Rapid detection and genomic characterisation of feline chaphamaparvovirus in southwestern China

Abstract

Objectives

This study aimed to develop a sensitive detection method and investigate feline chaphamaparvovirus (FeChPV) in cats from southwestern China.

Methods

A SYBR Green I-based qPCR assay targeting the VP1 gene was established and validated. It was then applied to 87 feline diarrhoeic faecal samples (2021–2023). Near-full-length genomes of positive samples were sequenced for phylogenetic, structural and selection analysis.

Results

The qPCR assay showed high sensitivity (50.9 copies/μl) and reproducibility (coefficient of variation <4.0%). FeChPv was detected in 22/87 (25.3%) cats with diarrhoea. Four strains shared 97.6–99.5% identity with global isolates and formed a distinct clade within Asian lineages. A consistent valine-to-isoleucine mutation at VP1-340 was identified under positive selection, which can induce conformational changes.

Conclusions and relevance

We provide a reliable tool for the detection of FeChPV and reveal unique evolutionary features of local strains, supporting further research into its pathogenesis and spread.

Introduction

The family Parvoviridae was originally divided by the International Committee on Taxonomy of Viruses (ICTV) into Parvovirinae, infecting vertebrates, and Densovirinae, infecting invertebrates. ¹ Recent metagenomic studies have shown that chaphamaparvoviruses (ChPVs) detected in vertebrate faeces share closer homology with Densovirinae than with Parvovirinae. Consequently, ICTV reclassified Densovirinae into two subfamilies: Densovirinae and Hamaparvovirinae, the latter encompassing ChPVs. ² ChPVs have been identified in diverse hosts, including humans, bears, dogs, cats, pigs, tilapia, birds and rodents.^{3 –5}

Feline chaphamaparvovirus (FeChPV) is a recently recognised parvovirus in cats, potentially associated with gastrointestinal and respiratory disorders. Since its initial discovery, FeChPV has been reported in Canada, Italy, Turkey and China, indicating broad geographic distribution. In 2019, Li et al ⁶ first identified FeChPV in a Canadian shelter during an outbreak of unexplained diarrhoea and vomiting, with a 47.1% (8/17) positivity rate in faecal samples, implicating the virus in disease emergence. Supporting this association, a 2021 Italian case-control study reported a 36.8% (14/38) FeChPV detection rate in diarrhoeic cats vs 2.0% (1/51) in healthy controls. ⁷ Notably, a 2022 study in Turkey detected FeChPV in asymptomatic cats, suggesting potential subclinical infections. ⁸ In a study from China, 30/37 (81.1%) cats with upper respiratory disease had FeChPV detected in ocular or nasal swabs, indicating a possible link to respiratory pathology, although other recognised upper respiratory pathogens were also detected. ⁹ Collectively, these studies highlight widespread circulation of FeChPV in cats with diverse clinical presentations, yet its epidemiology, pathogenicity and cross-species transmission remain poorly understood.

FeChPV is a small, non-enveloped icosahedral virus with a linear single-stranded DNA genome of approximately 4.0–4.5 kb, comprising two major open reading frames (ORFs). The 3′-end ORF encodes the non-structural protein NS1, whereas the 5′-end ORF encodes the structural protein VP1. The NS1 stop codon overlaps the VP1 start codon by 62 nucleotides, and an additional smaller ORF within the overlap region encodes a putative nuclear protein NP. ¹⁰

In this study, we designed universal and specific primers targeting conserved regions of the FeChPV VP1 gene and established a SYBR Green I-based real-time quantitative PCR (RT-qPCR) assay for its detection. This is the first study to apply this method to diarrhoeic cat samples from southwestern China and analysed the genetic variation and evolutionary characteristics of circulating FeChPV strains.

Materials and methods

Sample collection and viral nucleic acids

Between June 2021 and March 2023, 87 faecal samples were collected from diarrhoeic cats in Chengdu, Xichang and Chongqing. Samples were suspended in phosphate-buffered saline and centrifuged, and DNA was extracted and stored at –80 °C. To evaluate assay specificity, nucleic acids of feline panleukopenia virus (FPV), canine parvovirus type 2 (CPV-2), feline calicivirus (FCV) and feline herpesvirus type 1 (FHV-1), maintained in our laboratory, were included.

Ethical statement

Faecal samples were collected from cats with diarrhoea during routine diagnostic procedures at the veterinary clinic of Southwest Minzu University. The study was conducted in accordance with the guidelines of the Southwest Minzu University Animal Ethics Committee, and verbal informed consent was obtained from all pet owners for the use of residual samples for research purposes. Because only leftover samples were used and no additional procedures were performed on the animals, the committee waived the requirement for specific ethical approval.

Primer design and synthesis

All FeChPV genome, VP1 and NS1 sequences were retrieved from GenBank and analysed for sequence conservation. Nucleotide variation rates were 9.1% for NS1 and 7.2% for VP1, prompting us to select VP1 as the target gene. Using Primer Premier 5.0, a pair of universal primers was designed within a conserved region of VP1: forward 5′-ACAAGTAAGCAGTGAAACTGG-3′ (VP1 nt 141–161) and reverse 5′AATATCCAGAGACATGATAAG-3′ (VP1 nt 284–263), generating a 144-base pair (bp) amplicon. Primers were synthesised using Sangon Biotech.

Preparation of positive control plasmids

To generate positive control plasmids for assay development, clinical samples were first screened for FeChPV using conventional PCR primers as previously described by Liu et al. ¹¹ Nucleic acids from FeChPV-positive samples were subsequently amplified with the primer pairs designed in this study. The resulting PCR products were confirmed by agarose gel electrophoresis, and purified and ligated into the pMD19-T vector. Recombinant plasmids were then sequenced (Beijing Qingke Biotech) to verify the correct insertion.

Optimisation of reaction conditions

To determine the optimal conditions for the SYBR Green I qPCR assay, we performed a matrix of reactions using annealing temperatures in the range of 50–60 °C (in 1 °C gradients) and 10 μM primer stock solutions were added in volumes of 1.0, 0.8, 0.6 or 0.4 μl to achieve final concentrations of 0.5, 0.4, 0.3 and 0.2 μM in a 20 μl reaction. Amplification efficiency, quantification cycle values and specificity were compared to identify the optimal primer concentration and annealing temperature.

Standard curve construction

Ten-fold serial dilutions of the positive control plasmid were prepared in nuclease-free water. Each dilution was run in triplicate under the optimised SYBR Green I qPCR conditions, and the mean cycle threshold (Ct) values were plotted against the logarithm (log₁₀) of the plasmid copy number to construct the standard curve.

Specificity and sensitivity assessment

Assay specificity was tested against the nucleic acids of FPV, CPV-2, FCV and FHV-1. Sensitivity was determined using 10-fold serial dilutions of plasmid standards in the range of 5.09–5.09 × 10⁻³ copies, with nuclease-free water serving as a negative control to define the limit of detection.

Reproducibility testing

Assay reproducibility was evaluated using positive plasmid standards at 5.09 × 10⁸, 5.09 × 10⁷ and 5.09 × 10⁶ copies. For intra-assay variation, each concentration was tested in triplicate within a single run, and the mean Ct, SD and coefficient of variation (CV%) were calculated for each dilution. For inter-assay variation, the same dilutions were analysed in three independent runs performed on different days under identical conditions, and the mean Ct, SD and CV% across runs were determined.

Clinical sample screening

Genomic sequencing and analysis

To investigate genetic variation in southwestern FeChPV strains, overlapping PCR amplicons covering the viral genome were generated using primers and methods adapted from Hao et al. ⁹ Amplicons were assembled with the SeqMan module in DNAStar. Multiple sequence alignment of obtained FeChPV genomes, VP1 and NS1 sequences were performed against all FeChPV sequences available in GenBank as of 1 August 2025 (45 complete genomes, 46 VP1s, 45 NS1s) using ClustalW in MEGA 7.0. Phylogenetic trees for genome-wide nucleotide sequences and VP1/NS1 amino acid sequences were constructed using the maximum-likelihood method with the Jones–Taylor–Thornton model and 1000 bootstrap replicates. Recombination events were predicted with RDP 4.0 using multiple algorithms, including RDP, GENECONV, BootScan, MaxChi, Chimera, 3SEQ and SiScan.

Selection pressure analysis

Selection pressures were evaluated using the HyPhy online platform (https://www.hyphy.org/) under the Nei–Gojobori method. Synonymous substitutions per synonymous site (dS) and non-synonymous substitutions per non-synonymous site (dN) were computed for each codon pair with the Jukes–Cantor correction. Pairwise dN:dS ratios were calculated, with dN:dS >1 indicating potential positive selection.

Protein structural modelling

To assess the structural impact of mutations, VP1 proteins of strain M2 (GenBank OR413556) and reference strain BKK163 (GenBank PV189143), differing only at residue 340, were modelled using AlphaFold 3 (https://alphafold.ebi.ac.uk). Structural comparisons were visualised with ChimeraX v1.10 to assess conformational changes associated with the mutation.

Results

FeChPV detection in clinical samples

Using the previously reported PCR assay ¹¹ yielding a 620-bp amplicon, all 87 faecal swabs from diarrhoeic cats were screened. In total, 10/87 samples tested positive for FeChPV, corresponding to an 11.5% positivity rate, and were used as templates for subsequent assay development.

Primer specificity

PCR amplification using the primers designed in this study generated the expected fragment from positive sample DNA, which was cloned into pMD19-T and subsequently confirmed by sequencing. The resulting plasmid was used as the positive control (163 ng/μl, equivalent to 5.09 × 10¹⁰ copies/μl).

Optimised SYBR Green I qPCR conditions

Annealing temperatures of 50–60 °C and primer concentrations of 0.5–0.2 μM were tested; 55 °C and 0.5 μM of each primer were selected because they yielded specific amplification with low Ct values and no detectable primer-dimer. Under the optimised conditions, each 20 μl reaction contained 10 μl Hieff UNICON Universal Blue qPCR SYBR Green Master Mix, 1 μl of 10 μM forward and reverse primers (final concentration 0.5 μM of each primer), 1 μl of template DNA and nuclease-free water. Thermocycling conditions were 95 °C for 5 mins, followed by 40 cycles of 95 °C for 15 s and 55 °C for 30 s, and a melting-curve stage at 95 °C for 15 s, 60 °C for 1 min and 95 °C for 15 s.

Standard curve, melting curve and sensitivity

Amplification plots for 10-fold serial dilutions from 5.09 × 10⁷ to 5.09 × 10¹ copies/μl showed typical sigmoidal curves with well-separated Ct values, and the lowest dilution (5.09 × 10¹ copies/μl) was defined as the limit of detection (Figure 1a). Melting-curve analysis confirmed a single sharp peak without primer-dimer or non-specific products (Figure 1b). The corresponding standard curve was linear over this range, with a regression equation of y = –3.409x + 37.802, R² = 1 and an amplification efficiency of 96.5% (Figure 1c).

Figure 1

(a) Amplification plot, (b) melting curve and (c) standard curve of the SYBR Green I real-time PCR assay for feline chaphamaparvovirus. Ten-fold serial dilutions of the plasmid standard from 5.09 × 10⁹ to 5.09 × 10^–3 copies/μl were tested in triplicate (curves 1–13); N = negative control

Specificity and reproducibility

No fluorescence signal was observed with FPV, CPV-2, FHV-1 and FCV, confirming assay specificity (Figure 2). Intra- and inter-assay CV% were less than 4.0% across plasmid concentrations of 5.09 × 10⁸–5.09 × 10⁶ copies, demonstrating high reproducibility (Table 1).

Figure 2

(a) Amplification plot of clinical samples detected by the SYBR Green I qPCR assay. (b) Curve 1, feline chaphamaparvovirus plasmid standard; curves 2–5, feline panleukopenia virus, canine parvovirus type 2, feline herpesvirus type 1 and feline calicivirus positive samples

Table 1

Intra- and inter-assay reproducibility of the SYBR Green I real-time PCR assay for feline chaphamaparvovirus

The positive plasmid standards	Intra-assay Ct value		Inter-assay Ct value
	Mean ± SD	CV%	Mean ± SD	CV%
5.09 × 108	8.44 ± 0.07	0.87	8.72 ± 0.26	2.99
5.09 × 107	11.05 ± 0.09	0.77	11.49 ± 0.38	3.29
5.09 × 106	14.63 ± 0.07	0.47	14.82 ± 0.17	1.14

Ct = cycle threshold; CV% = coefficient of variation

Clinical screening

Application of the newly established SYBR Green I qPCR assay to 87 clinical samples detected FeChPV in 22 (25.3%) samples, whereas the previously reported SYBR Green I qPCR ¹¹ detected 19 (21.8%) positive samples. All 19 positives detected by the previously published SYBR Green I qPCR method were among the 22 positives identified by the new assay. Sequencing confirmed all positives as FeChPV.

Genome sequencing and phylogenetic analysis

Four near-complete FeChPV genomes were obtained from positive samples: M1 and M2 from Chengdu (GenBank OR413555–OR413556) and C13 and C14 from Xichang (OR413557–OR413558). Genome lengths were 3442 bp, encoding NS1 (1–1977 bp, 658 aa), NP (1352–1915 bp, 187 aa) and VP1 (1916–3442 bp, 508 aa). Intra-strain genome homology was 99.0–99.6%, and 97.6–99.5% compared with 41 FeChPV genomes from GenBank. VP1 nucleotide and amino acid identities were in the range of 98.9–99.8% and 99.0–99.8%, respectively, while NS1 identities were in the range of 98.9–99.7% (nt) and 98.8–99.7% (aa).

Phylogenetic analyses based on the complete genome, VP1 and NS1 aa sequences (Figures 3 and 4) revealed that FeChPV strains segregate into two major clades. One clade comprises the earliest reported Canadian strain together with genetically related isolates from Italy, the USA and some strains from China and Thailand, whereas the second clade is predominantly composed of Chinese and Thai isolates. Comparative analysis identified seven amino acid positions in VP1 (15, 45, 57, 208, 212, 419 and 444) and 11 in NS1 (124, 125, 196, 250, 271, 460, 478, 506, 550, 568 and 580) that consistently distinguished between the two clades. Notably, the four strains identified in this study clustered within the Asian lineage, distant from the Canadian clade, across all three phylogenetic trees. Within the VP1 phylogeny, these four strains formed a distinct sublineage characterised by a valine-to-isoleucine (Val-to-Ile) mutation at residue 340, highlighting a unique evolutionary signature (Figure 4a). A similar independent subclade, defined by the same amino acid mutation, was observed among four Italian isolates and one Thai strain within the Canadian-related lineage, suggesting that residue 340 may represent a critical site of adaptive variation. No evidence of recombination was detected in the four genomes characterised in this study.

Figure 3

Phylogenetic analysis of feline chaphamaparvovirus (FeChPV) genomes. The genomes of the FeChPV strains obtained in this study were aligned with all 41 FeChPV genome sequences available in GenBank. Phylogenetic trees were constructed using the maximum likelihood method based on the Jones–Taylor–Thornton model, with statistical support estimated from 1000 bootstrap replicates. Red circles indicate FeChPV strains detected in this study and red triangles represent the earliest reported Canadian FeChPV strain

Figure 4

Phylogenetic analysis of (a) feline chaphamaparvovirus (FeChPV) VP1 and (b) NS1 proteins. The VP1 and NS1 proteins of the FeChPV strains obtained in this study were aligned with all available sequences in GenBank. Phylogenetic trees were generated using the maximum likelihood method based on the Jones–Taylor–Thornton model, with 1000 bootstrap replicates for statistical support. Red circles denote FeChPV strains detected in this study and red triangles indicate the earliest reported Canadian FeChPV strain

Selection pressure analysis

Selection pressure analyses of the FeChPV VP1 and NS1 genes revealed mean dN:dS ratios of approximately 0.2074 and 0.2834 (dN:dS <1), respectively, indicating that both genes are subject to purifying selection, with most mutations likely being deleterious and eliminated. Despite this overall trend, several codons exhibited evidence of positive selection (dN:dS >1). Specifically, the seven VP1 residues and 11 NS1 residues that differentiated the two major phylogenetic clades showed strong signals of adaptive evolution, with VP1 sites tending towards infinite dN:dS and nine of the NS1 sites also approaching infinity (∞). These mutations may therefore confer selective advantages to the virus. Notably, residue 340 of VP1 also exhibited a strong positive selection signal (dN:dS → ∞), further supporting its potential role as an adaptive site. Codon-level selection results for VP1 and NS1 are provided in Tables 1 and 2 in the supplementary material.

VP1 structural modelling

Structural predictions generated with AlphaFold 3 indicated that the VP1 protein of strain M2 (GenBank OR413556, Ile340) differs from that of reference strain BKK163 (GenBank PV189143, Val340). Structural alignment revealed that the Val-to-Ile mutation at aa residue 340 altered the local conformation, converting the region spanning residues 271–272 from an alpha (α)-helix to a random coil (Figure 5).

Figure 5

Predicted tertiary structure of feline chaphamaparvovirus VP1 protein. The three-dimensional structures of VP1 from strain M2 (GenBank number OR413556; 340Ile) and reference strain BKK163 (GenBank number PV189143; 340Val) were modelled using AlphaFold 3. Structural comparison revealed that the valine-to-isoleucine substitution at position 340 induces a local conformational change in the 271–272 region, shifting from an alpha-helix to a random coil

Discussion

Comprehensive analysis of all FeChPV genomes in GenBank revealed NS1 and VP1 nucleotide variation rates of 9.1% and 7.2%, respectively. Owing to the relative conservation of VP1, it was selected as the detection target. Application of the new SYBR Green I qPCR assay developed here to 87 faecal swabs yielded a 25.3% positivity rate, higher than the 21.8% detected by the SYBR Green I qPCR assay by Liu et al. ¹¹ Primer conservation analysis indicated 100% sequence conservation of the primers designed in this study across 46 VP1 sequences, compared with 32.6% and 93.5% for Liu et al’s upstream and downstream primers, suggesting superior universality.

FeChPV is an emerging parvovirus in cats, and epidemiological data remain limited. The observed positivity rate in southwestern China suggests that FeChPV is circulating at moderate prevalence, yet more data are needed to confirm the prevalence and relevance of FeChPV infection. Global reports, including Canada, Italy, Turkey and USA, indicate wide geographic distribution and cross-regional spread potential.^{6 –9} FeChPV detection is associated with gastrointestinal and respiratory disease; however, coinfections with enteric pathogens (eg, FPV, kobuvirus, feline enteric coronavirus, feline bocavirus) and upper respiratory pathogens (eg, FCV, FHV-1), as reported in previous studies, complicate causal inference.^10,12 Given that other parvoviruses, such as CPV-2, FPV and raccoon parvovirus, are important enteric pathogens,^{13 –15} expanded surveillance and virological studies are warranted to elucidate FeChPV pathogenicity.

Four near-complete genomes obtained here clustered with most Chinese and Thai strains and diverged from the Canadian prototype. Selection pressure analysis indicated purifying selection across NS1 and VP1, but VP1 residue 340 exhibited strong positive selection and was predicted to induce 271–272 aa domain from α-helix to coil conformational change. Parallel emergence of the same mutation in geographically distant clades suggests convergent evolution, potentially linked to host adaptation or immune evasion. Similar structural changes in FPV VP2 (Ala 91 Ser) enhance viral virulence and epidemic potential. ¹⁶ The 271–272 aa domain are surface-exposed, suggesting that α-helix to coil transitions may increase binding flexibility to host receptors or immune evasion, facilitating host adaptation, transmission or virulence. Thus, the VP1-340 mutation may represent an adaptive marker with functional relevance.

Despite these findings, limitations include the modest sample size, lack of longitudinal clinical data and absence of experimental validation for predicted structural changes. Further in vitro and in vivo studies are required to determine the biological impact of VP1 mutations on viral fitness, host range and pathogenicity. Importantly, FeChPV has been reported in multiple continents and chaphamaparvoviruses infect diverse vertebrate hosts.^{3 –5} Given the close interactions between cats and humans, surveillance studies should also investigate the potential for cross-species transmission and zoonotic risk. Such efforts will be critical to fully understand the ecological and epidemiological significance of FeChPV.

Conclusions

A pair of FeChPV VP1-universal primers was designed, and a SYBR Green I real-time PCR assay was established, demonstrating high specificity, sensitivity and reproducibility. Epidemiological investigation in southwestern China revealed a 25.3% positivity rate, providing new evidence for its circulation in domestic cats. Genomic analysis identified residue 340 in VP1 as a site under positive selection, with predicted structural changes that may influence host adaptation or virulence. Together, these findings provide the first comprehensive insight into the epidemiology, genetic diversity and adaptive evolution of FeChPV in China, and lay the groundwork for future studies on its pathogenic potential and zoonotic risk.

Supplemental Material

Supplementary table 1

Codon-level selection results for VP1.

Supplemental Material

Supplementary table 2

Codon-level selection results for NS1.