A duplex SYBR green I-based real-time polymerase chain reaction assay for concurrent detection of feline parvovirus and feline coronavirus
Introduction
Feline parvovirus (FPV), also referred to as feline panleukopenia virus, is a non-enveloped single-stranded DNA virus that contains two open reading frames (ORF1 and ORF2) with genes encoding transcriptional regulatory proteins and capsid proteins, respectively. Capsid proteins comprise two structural proteins, namely, VP1 and VP2 proteins. VP2 is the most stable protein in FPV (Awad et al., 2018; Cheng et al., 2019; Christensen and Tattersall, 2002; Miranda et al., 2017). FPV, first reported by Verge in 1928 (Miranda et al., 2017), can cause acute, severe, and highly contagious infectious diseases, which, under natural conditions, may occur in cats, tigers, raccoons, mice, leopards, lions, lynxes, and other animals (Battilani et al., 2011; Demeter et al., 2009; Duarte et al., 2013; Sabshin et al., 2012). FPV infection typically manifests as fatal acute enteritis in adult cats, accompanied by severe leukopenia, and it is one of the main infectious diseases in cats (Fei-Fei et al., 2017). FPV predominantly replicates in the intestines of animals and may cause sudden high fever, intractable vomiting, severe diarrhea, dehydration, circulatory disturbance, and marked reduction in white blood cells. Taken together, this virus has a wide host spectrum and is highly contagious, and infections are frequently lethal (Leal et al., 2020).
Feline coronavirus (FCoV) belongs to the genus Alphacoronavirus in the Coronavirinae, a subfamily of Coronaviridae. And it is an enveloped RNA virus (Jaimes and Whittaker, 2018). FCoVs are mainly transmitted through digestive tract infections or through insect vectors. Feline infectious peritonitis virus (FIPV) can be transmitted vertically to the fetus through the placenta, or to healthy cats via blood-sucking insects. Infected cats can discharge a large amount of virus through the fecal route, and can be transmitted to healthy cats through the digestive tract or respiratory tract (Decaro et al., 2021; Paltrinieri et al., 2021; Stout et al., 2021). Among domestic cats kept in catteries of breeding facilities, 20 %–60 % were found to be coronavirus-positive, while approximately 80 %–100 % free-roaming cats were found to be coronavirus-positive. Under natural conditions, infected cats may shed viruses for long periods (Kipar and Meli, 2014; Pedersen, 2009, 2014; Rottier et al., 2005; Tekes and Thiel, 2016). Serology and gene sequencing showed that FCoVs comprise two groups: FIPV and feline enteric coronavirus (FECV) (Kipar and Meli, 2014). FIPV mainly infects monocytes and is chronic, systemic, and highly pathogenic (Kipar et al., 2010; Porter et al., 2014; Tekes and Thiel, 2016). The clinical signs include peritonitis and large ascites accumulation. Cats can be infected at different ages, but the incidence is higher in elderly cats and in cats under 2 years of age. Moreover, the incidence is higher in purebred cats than in mixed-breed cats. FECV is a non-pathogenic coronavirus that occurs in the intestines of healthy cats, and it mainly infects weaned kittens, while adult cats are mostly latently infected. The main clinical manifestations are vomiting, accelerated bowel motility, diarrhea, and dehydration, among others. FIPV and FECV can show viremia, however, only FIPV replicates in macrophages and causes feline infectious peritonitis (FIP) (Pedersen, 2009; Ward, 1970). The prevalence of both viruses is high in global cat populations, and the clinical manifestations of FPV and FECV are very similar. Therefore, it is necessary to have a method that can test both FPV and FCoV simultaneously.
Section snippets
Samples collection and nucleic acid isolation
Forty-eight fecal samples of cats were collected at different animal hospitals in Anhui Province, Jiangsu Province, and Shanghai City, China. The samples originated from cats of different ages that were asymptomatic or showed emesis or diarrhea (Table 5).
The fecal samples were homogenized, diluted with phosphate-buffered saline at a ratio of 1:10, and then centrifuged at 8000 × g for 3 min. The supernatant was used for nucleic acid extraction or stored at −40 °C for subsequent use. Viral DNA
Optimization of the duplex SYBR green I-based qPCR assay
The optimal qPCR reaction mix comprised 10 μL 2 × SuperReal PreMix Plus with SYBR green I (Tiangen), 8 μL ddH2O, 0.3 μL (10 μmol/L) of each of the four primers, and 1 μL template DNA. The reaction volume was 20 μL. Thermocycling was performed using 95 °C for 15 min, followed by 39 cycles of 95 °C for 10 s and 60 °C for 30 s. FPV and FCoV were distinguished according to disparate melting temperatures (Tm), which were 77.0 °C ± 0.5 °C (FPV) and 80.5 °C ± 0.5 °C (FCoV; Fig. 1a-b). Single peaks
Discussion
When FPV invades the digestive tract system, it will cause damage to the intestines, destroy the mucosal barrier, and harmful bacteria will easily enter the blood circulation system. If the cat has infected with FECV, it may mutate into FIP (Bank-Wolf et al., 2014; Barrs, 2019). Feline coronavirus infection is very common among cats, and approximately 20 %–60 % of pet cats carry antibodies (Sabshin et al., 2012). As shown in Table 5, FPV was only detected in fecal samples from cats less than 1
Authors' statement
Junhuang Wu and Yongqiu Cui were involved in performing experiments. Xu Guo, Yongdong Li, and Fazhi Xu were involved in data analysis, experimental design. Liting Sun, Zhiqing Xu and Yong Wang were involved drafting of the manuscript. Liting Sun and Yong Wang revised and examined manuscript. All authors read and approved the final manuscript. All authors have declared that no competing interests exist.
Funding
This study was supported by the Ningbo Health Branding Subject Fund (No. ppxk2018-10).
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgments
We would like to thank Editage (www.editage.cn) for English language editing.
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