Cachavirus, which belongs to the species Chaphamaparvovirus carnivran1, is a small, icosahedral, nonenveloped virus. It has a single-stranded DNA genome containing two major opening reading frames encoding nonstructural protein 1 (NS1) and the capsid protein (VP1) (1). In recent years, the family Parvoviridae has been re-organized by the International Committee on Taxonomy of Viruses, and the new subfamily Hamaparvovirinae now includes the genus Chaphamaparvovirus, which have been detected in several hosts (2). Chaphamaparvoviruses include bat (Eidolon helvum) parvovirus 2 (EHPV2) (3), common vampire bat (Desmodus rotundus) parvovirus (4), fruit bat (Eidolon helvum) parvovirus 1 (5), etc. Several studies have also detected chaphamaparvoviruses in mammals such as simians (6), Tasmanian devils (7), rat (8, 9), swine (10), and birds, including turkey (11), red-crowned crane (12), chicken (13), and peafowl (14).

Cachavirus, which belongs to the genus Chaphamaparvovirus, is a novel canine parvovirus first detected in dogs with diarrhea in the United States in 2019. Cachavirus infections have been associated with both asymptomatic infections and clinical signs such as diarrhea, and the virus tends to establish active infections in already-infected hosts (15). Notably, a recent study reported the detection of two strains of cachavirus in the feces of pet cats, which may have originated in dogs (16). Cachavirus was also found in wild coyotes in 2022, and a study indicated that the virus had been circulating in wild animals for at least 10 years (15, 17). These results confirm that cachavirus has multiple hosts and a capacity for cross-species transmission. Therefore, further research is required. Although cachavirus has been detected in several geographic locations, including the United States, Canada, and some provinces of China (18), our knowledge of the prevalence and genetic characteristics of the virus is relatively limited.

In this study, we collected 325 fecal samples from dogs in four provinces of northeastern China (Inner Mongolia Autonomous Region, Heilongjiang, Liaoning, and Jilin) and looked for cachavirus with PCR. We also examined the rate of cachavirus in mixed infections with other viruses. To examine the prevalence of and risk factors for cachavirus infection, we compared the collection date and location, and the host age of the positive samples. We subjected the detected cachavirus and known chapparvovirus sequences to a phylogenetic analysis, and investigated the codon usage bias in the cachavirus VP1 gene. We also performed a co-evolutionary analysis to investigate the development and transmission of cachavirus and other chaphamaparvoviruses.

At present, cachavirus infections are associated with clinical signs such as diarrhea, and the virus has been detected at limited geographic locations, including the United States and some provinces of China. However, the prevalence and genetic characteristics of the virus are relatively unknown. In the present study, we investigated the prevalence and coinfection rates of cachavirus in the entire northeastern region of China. We also conducted molecular and phylogenetic analyzes to better understand the evolution of cachavirus and to provide new directions for its control.

Cachavirus is highly prevalent in northeastern China, with a large geographic spread. The overall positivity rate in dogs was 6.8%, which is higher than previously reported in China (1.55%) (16) or in two recent studies in Canada [2.6% (15) and 3.3% (17)]. Cachavirus, a recently identified novel canine parvovirus, is spreading around the world and has been detected in the United States, Canada, Italy, and several provinces of China. Its geographic reach has increased our concern about this virus. Our results show that the positivity rate of cachavirus varies with the host’s age, immune status, and clinical symptoms and the collection date and location of the sample in northeastern China. Its preferred host age is clearly young dogs (puppies) and its seasonal preference is winter. Therefore, given the high prevalence of cachavirus, we must pay more attention to puppies, especially in winter.

The pathogenicity of cachavirus requires further study, especially with animal experiments. To date, reports of the pathogenicity of chaphamaparvoviruses in vertebrate hosts have been relatively limited. Mouse kidney parvovirus (MKPV) has been shown to cause a kidney disease called “inclusion body nephropathy” in a population of laboratory mice (31), and the prevalence of chaphamaparvoviruses in mouse liver tissues was high, suggesting that it may also be a gastrointestinal pathogen (9). Although there is no clear disease association, most infected dogs became ill, and one cachavirus-positive dog died. Therefore, the involvement of cachavirus in a small proportion of diarrheic dogs cannot be ruled out.

In this study, we have shown that cachavirus is often involved in coinfections with other viruses. All cachavirus-positive samples were coinfected with one or more other viruses. The coinfection rate was similar to that reported in China in 2018–2019 (17) and in Canada in 2022 (15). In those studies, the coinfection of cachavirus with CPV-2 was most frequent, whereas in our study, its coinfection with CCoV was most frequent (18). We also detected coinfections with CaAstV (18.2%) for the first time. Previous studies have shown that cachavirus easily establishes infections in already-infected hosts (15), suggesting that coinfection with cachavirus is widespread. It should be noted that dogs infected with cachavirus and multiple other viruses showed obvious clinical signs. Only four dogs coinfected with CCoV showed no symptoms, whereas the rest had diarrhea. The coinfecting viruses (CCoV, CaAstV, and CDV) are all clinically associated with severe diarrhea (32–34). Interestingly, both CCoV and cachavirus can be detected in asymptomatic dogs, but most coinfections of these two viruses cause diarrhea. Therefore, we infer that coinfection with other viruses increases the infectivity of cachavirus and aggravates its clinical symptoms. In particular, coinfections of CCoV and cachavirus may increase the pathogenicity of both. In daily life, diarrhea has always been a common disease affecting the health of dogs and can lead to death in severe cases. For a long time, there has been no preventive treatment for diarrhea in dogs, which may be related to complex infections of multiple viruses. Therefore, further study of the degree to which coinfection influences diarrhea in dogs, and the mechanism(s) by which coinfections affect clinical signs is required.

Cachavirus can infect multiple hosts, with potential transmission between them. Cachavirus was first detected in dogs, but was also detected in cats in 2018–2019 (16). In the latest study, cachavirus was found in wild animals, including coyotes (15, 17), and appears to have been circulating among wild animals for at least 10 years. Therefore, cachavirus may spread among wild animals, dogs, and cats, especially because both coyotes and dogs are canids, which may all be susceptible to this virus. Consequently, the hosts and transmission modes of cachavirus warrant intensive research.

With an amino acid analysis, we identified a clear hypervariable region in cachavirus NS1. The region is located at residues 252–255 (Ser252Cys, Gly253Leu, Gly254Thr, and Tyr255Phe), and is consistent with previous Chinese strains (16, 17). Therefore, we suspect that this mutation may be a unique change in the evolution of cachaviruses in China, but its role requires further study. In comparisons of the NS1 and VP1 sequences, strains CY56 and NWT-W78 showed different mutation sites from other strains, consistent with their positions on the phylogenetic trees.

To analyze the synonymous codon usage patterns in these cachavirus strains, we calculated the RSCU of the VP1 genes. To clarify the molecular evolution of individual cachavirus genes, we investigated the use of synonymous codons, to better understand the regulation of viral gene expression (35, 36). In most studies, the main factor causing variations in codon usage in genes with high A + T or G + C contents is mutation bias (37–40). Cachavirus VP1 shows the same preference for A/T-ending codons as the VP gene of CPV. However, the codon GGA (Gly) is preferred in cachavirus, whereas GGT (Gly) is preferred in CPV-2. The codon repertoire of cachavirus does not include GAG (Glu), whereas its RSCU in CPV-2 is 0.414 (41). This is an obvious difference between cachavirus and CPV-2. More comprehensive analyzes are required to determine the true extent of the codon usage bias changes within and among cachavirus strains and the effects of factors such as the dominant host species, transmission mechanisms, cell tropism, and the genetic structure of virus (42).

We conducted the first co-evolutionary analysis of cachavirus and other chaphamaparvoviruses, to understand their development and spread. Cachavirus showed significant cospeciation with Feline chapparvovirus, and the cachavirus has undergone host-switching events from dog to cat. Cats can be infected by both cachavirus and Feline chapparvovirus, although the divergence between the two viruses is nearly 30%, suggesting that they are two distinct species, and have been classified as Chaphamaparvovirus carnivoran 1 and 2. We speculate that the emergence of cachavirus in cats is probably the result of host-switching from dogs. In canine cachavirus, multiple cospeciation and host-switching events have occurred among wolves, coyotes, and dogs. From other co-evolutionary and transmission events, it can be inferred that chaphamaparvoviruses may have also been transmitted between wild and domestic animals. Therefore, appropriate measures must be taken to prevent the spread and evolution of cachavirus.

In summary, this is the first report of the presence of cachavirus in dogs in northeastern China. Cachavirus strains often coexist with other canine intestinal pathogens, and CaAstV was detected in all cachavirus-positive samples, with different incidence rates of enteritic symptoms. Our research also suggests that cachavirus continues to evolve in China, and extends our understanding of the epidemiological status of cachavirus in China. A codon usage analysis showed that most of the preferred codons in cachavirus are A- or T-ending codons, as in traditional canine parvovirus. A co-evolutionary analysis showed that cachavirus has undergone cospeciation and transmission among different host species. Large-scale studies are required to confirm the pathogenicity of this virus.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://www.ncbi.nlm.nih.gov/nuccore; OQ108871-OQ108876.

The animal studies were approved by Approval (NEAUEC20210326, 30 March 2021) was obtained from the Institutional Committee of Northeast Agricultural University for animal experiments. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.

YB, XY, ZG, KX, ZY, and HS contributed to sample collection and conducted experiments. NL drafted the manuscript and analyzed data. JG and LZ critically revised the manuscript and gave final approval. All authors contributed to the article and approved the submitted version.

This research was supported by the National Key R&D Program of China (2021YFF0703000), the SIPT program of Northeast Agricultural University (202210224166 and S202210224168).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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