Analysis of the vp2 gene sequence of a new mutated mink enteritis parvovirus strain in PR China
Virology Journal volume 7, Article number: 124 (2010)
Mink enteritis virus (MEV) causes a highly contagious viral disease of mink with a worldwide distribution. MEV has a linear, single-stranded, negative-sense DNA with a genome length of approximately 5,000 bp. The VP2 protein is the major structural protein of the parvovirus encoded by the vp2 gene. VP2 is highly antigenic and plays important roles in determining viral host ranges and tissue tropisms. This study describes the bionomics and vp2 gene analysis of a mutated strain, MEV-DL, which was isolated recently in China and outlines its homologous relationships with other selected strains registered in Genbank.
The MEV-DL strain can infect F81 cells with cytopathic effects. Pig erythrocytes were agglutinated by the MEV-DL strain. The generation of MEV-DL in F81 cells could infect mink within three months and cause a disease that was similar to that caused by wild-type MEV. A comparative analysis of the vp2 gene nucleotide (nt) sequence of MEV-DL showed that this was more than 99% homologous with other mink enteritis parvoviruses in Genbank. However, the nucleotide residues at positions 1,065 and 1,238 in the MEV-DL strain of the vp2 gene differed from those of all the other MEV strains described previously. It is noteworthy that the mutation at the nucleotide residues position 1,238 led to Asp/Gly replacement. This may lead to structural changes. A phylogenetic tree and sequence distance table were obtained, which showed that the MEV-DL and ZYL-1 strains had the closest inheritance distance.
A new variation of the vp2 gene exists in the MEV-DL strain, which may lead to structural changes of the VP2 protein. Phylogenetic analysis showed that MEV-DL may originate from the ZYL-1 strain in DaLian.
Parvoviruses are autonomous, single-stranded DNA viruses that have a genome length of approximately 5,000 bp [1–3]. Parvoviruses are capable of infecting a variety of hosts, for example, parvovirus B19 cause disease in humans, while others such as feline panleukopenia virus (FPV), canine parvovirus (CPV), raccoon parvovirus (RPV) and blue fox parvovirus (BFPV) infect carnivores, and sometimes even fatal to susceptible animals [4–10]. The mink enteritis virus (MEV) disease was first reported by Schofield (1949) . In 1952, Wills  isolated and identified the viral pathogen. MEV is a contagious disease can cause acute hemorrhagic enteritis in mink, in particular in younger animals, and it is frequently associated with leucopenia .
MEV is classified as a FPV subgroup, a classification which also includes CPV, RPV, and BFPV [13, 14]. A comparison of the sequence of amino acid residues between laboratory strains and isolated wild type (wt) strains showed homology of more than 98% [15–21]. The nucleotide sequence of the carnivorous animal parvovirus also has a high level of homology, and the FPV, MEV, RPV, and BFPV cannot be distinguished from each other by DNA sequence alone .
VP2 protein is the main structural protein of parvovirus capsid encoded by vp2 gene. That either residue 93 or residue 300 in VP2 protein of CPV binds to the cellular transferrin receptor (TfR) determines CPV infectivity of canine . Those capsid regions are also highly antigenic, and serves as the target of many neutralizing antibodies .
Tingxiu Jiang  reported the first incidence of MEV disease in China in 1981. Nearly 30 years later, this disease has now spread widely across China and affects almost all of the mink cultivation sites . In recent years, although a vaccine has been used to prevent further spread of the disease, the number of infections still continues to grow . This may be related to the capacity of the MEV to continuously mutate. In this study, the MEV-DL virus strain, which is a characteristic parvovirus, has been isolated with a mutation within VP2 protein.
Sample origin and bionomics
Samples of feces from minks with signs of illness were taken from DaLian in China and used for the isolation of MEV-DL. The fecal samples were manipulated according to the methods that have been described previously . The isolated viral particles were then inoculated into F81 cells. When the cytopathic effects of the virus on F81 cells reached 80%, cultures were scraped, then centrifuged after a freeze-thaw cycle twice, and the supernatant was collected [26, 27]. Electron microscopy, animal studies [28, 29], and hemagglutination tests  were used to analyze the bionomics of the MEV-DL strain.
vp2 gene cloning and sequencing
According to the vp2 gene sequence published in Genbank (accession number: M23999), a pair of specific primers were designed to amplify the vp2 gene of the isolated strain of MEV; the sequence of the forward primer was 5'-GCACCAATGAGTGATGGAGCAGTTC-3' (nt 294-318) and the reverse primer sequence was 5'-TCTAAGGGCAAACCAACCAACCACC-3' (nt 2,292-2,317). The size of the resulting product was 1,999 bp. The fecal samples from the mink that had been infected naturally were homogenized, frozen and thawed in normal saline before being subjected to centrifugation at 4,000 rpm for 20 min. The resulting supernatant was used as the template for PCR, for which the following conditions were applied: 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 45 s and polymerization at 72°C for 2 min 30 s. After electrophoresis on a 1.0% agarose gel and ethidium bromide staining, the PCR products were extracted from the gel and purified. The purified products were cloned into the PMD18-T vector, transformed into DH-5α, and then incubated at temperature of 37°C for 16 hours. The positive clones were sequenced by Shanghai Sangon Biological Engineering Technology & Services Co.,Ltd.
Phylogenetic analysis was performed using MEGA 4 , and seventeen vp2 gene sequences from MEV, CPV and FPV in Genbank were used in this study. A phylogenetic tree was constructed using the neighbor-joining method , and a bootstrap analysis with 500 replicates was performed to assess the confidence level of the branch pattern. The sequence distances were determined using the Jotun Hein method . The nt sequences of the vp2 gene of the analyzed parvovirus were as follows: MEV-e (U22191), Abashiri (D00765), ZYL-1 (GU272028), MEV-DL (HM015824), Suning (FJ712217), LYT-2 (FJ712221), Beregovoi-Biocentr (AY665656), Rodniki-Biocentr (AY665657), mink enteritis virus (M23999), 389/07 (EU145593), 933/07 (EU360958), ChangC2007 (FJ936171), 04S23 (DQ025992), K029 (EU009205), 128/08 (FJ005246), GR51/08 (GQ865518), 08-5-WH (FJ432717) and 11/09 (GU45715).
Sixty hours after inoculation of MEV-DL strain into normal F81 cells, these cells were integrated into a cell colony and cellular strings with the intracellular particles increased. The results from electron microscopy showed that the viral particles existed as a sphere with a diameter of approximately 20 nm. Hemagglutination assays showed that MEV-DL can agglutinate pig erythrocytes. Animal inoculation experiments demonstrated that the MEV-DL cultures infected minks and caused diarrhea 10 days after inoculation; 15 days after inoculation, clinic symptoms of the minks disappeared.
vp2 gene sequencing analysis and phylogenetic analysis
The PCR amplification of the MEV-DL strain vp2 gene products were cloned into PMD18-T and sequenced. A comparative analysis of the vp2 nucleotide sequence (1,755 bp) of this strain was performed against other MEV vp2 sequences that are stored in Genbank. This analysis showed that the mutated strain of MEV-DL was more than 99% homologous with the other strains of MEV cited above ( Figure 1). It was found that there were 33 different nt positions that existed in vp2 gene fragments among published vp2 sequences of MEV strains. (Additional file 1), but differences occurred at only 16 amino acid residues in the VP2 protein among the strains listed above (Table 1).
Specifically, the 1,065 and 1,238 nucleotide residues in the vp2 gene of the MEV-DL strain differed from those of all MEV strains that have been described previously. It is noteworthy that only the mutation at position 1,238 led to an Asp/Gly replacement mutation at the 413 amino acid residue of the VP2 protein, which may lead to structural changes, such as alterations in the alpha, amphipathic regions and tum regions forecasted by DNASTAR software.
At the phylogenetic level, the vp2 gene sequences of the MEV-DL and ZYL-1, as well as the vp2 gene sequences of FPV and MEV, formed clusters when compared to the vp2 gene sequences of CPV (Figure 2). The results also showed that the vp2 gene sequence of the ZYL-1 strain (accession number: GU2772028), which was isolated from DaLian in China, had a nucleotide sequence homology up to 99.9% with the relevant sequence of the MEV-DL strain, whereas the LYT-2 isolation strain (accession number: FJ712221) only had a nucleotide sequence homology up to 99.4% when compared to the MEV-DL strain ( Figure 1).
MEV, FPV, BFPV, RPV and CPV are all classified in the same family and more than 98% of their nucleotide sequences are shared . However, the evolutionary rate of parvoviruses varies between species; for instance, CPV had a nucleotide substitution rate that was similar to that of the RNA viruses, such as HIV, whereas FPV had a slow rate of nt substitution compared to CPV . The average annual replacement rates of CPV and FPV are 1.7×10-4 and 9.4×10-5, respectively . In the past 40 years, 33 nucleotide substitution positions have been found in the vp2 gene among the MEV strains Mutated bases in the vp2 gene in MEV-DL were included in this study. Additionally, a mutated base was also found in BFPV vp1 gene at position 296 . Therefore, it can be concluded that more nucleotide substitution positions may exist in the MEV genome than was previously thought, and MEV may have a greater rate of evolution.
Three antigenic variants of MEV have been identified, which differ by only small numbers of amino acid sequence changes in the capsid protein . Cross-immunity to these strains has protected mink from infection by both homologous and heterologous MEV strains . The vp2 gene encodes the major structural protein of parvoviruses . It determines the antigenicity of the parvovirus and its host specificity . Therefore, research into the vp2 gene is of great interest with regards to vaccine research and viral identification. In our study, analysis of the vp2 gene sequence showed that nt residues at positions 1,065 and 1,238 in MEV-DL strain differed from those of all MEV strains described previously. Furthermore, only a mutation at position 1,238 led to an Asp/Gly replacement in the VP2 protein, which is a new variant that has not been reported previously in MEV.
Different parvoviruses show differences in host tropism. For example, FPV can infect mink, but cannot infect canines ; likewise, CPV-2a can cause disease in cats, but cannot infect mink . FPV virus could bind with canine transferrin receptors and cause the subsequent infection of canine cells if the 93 and 323 amino acid residues of FPV VP2 protein changed to be the same as that of CPV . In contrast, changes to residues in the vicinity of residue 300 of the amino acid sequence can reduce the amount of adsorption of the virus into canine cells . In the late 1980 s and early 1990 s, the original CPV-2a and CPV-2b strains were replaced by the new CPV-2a and CPV-2b strains, which had resulted from a change in residue 297 of the amino acid sequence of the VP2 protein [40–43].
Sixteen amino acid residues are known to be variable in VP2 proteins of nine MEV strains listed in table 1, and these include in particular the amino acid residues in the vicinity of residue 300 (Table 1). This may lead to changes in MEV-specific adsorption by the host, and further studies should be developed to explain whether the mutation that affects position 413 (Asp/Gly replacement) would lead to changes in host adsorption of MEV and subsequent pathogenicity.
The VP2 protein region (between residues 267 and 498) forms the GH loop located between the βG and βH strands and is affected by the greatest variability among parvoviruses due to its exposure on the capsid surface . In the present study, a strain of MEV has been shown to have a difference at position 413 (Asp/Gly). Residue 413 was not exposed on the capsid surface, as forecasted by DNASTAR, and the change of this position led to alterations in the structure of the alpha, amphipathic and tum regions. This change would be likely to have a significant effect on the structure of the VP2 protein, and so influence its antigenicity and bionomics. This may help to explain the differences between other MEV strains and the variation in response to vaccines that are presently used in China.
In this paper, the vp2 nucleotide sequence of the MEV-DL strain has been shown have a homology of up to 99.9% with the ZYL-1 strain (accession number: GU272028). Phylogenetic analysis indicates that only minor changes have occurred between the MEV-DL and ZYL-1 strains. These results infer that the MEV-DL strain may originate from the ZYL-1 strain that was already present in China and, therefore, have little or no relationship to other MEV strains found elsewhere. Due to the high rate of nt substitution in MEV strains, it is necessary to isolate the current MEV strains to understand and prevent the disease caused by MEV.
Johnson RH, Siegl G, Gautschi M: Characteristics of feline panleukopenia virus strains enabling definitive classification as parvoviruses. Arch Gesamte Virusforsch. 1974, 46: 315-324. 10.1007/BF01240073.
Ohshima T, Mochizuki M: Evidence for Recombination Between Feline Panleukopenia Virus and Canine Parvovirus Type 2. J Vet Med Sci. 2009, 71 (pt4): 403-408. 10.1292/jvms.71.403.
Tingxiu Jiang, Houkun Pu, Long Wang: Preliminary report about Mink viral enteritis disease. Fur animals. 1981, 2: 224-
Brown TT, Paul PS, Mengeling WL: Response of conventionally raised weanling pigs to experimental infection with a virulent strain of porcine parvovirus. Am J Vet Res. 1980, 41: 1221-1224.
Jacoby RO, Johnson EA, Ball-Goodrich L, Smith AL, McKisic MD: Characterization of mouse parvovirus infection by in situ hybridization. J Virol. 1995, 69: 3915-3919.
Mengeling WL, Cutlip RC: Pathogenesis of in utero infection: experimental infection of five-week-old porcine fetuses with porcine parvovirus. Am J Vet Res. 1975, 36: 1173-1177.
Parrish CR, Kawaoka Y: The origins of new pandemic viruses: the acquisition of new host ranges by canine parvovirus and influenza A viruses. Annu Rev Microbiol. 2005, 59: 553-586. 10.1146/annurev.micro.59.030804.121059.
Reynolds H: Pathological changes in virus enteritis of mink. Can J Comp Med. 1970, 34: 155-163.
Uttenthal ÅS, Larsen E, Lund ME, Bloom T, S Alexandersen: Analysis of experimental mink enteritis virus infection in mink: in situ hybridization serology, and histopathology. J Virol. 1990, 64: 2768-2779.
Young NS, Brown KE: Parvovirus B19. N Engl J Med. 2004, 350: 586-597. 10.1056/NEJMra030840.
Schofield FW: Virus enteritis in mink. J Am Vet. 1949, 30: 651-654.
Wills GC: Notes on infectious enteritis of mink and its relationship to feline enteritis. Can J Comp Med Vet Sci. 1952, 16 (pt 12): 419-420.
Parrish CR: The emergence and evolution of canine parvovirus-an example of recen host range mutation. Semin Virol. 1994, 5: 121-132. 10.1006/smvy.1994.1013.
Siegl G, Bates RC, Berns KI, Carter BJ, Kelly DC, Kurstak E, Tattersall P: Characteristics and taxonomy of Parvoviridae. Intervirology. 1985, 23: 61-73. 10.1159/000149587.
Carlson J, Rushlow K, Maxwell I, Maxwell F, Winston S, Hahn W: Cloning and sequence of DNA encoding structural proteins of the autonomous parvovirus feline panleukopenia virus. J Virol. 1985, 55: 574-582.
Kariatsumari T, Horiuchi M, Hama E, Yaguchi K, Ishigurio N, Goto H, Shinagawa M: Construction and nucleotide sequence analysis of an infectious DNA clone of the autonomous parvovirus, mink enteritis virus. J Gen Virol. 1991, 72: 867-875. 10.1099/0022-1317-72-4-867.
Mattyn JC, Davidson BE, Studdert MJ: Nucleotide sequence of feline panleukopenia virus: comparison with canine parvovirus identifies host-specific differences. J Gen Virol. 1990, 71: 2747-2753. 10.1099/0022-1317-71-11-2747.
Parrish CR, Aquadro CF, Carmichael LE: Canine host range and a specific epitope map along with variant sequences in the capsid protein gene of canine parvovirus and related feline, mink and raccoon parvoviruses. Virology. 1988, 166: 293-307. 10.1016/0042-6822(88)90500-4.
Reed AP, Jones EV, Miller TJ: Nucleotide sequence and genome organization of Canine Parvovirus. J Virol. 1988, 62: 266-276.
Rhode SL: Nucleotide sequence of the coat protein gene of canine parvovirus. J Virol. 1985, 54: 630-633.
Tratschin JD, Mcmaster GK, Kronauer G, Siegl G: Canine parvovirus: relationship to wild-type and vaccine strains of feline panleukopenia virus and mink enteritis virus. J Gen Virol. 1982, 61: 33-41. 10.1099/0022-1317-61-1-33.
Truyen U, Parrish CR: The evolution and control of parvovirus host ranges. Semin Virol. 1995, 6: 311-317. 10.1006/smvy.1995.0037.
Jeoung SY, Kim D, Ahn SJ, Pak SI: Epidemiological observation on recent outbreaks of canine parvoviral enteritis in Korea. J Vet Clin. 2006, 23: 223-229.
Guo liang Luo, Jianjun Zhao, Hailing Zhang, Fengxue Wang, Xiuli Chai, Wei Wu, Yiqun Shao, Li Yi: Survey and analysis of main infectious disease of mink, fox and raccoon dog in China during period of 2006-2007. sepecial wild economic animal and plant research. 2008, 4: 71-74.
Mochizuki M, San Gabriel MC, Nakatani H, Yoshida M, Harasawa R: Comparison of polymerase chain reaction with virus isolation and haemagglutination assays for the detection of canine parvoviruses in faecal specimens. Res Vet Sci. 1993, 55: 60-63.
Jiahui Rao, Yuping Wang, Lianchang Lei, Bo Li, Jing Zuo: Isolation and identif ication of feline panleukopenia virus strain JL1205. Chin J Vet Sci. 2009, 29 (PT6): 716-720.
Xijun Yan, Wei Wu, Wang Fengxue, Xiqun Shao, Chuangfang Zhao, Lili Jiang: islation and identification of a mink enteritis parvovirus strain. Animal Husbandry & Veterinary Medicine. 2007, 39 (pt3): 25-26.
Krunajevic T: Experimental virus enteritis in mink: apathologic-anatomical and electron microscopical study. Acta Vet Scand Suppl. 1970, 30: 1-88.
Sheilah MF, Wobesert G: A technique for save multiple bleedings or intravenous injections in mink. Can Vet Jour. 1970, 11 (pt2): 33.-
Kumar S, Tamura K, Nei M: MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Beief Bioinform. 2004, 5: 150-163. 10.1093/bib/5.2.150.
Saitou N, Nei M: The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Bio Evol. 1987, 4: 406-425.
Higgins DG, Sharp PM: CLUSTAL: a package for performing multiple sequence alignment on a microcomputer. Gene. 1988, 73: 237-244. 10.1016/0378-1119(88)90330-7.
Shackelton LA, Parrish CR, Truyen U, Holmes EC: High rate of viral evolution associated with the emergence of canine parvovirus. Proc Natl Acad Sci USA. 2005, 102: 379-384. 10.1073/pnas.0406765102.
Haifang Liu, Chuangwei Hu, Zhijing Xie, Changpeng Ni, Yun Jia, Shijin Jiang, Xinxiao Zhang, Dubao Yang: Identification and vp1 gene analysis of a Blue Fox strain. Acta Theriologica sinica. 2009, 29 (1): 81-85.
Parrish CR, Gorham JR, Schwartz TM, Carmichael LE: Characterisation of antigenic variation among mink enteritis virus isolates. Am J Vet Res. 1984, 45: 2591-2599.
Karin H, Laura AS, Parrish CR, Edward C: Holmes: Phylogenetic analysis reveals the emergence, evolution and dispersal of carnivore parvoviruses. J Gen Virol. 2008, 89: (Pt 9): 2280-2289.
Truyen U, Parrish CR: Canine and feline host ranges of canine parvovirus and feline panleukopenia virus:distinct host cell tropisms of each virus in vitro and in vivo. J Virol. 1992, 66: 5399-5408.
Barker IK, Povey RC, Voigt DR: Response of mink, skunk, red fox and raccoon to inoculation with mink virus enteritis, feline panleukopenia and canine parvovirus and prevalence of antibody to parvovirus in wild carnivores in Ontario. J Comp Med. 1983, 47: 188-197.
Hueffer K, Parker JS, Weichert WS, Geisel RE, Sgro JY, Parrish CR: The natural host range shift and subsequent evolution of canine parvovirus resulted from virus-specific binding to the canine transferrin receptor. J Virol. 2003, 77: 1718-1726. 10.1128/JVI.77.3.1718-1726.2003.
Battilani M, Scagliarini A, Tisato E, Turilli C, Jacoboni I, Casadio R, Prosperi S: Analysis of canine parvovirus sequences from wolves and dogs isolated in Italy. J Gen Virol. 2001, 82: 1555-1560.
Martella V, Decaro N, Elia G, Buonavoglia C: Surveillance activity for canine parvovirus in Italy. J Vet Med. 2005, 52: 312-315. 10.1111/j.1439-0450.2005.00875.x.
Parrish CR, Aquadro CF, Strassheim ML, Evermann JF, Sgro JY, Mohammed HO: Rapid antigenic-type replacement and DNA sequence evolution of Canine parvovirus. J Virol. 1991, 65: 6544-6552.
Truyen U, Steinel A, Bruckner L, Lutz H, Möstl K: Distribution of antigen types of canine parvovirus in Switzerland, Austria and Germany. Schweiz Arch Tierheilkd. 2000, 142: 115-119.
Decaro N, Desario C, Parisi A, Martella V, Lorusso A, Miccolupo A, Mari V, Colaianni ML, Cavalli A, Di Trani L, Buonavoglia C: Genetic analysis of canine parvovirus type 2c. Virology. 2009, 385: 5-10. 10.1016/j.virol.2008.12.016.
This work was supported by Jilin five star animal health pharmaceutical company.
The authors declare that they have no competing interests.
L-CL conceived the study, JZ and J-HR planned the experimental aspects of the study, JZ, H-HX and L-MM performed the sequence studies. BL and JZ carried out the cell cultures, and JZ, BL and X-HC contributed to the discussion of all results in this work and drafted the manuscript. W-YH and Y-PW made an equal contribution as L-CL. All of the authors read and approved the final manuscript.
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Zuo, J., Rao, J., Xu, H. et al. Analysis of the vp2 gene sequence of a new mutated mink enteritis parvovirus strain in PR China. Virol J 7, 124 (2010). https://doi.org/10.1186/1743-422X-7-124