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. 2024 Oct 1;44(1):1–13. doi: 10.1080/01652176.2024.2408742

Canine parvovirus in North-East India: a phylogenetic and evolutionary analysis

Kiran Jayappa a, Tridib Kumar Rajkhowa a,✉, Satish S Gaikwad b
PMCID: PMC11445921  PMID: 39350725

Abstract

Canine parvovirus type 2 (CPV-2) infection in dogs is considered as one of the most common cause of morbidity and mortality in young dogs and continues to occur with high incidence worldwide. Despite a single-stranded DNA virus, CPV-2 possesses a high mutation rate which has led to the development of new variants from time to time. These variants are classically classified based on the amino acid markers present in the VP2 gene. In this study, we examined 20 different cases of CPV-2 infection from seven different states of the North East region (NER) of India. The near-complete genome sequences of all these isolates were subjected to phylodynamic and phylogeographic analysis to evaluate the genetic diversity and geographical spread of CPV-2 variants. Analysis of the deduced amino acid sequences revealed residues characteristic of the ‘Asian CPV-2c lineage’ in all the 20 sequences confirming it as the dominant strain circulating in NER, India. The phylogenetic analysis based on the whole genome showed that all 20 sequences formed a monophyletic clade together with other Asian CPV-2c sequences. Furthermore, phylogeographic analysis based on the VP2 gene showed the likely introduction of Asian CPV-2c strain to India from China. This study marks the first comprehensive report elucidating the molecular epidemiology of CPV-2 in India.

Keywords: Asian CPV-2c, canine parvovirus type 2, North East India, molecular surveillance, phylogenetic analysis, phylogeographic analysis

Introduction

Canine parvovirus type 2 infection first emerged in the late 1970s and continues to be one of the most common viral diseases in the canine population, characterized by high fever, severe haemorrhagic diarrhoea, vomiting, dehydration and lymphopenia (Truyen 2006; Decaro and Buonavoglia 2012). Despite the practice of regular vaccination, the canine parvovirus infection still prevails to be a main cause of mortality in pups worldwide. The etiology, canine parvovirus type 2 (CPV-2) is a single-stranded DNA virus belonging to the family Parvoviridae, subfamily Parvovirinae, genus Protoparvovirus, and species Protoparvovirus carnivoran1 (Cotmore et al. 2019). The genome of CPV-2 consists of 5.2 kb single-stranded DNA with two open reading frames (ORFs), respectively encoding for two structural (VP1 and VP2) and two nonstructural (NS1 and NS2) proteins, through alternative splicing of the mRNA (Hoelzer et al. 2008). Among the structural proteins, VP2 protein is the major component of icosahedral capsid and plays an important role in receptor binding, host-range determination and antigenicity (Hueffer et al. 2003; Allison et al. 2015).

CPV-2 is included in the unique Protoparvovirus carnivoran1 species along with other related viral species, such as feline panleukopenia virus, mink enteritis virus, and raccoon parvovirus (Tijssen et al. 2011; Cotmore et al. 2014). After being discovered in the USA in 1978, it quickly spread throughout the world (Appel et al. 1979). It is believed to have originated from the feline panleukopenia virus or a closely related virus infecting other carnivores (Truyen 2006). CPV-2 shares over 98% nucleotide homology with FPV, with six amino acid mutations in VP2 which enabled it to gain canine host range while losing its ability to infect felines (Miranda and Thompson 2016). In 1980 and 1984, the original CPV-2 type was rapidly replaced by new antigenic variants which were termed as CPV-2a and CPV-2b respectively, that regained the feline host range (Parrish et al. 1988). In 2000, yet another antigenic variant (CPV-2c) was identified in Europe, which soon spread to different countries across the globe. Although these variants showed several mutations, they are mostly defined based on the amino acid at VP2 residue 426 (Asn in CPV-2a, Asp in CPV-2b, and Glu in CPV-2c). These variants have been distributed in varying proportions in different countries (Miranda and Thompson 2016). Interestingly, till the last decade, CPV-2a was the dominant variant circulating in Asia and has been rapidly replaced by CPV-2c in recent years (Hao et al. 2022). This can be mainly attributed to the emergence of a CPV-2c variant, named in the literature (Mira et al. 2017) as ‘Asian CPV-2c’, characterized by a set of unique mutations in its NS1 and VP2 proteins. This strain originated in Southeast Asian countries (Zhao et al. 2017; Charoenkul et al. 2019; Moon et al. 2020) and soon its presence was reported in European and African countries (Mira et al. 2017; Ogbu et al. 2020). The recent phylogenetic analyses based on the whole genome sequences (WGS) showed that all the Asian CPV-2c sequences grouped together and formed a single monophyletic clade, now commonly termed as ‘Asian CPV-2c lineage’ (Nguyen Manh et al. 2021; Franzo et al. 2023).

First case of CPV-2 infection in India was documented in 1982 (Ramadass and Khadher 1982). Subsequently, all the different variants of CPV-2 (CPV-2a, CPV-2b and CPV-2c) were detected from different parts of the country. Since the early 2000s, CPV-2a has been the predominant variant circulating in India (Chinchkar et al. 2006; Mohan Raj et al. 2010; Srinivas et al. 2013; Mukhopadhyay et al. 2014; Kulkarni et al. 2019), whereas CPV-2c has been less frequently reported (Nandi et al. 2010). However, all these data are based on the VP2 gene sequences (mostly partial VP2 gene nucleotide sequences) of CPV-2, largely sourced from mainland India. Consequently, comprehensive information about the whole genome of circulating CPV-2 is still lacking.

Since the past 3–4 years, there has been increased CPV-2 incidence and mortality in the dog population of the North East region (NER) of India (based on personal consultation with the clinicians). In our initial study (Kiran and Rajkhowa 2023) we detected the circulation of a divergent CPV-2c strain within the canine populations of Mizoram and Manipur. The current study extends beyond this initial scope, encompassing field cases of CPV-2 from the entire NER, India, thus including seven states. Here, we have analysed the genetic diversity and geographic distribution of CPV-2 variants by examining the near-complete genome of 20 isolates. This broader analysis is intended to enhance our understanding of the dissemination of CPV-2 strains within this region and to compare these with the recently defined Asian CPV-2c lineage (Franzo et al. 2023). The present study provides new insights into the spatial distribution and genomic variations of CPV-2, which may be crucial for the control and prevention strategies of CPV-2 infection in India.

Materials and methods

Sample collection

A total of 58 necropsies of dead pet dogs referred with a clinical history of fever, dehydration, haemorrhagic diarrhoea and vomiting, were performed to confirm the CPV-2 infection (Supplementary Table S1). The samples were collected over a period from February 2020 to May 2022 from the entire North Eastern Region of India, covering seven different states namely Sikkim, Assam, Arunachal Pradesh, Meghalaya, Manipur, Tripura and Mizoram (Figure 1). Detailed necropsy was conducted on all the dead dogs with the owner’s consent, and gross lesions were recorded. Representative tissue samples (comprising of lung, intestine, spleen, liver, mesenteric lymph nodes, heart and kidney) were collected and preserved in 10% neutral buffered formalin for histopathological examination and also at −80 °C for molecular diagnosis. The frozen tissue samples were packed in triple packaging system with ice packs and transported to the College of Veterinary Sciences and Animal Husbandry, Selesih, Aizawl, Mizoram. Fixed tissues were processed for routine paraffin embedding technique. Sections were cut at 4–5 μm thickness and stained with hematoxylin and eosin (H & E) (Suvarna et al. 2019). The stained sections were observed under the light microscope and lesions were recorded. The details of the samples are summarized in Table 1.

Figure 1.

Figure 1.

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The map showing the North East region of India covered under the present study. Samples were collected from all the North Eastern states, except from Nagaland.

Table 1.

Detail of the isolates included in the study.

Sl No. Isolates Date of sampling Breed Age Vaccination status Place Accession No.
1 CPV/INDIA/MN01 17-Jun-21 Non-descript 4 months Not vaccinated Imphal, Manipur OQ092723
2 CPV/INDIA/MN03 15-Jul-21 Crossbreed 6 months Complete vaccination Imphal, Manipur OQ092724
3 CPV/INDIA/MN17 30-Dec-21 Crossbreed 6 months Complete vaccination Imphal, Manipur OQ092725
4 CPV/INDIA/MZ02 03-Feb-20 Crossbreed 1 months Complete vaccination Aizawl, Mizoram OQ427367
5 CPV/INDIA/MZ03 16-Mar-20 German Shepherd 3 months Complete vaccination Aizawl, Mizoram OQ427368
6 CPV/INDIA/MZ04 22-Jul-20 St. Bernard 2 months Incomplete vaccination Aizawl, Mizoram OQ092726
7 CPV/INDIA/MZ01 29-Feb-20 Crossbreed 3 weeks Not vaccinated Aizawl, Mizoram OQ092727
8 CPV/INDIA/SK06 13-Feb-22 Crossbreed 4 months Not vaccinated Gangtok, Sikkim OQ092728
9 CPV/INDIA/SK22 17-Jan-22 Labrador 6 weeks Not vaccinated Gangtok, Sikkim OQ427369
10 CPV/INDIA/SK35 01-Jan-22 Non-descript 2 months Incomplete vaccination Gangtok, Sikkim OQ092729
11 CPV/INDIA/TR02 12-Mar-22 Spitz 6 weeks Not vaccinated Agartala, Tripura OQ092730
12 CPV/INDIA/TR06 21-Mar-22 Non-descript 4 months Not vaccinated Agartala, Tripura OQ092731
13 CPV/INDIA/TR15 14-Mar-22 Non-descript 2 months Incomplete vaccination Agartala, Tripura OQ092732
14 CPV/INDIA/ML31 25-Apr-22 Crossbreed 3 months Complete vaccination Shillong, Meghalaya OQ092733
15 CPV/INDIA/ML35 26-Mar-22 Non-descript 1 month Not vaccinated Shillong, Meghalaya OQ092734
16 CPV/INDIA/ML22 02-May-22 Crossbreed 8 months Not vaccinated Shillong, Meghalaya OQ092735
17 CPV/INDIA/AM36 02-Feb-22 German Shepherd 3 months Complete vaccination Guwahati, Assam OQ092736
18 CPV/INDIA/AM26 12-Mar-22 Labrador 2 months Incomplete vaccination Guwahati, Assam OQ092737
19 CPV/INDIA/AP49 25-Feb-22 Golden Retriever 3 months Not vaccinated Seppa, Arunachal Pradesh OQ092739
20 CPV/INDIA/AP45 20-Feb-22 Labrador 8 months Not vaccinated Seppa, Arunachal Pradesh OQ092740
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Detection of CPV-2 by using PCR assay

Total DNA was extracted from tissue samples by using the Phenol-Chloroform-Isoamyl alcohol solution (VWR-Amresco, USA). CPV-2 infection was confirmed by a PCR assay targeting the VP2 gene by using a previously described primer set (Buonavoglia et al. 2001). All the PCR assays were carried out with Phusion Flash High-Fidelity PCR Master Mix (Thermo Scientific, USA) according to the manufacturer’s instructions. To rule out any possible coinfection cases, all the CPV-2 positive samples were tested for other different enteric viral pathogens by PCR/reverse transcription PCR assays. For this, total RNA was extracted by using RiboZolTM solution (VWR-Amresco, USA) and cDNA was synthesized by using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA) according to manufacturer’s instructions. For the detection of other canine viral pathogens, a multiplex PCR assay (Hao et al. 2019) was used targeting canine circovirus, canine adenovirus type 2, canine distemper virus and canine coronavirus. The PCR products were visualised in 1.5% agarose gel and the size of the PCR product was compared to the Gene RulerTM 100 bp DNA Ladder (Thermo Scientific, USA).

Cloning and sequencing of CPV-2

The CPV2 positive cases from different cities from each state were selected for near-complete genome sequencing with particular consideration on their age and vaccinal status. For the analysis of the whole coding regions of CPV-2 covering both the VP and NS genes, the CPV-2 positive samples were subjected to PCR assays by using two overlapping primer pairs (Pérez et al. 2014). The generated amplicons were purified by using GeneJET Gel Extraction Kit (Thermo Scientific, USA) and were cloned into pTZ57R/T vector using InsT/Aclone™ PCR product cloning kit (Fermentas Life Sciences, Canada). The recombinant plasmids containing gene fragments were subjected to DNA sequencing by outsourcing (Eurofins Genomics India Pvt. Ltd.). The overlapping fragments were assembled to obtain the near full length (4,556 bp) sequence and analysed. All 20 CPV-2 sequences were submitted to the GenBank database, with accession numbers reported in Table 1.

Dataset preparation

The obtained sequences were submitted to nBLAST to search for related sequences in the GenBank database. A total of 195 reference sequences encompassing both ORFs were retrieved from the NCBI database covering a period from 1978 to 2022 (Supplementary Table S2). Metadata like country and year of sample collection were also collected for the reference sequences. The 20 sequences obtained from field cases were added to get the full dataset of a total of 215 sequences which were aligned by using Clustal W implemented in MEGA 10 software (Kumar et al. 2018). To exclude recombinants from Bayesian and selection pressure analyses, recombination analysis was performed on the aligned dataset of a total of 215 sequences using RDP-4 software (Martin et al. 2015). For the detection of recombination signals, algorithms such as Chimaera, Bootstcan, RDP4, GENECONV and Maxchi were used and recombinants were removed from the dataset. Recombination events that were recognised by at least three models and had a p-value <0.05 were considered valid. The recombination analysis showed no recombination events so the dataset of 215 sequences was finalised.

Phylogenetic analysis

All the 20 sequences obtained from this study were compared with 195 reference sequences to assess time-calibrated phylogeny and tMRCA (time to Most Recent Common Ancestor) by using BEAST package (v2.7) (Bouckaert et al. 2019). The best-fit nucleotide substitution model was determined as HKY+G + I by using the model finder package in MEGA 10 based on Akaike information, and Bayesian information criterion tests (Kumar et al. 2018). Coalescent Bayesian Skyline was selected as the tree prior. For the determination of clock model Path sampling/Stepping stone sampling model selection analysis was performed for strict, relaxed and random clock models. The fit model was selected by comparing Bayes factors (BF) which were calculated by using MLEs (marginal likelihood estimate) of different models (Kass and Raftery 1995) and Relaxed log normal was determined as the best-fit model. MCMC (Markov chain Monte Carlo) of 300 million chains was performed to ensure adequate ESS (effective sample size) value (>200). Tracer v1.7.2 (Rambaut et al. 2018) was used to visualise statistical support for different parameters. A 95% HPD (highest posterior density) interval was used to determine the uncertainty in parameter estimates. Obtained trees were summarised to get the maximum clade credibility tree by using TreeAnnotator v2.5.2 and this tree was visualised in FigTree v1.4.4.

Phylodynamic and phylogeographic analysis of the Asian CPV-2c lineage

Phylogeographic analysis based on the complete VP2 gene of Asian CPV-2c lineage sequences was carried out to know the spread of Asian CPV-2c strains into India and their origins. Based on previous reports (Mira et al. 2017; Zhao et al. 2017; Charoenkul et al. 2019; Moon et al. 2020; Ogbu et al. 2020; Nguyen Manh et al. 2021; Franzo et al. 2023), in this study, Asian CPV-2c lineage has been defined by a combination of mutations characterised by amino acid residues 5Ala/Gly, 267Tyr, 324Ile, 370Arg and 440Thr in VP2 and 60Val, 544Phe, 545Val, and 630Pro in NS1. Representative Asian CPV-2c lineage sequences were retrieved from NCBI database from different countries collected during the time period from 2013 to 2022. A total of 68 reference sequences along with 20 sequences from this study were subjected to phylogeographic analysis (Supplementary Table S3). The evolutionary rate and tMRCA were analysed through MCMC-implemented BEAST package v2.5.2 by using the parameters and tools as mentioned in the above section. The geographical centroid point of each country from which the Asian CPV-2c strain has been reported was chosen as the sample collection location. The location tree was converted into a Javascript object file and was visualised in Spread3 (Bielejec et al. 2016). Spread3 was used to visualise the results of the Bayesian stochastic search variable selection (BSSVS) values and to calculate BF support for the transitions, and R Studio was used to plot them on a chord diagram.

Selection pressure analysis

Selection pressure on VP2 and NS1 gene codons of CPV-2 was analysed by using Datamonkey server (http://www.datamonkey.org) (Weaver et al. 2018). Two datasets were prepared for each gene by removing the stop codon and the ω ratios or dN/dS (non-synonymous to synonymous) ratio was calculated. To identify the non-neutral selection different methods were applied including, FEL (Fixed-Effects Likelihood), FUBAR (Fast, Unconstrained Bayesian AppRoximation), SLAC (Single Likelihood Ancestor Counting) and MEME (Mixed Effects Model of Evolution). A posterior probability value of >0.9 for FUBAR and a p-value < 0.1 for FEL, SLAC and MEME was set as significant for sites under selection. The values of ω = 1 were defined as neutral selection, ω > 1 as positive selection, and ω < 1 as negative selection.

Result

Clinico-pathological findings

Necropsy cases with a clinical history of gastrointestinal signs from different NER, India were investigated. All the CPV-2 positive cases showed severe haemorrhagic diarrhea, vomiting, and fever and subsequently died. Of the 20 dogs included in this study, only six dogs completed the routinary vaccinal protocol (Table 1). Detailed necropsy examination revealed thickening of the serosal layer of intestine with segmental fibrin deposition with petechial to ecchymotic hemorrhages (Figure 2(a)). The intestinal lumen was filled with bloody watery fluid and mucosa showed linear hemorrhages. A thick coating of blood-mixed mucus exudates covered the highly congested to haemorrhagic gastric mucosa, with haemorrhages on the serosal surface noted in a few cases (Figure 2(b)). Mesenteric lymph nodes were swollen, hemorrhagic and edematous. Haemorrhagic myocarditis was observed in a few cases (Figure 2(c)).

Figure 2.

Figure 2.

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Gross and microscopic findings in CPV-2 infection. 2a: Small intestine showing petechial to ecchymotic haemorrhages on its serosal layer and the inner picture showing haemorrhagic mucosa. 2b: Stomach showing streaks of haemorrhages on its serosal surface. 2c: Heart showing haemorrhagic myocarditis. 2d: Jejunum showing denuded mucosa, severe haemorrhage in the lamina propria with infiltration of inflammatory cells and necrotic intestinal crypts. 2e: Cortical region of MLN showing depleted lymphoid follicles with severe haemorrhages in paracortex and medullary areas. 2f: Myocardium showing enlarged and congested blood vessels with thrombus and degenerating muscle fibers showing loss of cross striations.

Histopathological examination showed haemorrhagic enteritis with shortened and sloughed-off villi, infiltration of mononuclear inflammatory cells and hemorrhages in lamina propria. Intestinal crypts were dilated, filled with necrotic debris and often were empty (Figure 2(d)). Mesenteric lymph nodes showed hemorrhages and lymphoid depletion in follicles (Figure 2(e)). Severe congestion and vasculitis with the formation of thrombus and focal areas necrosis were observed in the myocardium (Figure 2(f)).

PCR detection of CPV-2 and sequence analysis

The PCR assay targeting the CPV-2 VP2 gene yielded positive results for CPV-2 in 39 cases out of a total of 58 cases included in this study. All 39 CPV-2 positive cases tested negative for CDV, CAdV-2, CCiV and CCoV (the PCR results for all the analysed cases are given in Supplementary Table S1). From these 39 cases, 20 representative cases were selected and a near-complete genome sequence was obtained. The near-complete genome reciprocal nucleotide identity within the 20 sequences ranged from 99.12 to 99.88% (Supplementary Table S4). The NS1 and VP2 sequences showed 99.99 to 98.94% and 99.94 to 98.2% reciprocal nucleotide identity, respectively (Supplementary Tables S5 and S6). Comparison of the complete genome sequences with other reference sequences showed maximum nucleotide similarity with CPV-2c sequences collected in China in 2016 (99.86%; Acc no. MF805796), Vietnam in 2020 (99.72%; Acc no. OK806280) and Thailand in 2016 (99.70%; Acc no. MH711902).

The analysis of deduced amino acid sequences revealed distinctive mutations in both nonstructural and structural proteins. The analysis of VP2 protein in all the 20 sequences revealed Glu at VP2-426 residue, which is considered as a characteristic marker for CPV-2c. All the sequences showed 60Val, 544Phe, 545Val and 630Pro in the NS1 sequences and 5Ala/Gly (five sequences showed Ala and 15 of them showed Gly), 267Tyr, 324Ile,370Arg and 440Thr in VP2.

Selection pressure analysis

Selection pressure analysis by different methods (FEL, FUBAR, SLAC and MEME) on both VP2 and NS1 genes indicated that most of the sites were under negative selection. Analysis by the FEL method showed a synonymous to the non-synonymous ratio of 38.5:1 for NS1 and 78.5:1 for VP2 gene. Site-by-site analysis by FEL, FUBAR, SLAC, and MEME showed four, seven, three and six positively selected sites for the NS1 gene and two, seven, four and three for the VP2 gene, respectively. However, only five sites (544, 572, 582, 583, 597) for NS1 and five sites (5, 297, 324, 426, 440) for VP2 were considered as positive as they were identified by at least two methods (Supplementary Table S7).

Phylogenetic analysis based on the whole genome of CPV-2

We performed a comparative phylogenetic analysis of the 20 sequences obtained in this study with 195 reference sequences using the Bayesian inference technique. Initially, the phylogenetic tree branched into two primary clades, labeled as Clade I and Clade II, with the older CPV-2 and CPV-2a sequences grouped in clade origin or Clade-O (Figure 3). All the sequences from this study were grouped in the Clade-II, along with other Asian CPV-2c sequences originating from East Asian nations such as China, Vietnam, Thailand, South Korea, Mongolia, and other countries including Italy, Nigeria, and Canada. A distinctive feature of Clade-II was the clustering of all worldwide Asian CPV-2c sequences into a single subclade here designated as the Asian CPV-2c lineage (Figure 3). In contrast, Clade-I included European CPV-2c sequences which are characterized by 5Ala, 267Phe, 324Tyr, and 370Gln in VP2 and 60Ile, 544Tyr, 545Glu, and 630Leu in NS1. The 20 Asian CPV2c sequences from NER, India, further arranged into three separate branches within the Asian CPV2c lineage. The isolate (CPV/INDIA/MN17) from Manipur state segregated along with sequences originating from China, Italy, Mongolia, and Canada. Sequences from Meghalaya, Assam, and Arunachal Pradesh formed a unified sub-clade, while sequences from Manipur, Sikkim, Mizoram, and Tripura composed another separate sub-clade.

Figure 3.

Figure 3.

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Phylogenetic Bayesian analysis based on 215 whole coding regions of CPV-2 with posterior probability. The sequences are identified with the accession number, country, and date of collection. The sequences from the current study are labeled in Red. The branches of Clade-I are colored blue and Clade-II with green.

Phylodynamic and phylogeographic analysis of Asian CPV-2c lineage

We have carried out the phylogeographic analysis of Asian CPV-2c to elucidate the possible viral introduction into India and its subsequent dissemination worldwide. The analysis is based on the complete VP2 nucleotide sequences from 83 representative Asian CPV2c isolates, as illustrated in Figure 4. All sequences, inclusive of those from NER, India, appear to have their origin traced back to China. The phy­logenetic reconstruction showed that the time to the most recent common ancestor (tMRCA) of Asian CPV-2c was in 2007.81 (95% HPD: 2002.25 to 2012.85) and the evolutionary rate was estimated to be 3.405 × 10−4 nt/site/year (95% HPD: 1.815 to 5.288 × 10−4 nt/site/year).

Figure 4.

Figure 4.

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Bayesian evolutionary tree of Asian CPV-2c based on 85 VP2 sequences. The sequences are identified with the accession number, country, and date of collection. The sequences from the current study are labeled in Red. The tree branches are labeled based on the location and the thickness represents the posterior probability.

The geospatial distribution and the temporal progression of the Asian CPV-2c virus, according to the location-based discrete trait, are depicted in Figure 5. Lines interconnecting various countries represent distinct branches of the Maximum Clade Credibility tree. The circles surrounding geographic locations reflect the quantity of branches sustained at that location over a specific time. The BF values, in the BSSVS analysis, corroborate these findings, supporting the tracked viral spread. Detailed results are provided in the form of a chord diagram (Figure 6). Our data show that the earliest known sequence of Asian CPV-2c originated in Indonesia in 2013. The analysis suggests a pattern of spread where the virus first spread from Indonesia to other Southeast Asian countries. Subsequently, China became a likely hub, facilitating the distribution of Asian CPV-2c to nations such as Thailand, Taiwan, Vietnam, and South Korea. From these East and Southeast Asian countries, the virus migrated to other Asian territories like Myanmar and India, even reaching Turkey. Its footprint extended beyond Asia, with detection in Italy and Romania in Europe, Nigeria in Africa, and Canada in North America. Additionally, it shows multiple entries of Asian CPV-2c into India from China and Taiwan.

Figure 5.

Figure 5.

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Map showing spatial-temporal distribution of Asian CPV-2c across the world as predicted from the phylogeographic analysis of VP2 sequences by using BEAST.

Figure 6.

Figure 6.

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Chord diagram showing the transmission events of Asian CPV-2c between different countries, where the thickness of the chord is indicating the Bayes factor support.

Discussion

This study carried out in NER, India, delves into the molecular epidemiology of CPV-2. The strategic geographical location of NER, India, which is surrounded by five neighboring countries – China, Bhutan, Nepal, Myanmar, and Bangladesh (Figure 1), makes the region a crucial hotspot for the influx of transboundary diseases. This has accentuated the necessity for continuous molecular surveillance of various infectious agents in the region.

We investigated field cases of CPV-2 from seven different states of NER, India, namely Assam, Sikkim, Arunachal Pradesh, Meghalaya, Manipur, Tripura and Mizoram (Figure 1). Clinical examination and detailed necropsy examination of CPV-2 cases showed characteristic findings that are typical of CPV-2 infection (Greene and Decaro 2012; Sykes 2014). Vaccination failure is a frequent occurrence in CPV-2 infection and the role of CPV-2 variants in vaccination failure is a matter of debate (Decaro et al. 2020). At present, vaccines commonly used in India are prepared using the original CPV-2. While it has been demonstrated that vaccines prepared using the older variant can protect against all other variants, there are disagreements over this (Decaro et al. 2020). Many previous reports have shown concerns about the efficacy of these vaccines against the Asian CPV-2c lineage (Chiang et al. 2016; Charoenkul et al. 2019; Balboni et al. 2021; Hao et al. 2022). Additionally, a study (Mittal et al. 2014) from India speculated that the CPV-2 vaccines failed to generate protective antibody titers against the circulating strains. In the present study, six of the dogs (which were included in the near-complete genome analysis) received a full course of vaccination (3 doses) against CPV-2 which warrants further detailed investigation to identify the potential of Asian CPV-2c lineage as immunization escape strain. Further investigation with tests like virus neutralization test (VNT) would be crucial to explore this possibility. However, this study was limited only to characterize the field strains of CPV-2 and virus isolation was not a part of it. The study was conducted by using samples collected from post-mortem cases. Due to the nature of the sample collection, we unfortunately did not have access to the serum samples necessary for performing VNT.

In recent years, there have been attempts to delineate the CPV-2 lineages by using whole genome and/or complete VP2 gene phylogenetic clustering (Chung et al. 2020; de Oliveira Santana et al. 2022) and, in some cases, to correlate these clusters with their geographic origin (Grecco et al. 2018; Nguyen Manh et al. 2021). Our phylogenetic analysis based on the whole genome of 215 CPV-2 strains by using the Bayesian inference method has produced two large clades (Figure 3), similar to the recent reports (Nguyen Manh et al. 2021; de Oliveira Santana et al. 2022). Most of the sequences grouped in Clade-I were from America and Europe, while Clade-II contained a majority of the sequences from Asia. Some unique mutations were observed between the Clade-I and Clade-II CPV-2 strains. As described earlier (Nguyen Manh et al. 2021), our analyses also found that the amino acid position 324 differentiates Clade-I (Western) (324Tyr) and Clade-II (Asian) (324Ile). These findings further underscore the constraints existing in the current approach of defining variants solely based on amino acid markers.

All the 20 sequences from NER, India, showed the characteristic amino acid residues of Asian CPV-2c defined in this study (5Ala/Gly, 267Tyr, 324Ile, 370Arg and 440Thr in VP2 and 60Val, 544Phe, 545Val, and 630Pro in NS1) and were clustered together with other Asian CPV-2c reference sequences to from Asian CPV-2c lineage. Apart from CPV-2c variants, this clade also included three CPV-2b variants from Italy, Hungary and Australia (Supplementary Table S2). Amino acid residue 426 is under positive selection pressure (Supplementary Table S7) and a reversion at this position may confer more advantages in a specific environment. We speculate that this may be the reason for the emergence of these sporadic CPV-2b strains. Further, the sequences from this study formed three distinct subgroups within the Asian CPV2c sub-clade with almost 100% posterior probability (Figure 3). One of the subgroups contained sequences from Manipur, Sikkim, Tripura and Mizoram states; another contained sequences from Assam, Arunachal Pradesh and Meghalaya states, which may indicate minor regional adaptation or multiple introductions of the virus into these states.

CPV-2 was identified as early as in the 1980s in India (Ramadass et al. 1982). Over the years, the original CPV-2 was replaced by CPV-2a and CPV-2b variants, which still remain prevalent in the country. CPV-2 has been reported from different regions of India including the North (Chinchkar et al. 2006; Mittal et al. 2014; Nandi et al. 2010), South (Mukhopadhyay et al. 2014; Srinivas et al. 2013) and Western regions (Kulkarni et al. 2019; Nandi, 2010). The previously published works from mainland India have reported CPV-2a as the dominant variant followed by CPV-2b (Chinchkar et al. 2006; Kulkarni et al. 2019; Mittal et al. 2014; Mohan et al. 2010; Srinivas et al. 2013). The first detection of CPV-2c variant in India was reported from samples collected in 2006 (Nandi et al. 2010). However, reports of CPV-2c have been infrequent since then. Contrary to this trend, in the present study, we observed the CPV-2c as the predominant variant circulating in the NER, India with all the isolates characterised as the Asian CPV-2c lineage.

According to our phylogeographic analysis Asian CPV-2c lineage originated in Indonesia or China (Figure 4), with an estimated tMRCA of 2007.81. This finding aligns closely with a recent study by (Franzo et al. 2023), where they determined the origin to Indonesia with an estimated tMRCA in 2006.43. Further, the Asian CPV-2c lineage has shown a high evolutionary rate of 3.405 × 10−4 nt/site/year. In comparison (Franzo et al. 2023), estimated a slightly higher evolutionary rate of 4.62 × 10−4 nt/site/year. This bias might be because, in the present study, the dataset contained a limited number of Asian CPV-2c lineage sequences. Nevertheless, the evolutionary rate showed by Asian CPV-2c lineage (Franzo et al. 2023) is much higher than the mutation rates observed in other CPV-2 variants (Shackelton et al. 2005; Hoelzer et al. 2008). This high rate of mutation in Asian CPV-2c lineage may have implications on the genetic diversity of the strain within this lineage in the near future. Our phylogeographic analysis reveal that the earliest known sequence of Asian CPV-2c originated in Indonesia in 2013. However, currently, China (Zhuang et al. 2019; Chen et al. 2021; Jiang et al. 2021) has become a hub facilitating the distribution of Asian CPV-2c to nations such as Singapore, South Korea (Moon et al. 2020), Thailand (Charoenkul et al. 2019), Vietnam (Nguyen Manh et al. 2021), Taiwan (Chiang et al. 2016), Mongolia (Temuujin et al. 2019) and Myanmar (Mon et al. 2022). BSSVS analysis showed several transition pathways between East and Southeast Asian countries. There are multiple entries of Asian CPV-2c to India from China, which is well supported with >200 BF values. The footprint of the variant has now extended beyond Asia, with detection from Turkey (Temizkan and Sevinc Temizkan 2023), Italy (Mira et al. 2017) and Romania (Balboni et al. 2021) in Europe, Nigeria (Ogbu et al. 2020) and Namibia (Franzo et al. 2022) in Africa, and Canada (Canuti et al. 2022) in North America. The spreading pattern of Asian CPV-2c lineage reflects the emergence and subsequent worldwide establishment of this CPV-2c lineage. This also shows the dynamics of international travel and trade and highlights the genetic adaptability of the virus which needs further investigation.

The selection pressure analysis of the whole genome dataset showed that CPV-2 mostly underwent negative selection with few positively selected sites in VP2 and also in NS1. Intriguingly, four of the positively selected sites in VP2 (5, 297, 324 and 440) were the markers for the Asian CPV-2c lineage, suggesting that mutations at these sites may be associated with adaptations unique to this strain, potentially influencing their virulence and antigenicity. Also, in the case of NS1, one of the positively selected sites (544) was a marker for this strain, suggesting that it could impact its ability to replicate or evade the host immune system, which requires further investigation. Many of the characteristic markers of Asian CPV-2c lineage have been observed since many years in the Asian CPV-2 population. The 5Gly was first seen in Chinese CPV-2c strains in 2014 (Wang et al. 2016). The 267Tyr (Nakamura et al. 2004; Zhang et al. 2010; Pérez et al. 2012) and 324Ile (Jeoung et al. 2008; Zhang et al. 2010; Pérez et al. 2012) changes were first identified in East and Southeast Asian and Uruguayan strains and the 370Arg was first reported from China in CPV-2a strains (Guo et al. 2013). Although the function of 5Gly is yet to be elucidated, the 324Ile and 370Arg are known to be involved with host range specificity. The 370Arg is also described to be involved with the hemagglutination property (Hueffer et al. 2003).

The current study improves the existing literature on the molecular epidemiology of CPV-2 by providing a comprehensive genomic characterization of the CPV-2 variants in the NER, India. Unlike previous studies that have reported a high prevalence of CPV-2a and CPV-2b variants in mainland India based on VP2 gene sequences (Chinchkar et al. 2006; Mohan Raj et al. 2010; Nandi et al. 2010; Srinivas et al. 2013; Mukhopadhyay et al. 2014; Kulkarni et al. 2019), our study confirms the predominance of the Asian CPV-2c lineage in NER, India, through whole genome sequencing. These results also support the findings from neighboring East and Southeast Asian countries and provide insights into the evolutionary trajectory of this strain. The evidence of state-wise clustering within the NER, India, indicates localized variations that need further investigation to understand their epidemiological significance. This study emphasises the importance of WGS analysis over partial VP2 gene analysis to gain a more comprehensive understanding of the genetic dynamics of CPV-2 in India.

Conclusion

The different states of NER, India shares a total of 5,182 kilometers of porous international border with Bhutan, China, Nepal, Myanmar and Bangladesh. The region is also located at the junction of the Eastern Himalayas biodiversity hotspot and the Indo-Burma biodiversity hotspot. This strategic geographical location of NER, India, puts the entire region at risk for transboundary diseases. Our studies on field outbreaks of CPV-2 in the canine population in the entire NER, India revealed the occurrence of severe clinical disease that resulted in mortality in affected dogs. The phylogeographic analysis shows the transboundary nature of the Asian CPV-2c lineage, with probable multiple introductions to India. Finally, given the high mutation rate, positive selection of markers within the Asian CPV-2c lineage, low vaccination coverage, and the rapidity through which it is spreading to different regions and displacing other variants, we speculate that Asian CPV-2c lineage could become the dominant lineage in India in near the future.

Supplementary Material

Supplemental Material
TVEQ_A_2408742_SM5151.zip (71.6KB, zip)

Acknowledgments

We are thankful to the Dean, C. V. Sc. & A. H., CAU (I), Selesih, Aizawl, Mizoram for providing necessary facilities to carry out the investigation.

Funding Statement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Ethical approval

This article does not contain any studies with live animals and was approved by the Institutional Animal Ethics Committee (Approval number: CVSC/CAU/IAEC/20-21/P-28).

Authors contributions

Kiran Jayappa – Conceptualization, Methodology, Data curation, Formal Analysis, Investigation, Writing – Original Draft Preparation. Tridib Kumar Rajkhowa – Conceptualization, Validation, Writing – Review & Editing, Supervision, Resources. Satish S. Gaikwad – Formal Analysis, Writing – Review & Editing. All authors declare hereby that they agree to be accountable for all aspects of the work.

Disclosure statement

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

Data availability statement

The data that support the findings of this study are available in the Supplementary Material of this article.

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Associated Data

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

Supplementary Materials

Supplemental Material
TVEQ_A_2408742_SM5151.zip (71.6KB, zip)

Data Availability Statement

The data that support the findings of this study are available in the Supplementary Material of this article.

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