012). Further, highest number of non-synonymous mutations (12) were observed by CPV-2c isolates such as Ser292Asn, Val294Leu, His309Gln, His318Gln, Gln370Arg, His404Gln, Leu404Gln, Glu424Val, Gln426Glu, Tyr427Asp, Val436Ile and Leu447Ile compared to six in 2a and two in FPV isolates. This could be attributed to higher sequence variability of CPV-2c due to its acquisition of multiple nucleotides and amino acid changes over time (Battilani et al. 2019). Mutation Gln370Arg was shown by 2c sequence (MNC 29) from the present study had also been reported from Italian, Chinese, Taiwanese, Thai, and Japanese canine 2c strains (Yi et al. 2016, Geng et al. 2017, Zhao et al. 2017, Mira et al. 2018, Jiang et al. 2021). As per the existing information, residue 370 may be necessary for a conformational shift or may influence receptor binding via neighboring residues (Buonavoglia et al. 2001). Non-synonymous mutations observed by FPV isolates were Val401Ile and Ile466Asn. Similarly, non-synonymous mutations observed by CPV-2a isolates were Ser292Asn, Pro293Ser, Ile336Val, His404Gln, His426Asn and Ala440Thr. Amino acid mutation 440 Thr→Ala was reported in both the 2a isolates (MHC 20 and MZC 2) from the present study and had been reported in canine CPV-2a, 2b and 2c sequences from different parts of the world (Chinchkar et al. 2006, Clegg et al. 2012, Calderón et al. 2011, Battilani et al. 2019). The 440 residue sits at the peak of the threefold spike and considered to be the primary antigenic site of the virus (Chapman and Rossmann 1993). The remaining non-synonymous mutations were unique to this study and had not been reported till date. These non-synonymous mutations were recorded in the GH loop of VP2 protein (267–498 residues located between βG and βH strands) of the virus which were exposed on the surface of capsid and likely to undergo mutation aiding in evolvement of new variants (Kang et al. 2008).

Presence of CPV variants (CPV-2a and CPV-2c) in cat from the present study proves the existence of interspecies transmission. Further, close similarity of the present cat FPV sequences with dog FPV sequence (OP778053) from our previous study was indicative of possible transmission of FPV between cats and dogs.

This study confirms that cat population is susceptible to both CPV variants and FPV facilitating interspecies transmission and high genetic heterogeneity. Complete lack of vaccination in the cat population once again highlights the urgent need of educating the owners regarding compulsory vaccination against FPV. Further, studies evaluating the cross protection between FPV vaccines against CPV infection in cats is need of the hour.

In conclusion, the present study provided important update with respect to the evolutionary phylodynamics of parvovirus infections in cats from four states of India. However, further studies with larger sample size over a wider geographical regions incorporating complete VP2 gene analysis are imperative for in-depth understanding of the problem helping in early diagnosis, timely intervention and the development of strategies for feline vaccination in lieu of predominance of CPV‐2c. The present finding will also sensitize veterinary practitioners to put more attention on both CPV and FPV infections in light of interspecies transmission. Last but not the least, the role of cats as the source of new variants of CPV-2 needs to be constantly monitored in the country to rule out mutations along with recombination thereby causing emergence and evolution of parvoviruses.