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Molecular characterization of feline panleukopenia virus isolated from mink and its pathogenesis in mink
Veterinary Microbiology 205 (2017) 92–98
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Original Research
Molecular characterization of feline panleukopenia virus isolated from mink and its pathogenesis in mink
MARK
Diao Fei-feia,b,1, Zhao Yong-fenga,b,1, Wang Jian-lia,b, Wei Xue-huaa,b, Cui Kaic, Liu Chuan-yia,b, ⁎ Guo Shou-yua,b, Shijin Jianga,b, Xie Zhi-jinga,b, a b c
Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Taian, Shandong, 271018, China College of Veterinary Medicine, Shandong Agricultural University, Taian, Shandong, 271018, China College of Animal Science and Veterinary Medicine, Qingdao Agricultural University, Qingdao, Shandong, 266109, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Feline panleukopenia virus Mink Animal experiment
Six feline panleukopenia viruses (FPV) were detected in the intestinal samples from the 176 mink collected in China during 2015 to 2016, named MEV-SD1, MEV-SD2, MEV-SD3, MEV-SD4, MEV-SD5 and MEV-SD6. The VP2 genes of the isolates shared 98.9%–100% identity with the reference sequences. The substitution of residue V300A in VP2 protein differentiates the isolates from the reference MEVs, and A300 is a characteristic of FPV. Furthermore, phylogenetic analysis of VP2 genes indicated that the six isolates were clustered into the same branch of all the reference FPVs. The NS1 genes of the isolates shared 98.2%–100% identity with the reference sequences. The NS1 genes of the six isolates and the three reference FPVs formed one unique evolutionary branch. To clarify the pathogenicity of the isolates, animal experiments were performed on healthy mink, using MEV-SD1. As a result, the morbidity of the inoculated animals was 100% and the mortality was as high as 38.9%. It was implied that the FPV infection caused a high morbidity and mortality in mink and the inoculation dose had an effect on pathogenicity of MEV-SD1 in mink.
1. Introduction Feline panleukopenia virus (FPV), mink enteritis virus (MEV) and canine parvovirus (CPV) are very closely related viruses, showing a genome identity of more than 98% (Mcmaster et al., 1981), and belong to the Protoparvovirus genus within the Parvovirinae subfamily of the Parvoviridae family of single-stranded DNA viruses. FPV-induced disease in cats has been known since the 1920s (Steinel et al., 2000) and can infect many species within the order Carnivora, including large and small cats, mink, raccoons and foxes (Steinel et al., 2001). MEV was first reported in southern Canada (Wills, 1952), and causes intestinal enteritis, myocarditis and lymphopenia in mink, especially in neonates and young mink (Uttenthal et al., 1990; Yuan et al., 2014). Whereas CPV-2 and its associated disease in dogs were recognized in the late 1970s and spread globally within a few months (Appel et al., 1979). CPV has undergone a series of evolutionary selections in nature which have resulted in the global distribution of new virus variants, including CPV-2a, CPV-2b and CPV-2c (Parrish et al., 1999; Buonavoglia et al., 2001 Buonavoglia et al., 2001). The viruses show different biological characteristics such as the pH dependence of haemagglutination (HA)
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and host cell specificity in vitro and in vivo. CPV can replicate in canine and feline cell lines, however, FPV and MEV do not replicate or replicate only poorly in canine cell lines such as A72 canine fibroma, MDCK and Cf2Th (Horiuchi et al., 1994). FPV, CPV and MEV are linear, single-stranded DNA viruses with an average genome size of approximately 5000 bp, containing two large open reading frames (ORFs), one in the 5′ half and the other in the 3′ half of the genome, with encoding two nonstructural proteins (NS1 and NS2) and two capsid proteins (VP1 and VP2), respectively (Kariatsumari et al., 1991; Langeveld et al., 1995; Christensen and Tattersall, 2002). Several amino acid residues in VP2 protein influence the antigenicity and host range of both CPV-2 and FPV and have subsequent effects on the viral surface structure (Chang et al., 1992; Truyen et al., 1995, 1996; Langeveld et al., 1995; Parrish, 1999; Buonavoglia et al., 2001; Govindasamy et al., 2003). The amino acids N93, A103, and N323 are critical for CPV-2 replication in dogs, whereas K80, N564, and A568 are critical for FPV replication in cats. Amino acid differences at positions 87, 300, and 305 of VP2 protein distinguish CPV-2a and CPV-2b from CPV-2. The feline host range of the new antigenic types CPV-2a and CPV-2b is most likely determined by the
Corresponding author at: College of Veterinary Medicine, Shandong Agricultural University, Taian, Shandong, 271018, China. E-mail address: [email protected] (X. Zhi-jing). The authors contributed equally to this work.
http://dx.doi.org/10.1016/j.vetmic.2017.05.017 Received 8 March 2017; Received in revised form 10 May 2017; Accepted 20 May 2017 0378-1135/ © 2017 Elsevier B.V. All rights reserved.
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were compiled and edited using the Lasergene sequence analysis software package (DNASTAR, Madison, WI, USA). The deduced amino acid sequences were also compared using DNASTAR software. Multiple sequence alignment was carried out by using CLUSTAL W. Phylogenetic trees were constructed using MEGA6.0 software by the neighborjoining method and the maximum composite likelihood model was used to calculate distances between sequences. Bootstrap values were calculated on 1000 replicates of the alignments.
amino acid changes M87L, A300G, and D305Y. The substitution of N426D differentiates CPV-2b from CPV-2a. In 2000, a new mutant with VP2 D426E, a strategic residue for the antigenicity of CPV, was reported in Italy and was designated CPV-2c (Buonavoglia et al., 2001). MEV vaccines have been used with some success to prevent further spread of the viral disease with significant decreases in morbidity and mortality (Sun et al., 2013). However, due to the genetic variability of MEV, these vaccines are becoming increasingly inadequate (Sun et al., 2013). The objectives of the study were to clarify molecular characterization of the parvoviruses isolated from the mink in China, and whether experimental oral gavage infection of the mink results in clinical signs and leads to virus shedding.
2.4. Mink pathogenesis experiments To determine the pathogenicity of the isolates, animal experiments were performed on 42 healthy mink (2 months of age) which were negative for parvovirus antigen and anti-parvovirus antibody. Fortytwo mink were divided into 7 groups on average. MEV-SD1 was titrated by 50% tissue culture infectious dose (TCID50) assay (Yuan and Parrish, 2000) in CRFK cells. The mink in group 1 to 6 were lightly anesthetized with ketamine chloride (Ketalar, Parke-Davis) and were inoculated via oral gavage with 106.0 TCID50, 105.0TCID50, 104.0 TCID50, 103.0 TCID50, 102.0 TCID50 and 101.0 TCID50, using MEV-SD1. Six mink in group 7 were inoculated via oral gavage with 0.9% NaCl solution, serving as the unchallenged group. The animals were housed individually and fed twice daily on a commercial meat-based diet, and water was freely available at all times. The animal experiments were performed in accordance with regulatory standards and guidelines approved by the Shandong Agricultural University’s Animal Care and Use Committee, and the approved NO. is SDAUA-2015-002. From postinfection (p.i.) onwards, clinical signs of the mink were monitored and scored daily for 15 days or until the inoculated mink died from MEV-SD1 infection. To determine virus shedding, rectal swabs were collected from the animals for 15 days and were confirmed by PCR as above. The tissue samples were collected from the mink either killed by MEV-SD1 infection or euthanized on days 15 after MEVSD1 inoculation, including cerebrum, cerebellum, tonsil, retropharyngeal lymph node, thymus, lung, myocardium, bone marrow, liver, spleen, kidney, bladder, mesenteric lymph node, ileum, jejunum, colon and rectum. The samples were rapidly immersed in 10% neutral formalin buffer to prevent autolysis, and then processed into paraffin, sectioned at 4 um using the microtome Leica RM2235 (Leica Microsystems Ltd.), and stained with hematoxylin and eosin (HE) for the detection of histological lesions by light microscopy. Serum samples were collected on day 15 p.i. for serological testing using HI (Yuan and Parrish, 2000). The LD50 of MEV-SD1 in mink was titrated using Reed and Muench.
2. Material and methods 2.1. Samples During 2015–2016, the intestinal samples from 176 mink affected by enteritis at the age of 90–110 days were collected from seven mink farms throughout an east coast of China. The 176 mink were all vaccinated at the age of 50–60 days, using licensed inactivated cellderived MEV vaccine in China. The samples were transported on ice to the laboratory and stored frozen at −20 °C until further use. The study was approved by the Shandong Agricultural University’s Animal Care and Use Committee and also complied with the European Union (EU) Animal Welfare legislation. 2.2. Virus isolation DNA was extracted from the 176 intestinal samples by using the EasyPure Genomic DNA Kit (TransGen Biotech, China). The samples were tested by PCR using the primers for FPV-like parvovirus, VP2-P1: 5′- CTTTGCCTCAATCTGAAGGAG-3′, VP2-P2: 5′- GAATTGGATTCCAAGTATGAG-3′. The PCR conditions were available upon request. The 6 of the 176 intestinal samples were positive for VP2 gene by PCR and were homogenized in phosphate-buffered saline solution supplemented with 2000 unit/ml penicillin and 2000 mg/ml streptomycin, immediately centrifuged at 5000 × g for 5 min to precipitate debris. Subsequently, the intestinal suspensions were filtered through a 0.22μm Millipore filter (Millipore, Bedford, MA, U.S.A.) for virus isolation in Crandell feline kidney (CRFK) cells. The cells were cultured at 37 °C in a humidified 5% CO2 incubator in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal calf serum. A typical cytopathic effect of parvovirus infection was observed in the cell monolayer on day 4 postinoculation.
3. Results 2.3. Nucleotide sequencing and phylogenetic analysis 3.1. Six parvoviruses isolated from the mink DNA was extracted from the 6 intestinal samples by using the EasyPure Genomic DNA Kit (TransGen Biotech, China). PCR was used to amplify a 1755 bp segment of VP2 gene using primer pair VP2-F 5′ATGAGTGATGGAGCAGTTCAACCAG-3′ and VP2-R 5′-TTAATATAATTTTCTAGGTGCTAG-3′, and a 2007 bp segment of NS1 gene using primer pair NSI-F 5′-ATGTCTGGCAACCAGTATACTGAG-3′ and NS1-R 5′-TTAATCCAAGTCGTCTCGAAAATC-3′. PCR conditions used were available upon request. The PCR products were extracted from agarose gels, using a GenScript QuickClean gel extraction kit (GenScript, Piscataway, NJ, USA), and sequencing was performed in Sangon Biological (Shanghai) Co., Ltd (Shanghai, China). The sequences of the VP2 and NS1 genes of the 6 isolates were submitted to GenBank, and were assigned GenBank accession number individually, accession numbers KY094112 to KY094117 and KY094120 to KY094125. BLAST analyses (http://blast.ncbi.nlm.nih.gov/Blast.cgi) were used on each sequence to identify related reference viruses. To investigate more precisely the genotype and genetic origin, the DNA sequences
In the study, six parvoviruses were isolated from the mink exhibiting diarrhea disease, named MEV-SD1, MEV-SD2, MEV-SD3, MEV-SD4, MEV-SD5 and MEV-SD6, respectively. 3.2. Molecular characterization of the six parvoviruses The VP2 genes of the 6 isolates showed 99.8%–100% identity at the nucleotide level, which shared 98.9%–100% identity with the reference strains. Phylogenetic analysis of VP2 genes revealed that the isolates and the reference were clustered into 4 branches (Fig. 1). The isolates were clustered into one evolutionary branch and shared the identical branch with the reference FPVs. To identify the molecular characteristics of the isolates in detail, the deduced amino acid sequences of the VP2 proteins of the isolates were analyzed and aligned using DNASTAR software, and had 99.3%–100% similarity, which shared 98.1%-100% similarity with the reference strains. The mutations were shown in Table 1. The NS1 genes of the six isolates shared 99.9%–100% at the 93
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Fig. 1. Phylogenetic tree of the VP2 gene sequences of the 37 parvoviruses. The tree was constructed with MEGA 6.0 using neighbor-joining method. The origin of the reference sequences was also stated in Table 1.
weak, and developed diarrhea. By the fourth day after inoculation, the clinical signs were observed in the thirty-six inoculated mink. Following the appearance of diarrhea, the feces contained large quantities of mucus intestinal casts were seen frequently in the droppings. Thereafter, the feces consisted mostly of yellowish green watery fluid, even bloody fluid. Some of the animals died after inoculation. The survived animals were severely dehydrated and debilitated, but resumed eating and achieved complete clinical recovery. The serum samples that collected from the survived mink on day 15 p.i. were antibody-positive for MEV-SD1, HI titre 1:128-1:512. The control mink
nucleotide level, which showed 98.2%–100% identity with the reference strains. The NS1 proteins of the six isolates shared 99.6%–100% similarity, which had 98.1%–100% similarity with the reference strains. Phylogenetic analysis of NS1 genes indicated that the 6 NS1 genes and the three reference FPV NS1 genes formed one unique evolutionary branch (Fig. 2). 3.3. Pathogenesis of MEV-SD1 in mink Twenty hours p.i., some of the inoculated mink were anorectic and 94
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Table 1 Amino acid sequence variations in the VP2 proteins of FPV and the reference viruses.
lymphocytopenia and necrosis in spleen, lymphocytopenia and reticulocytosis in mesenteric lymph nodes, intestinal vilus breakageand inflammatory cells infiltration of the intestinal lamina propria (Fig. 3).
showed no clinic signs, virus shedding and seroconversion. The major differences in clinical symptoms of the mink infected with the six different virus titers were shown in Table 2. The mean time to loss of appetite (hours), the mean time to show watery diarrhea (days) and the mean time to death after showing clinical signs (hours) were shorter and shorter with the increase of the inoculation doses. Virus shedding from the inoculated mink was confirmed by PCR from on days 1–13 p.i. The morbidity rate of the inoculated animals was 100%, the mortality rate 38.9%. The LD50 of MEV-SD1 in mink was 104.75 TCID50. Histologic lesions were found in the mink that died from infection, including
4. Discussion The diseases caused by FPV were known to occur only in cats or raccoons until the mid-1940s, when a similar disease with a high mortality was observed in mink, named MEV, which was classified into three antigenic types, MEV type 1 (MEV-1), MEV type 2 (MEV-2) and 95
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Fig. 2. Phylogenetic tree of the NS1 gene sequences of the 32 parvoviruses. The tree was constructed with MEGA 6.0 using neighbor-joining method.
antigenically and genetically very close to FPV (Appel et al., 1979; Parrish et al., 1999; Buonavoglia et al., 2001). FPV and CPV are classified as host-range variants of FPV. In the study, six parvoviruses were isolated from the mink suffering from enteritis in China. The rest of the 176 sample were negative for parvovirus, but positive for Escherichia coli or salmonella, which also cause diarrhea disease. Residues 80, 93, 103, 323, 564 and 568 of VP2 protein affect the antigenic properties of parvoviruses isolated from carnivores (Parrish, 1999), and were conserved in the isolates. Four mutations in VP2 protein at residues 87, 300, and 305 have been shown previously to alter host range, TfR binding, and antigenic structure in parvoviruses (Stucker et al., 2012), whereas residues 87, 101 and 305 were also conserved in the isolates. Residue 300 in VP2 protein exhibits extensive amino acid variation among the carnivore parvoviruses and the VP2 300 loop region (299, 300 and 301 residues) of the threefold spike is a key determinant of host range via control of TfR binding (Jing et al., 2010; Wang et al., 2012). The capsid regions are also highly antigenic, and serves as the target of many neutralizing antibodies (Carlson et al., 1985), A holds the position in CPV-2, G in CPV-2a and CPV-2b, D in CPV-2c, and V in RRPV. BFPV from a blue fox was antigenically similar
Table 2 Clinical symptoms among mink pathogenesis experiments of MEV-SD1. Groups No. ID MTA MTD MTH M/M RI
6 106.0 22 ± 4 1.5 ± 0.5 24 ± 7 100/100 0
6 105.0 22 ± 4 1.5 ± 0.5 24 ± 7 100/100 0
6 104.0 26 ± 6 2 ± 0.5 36 ± 5 100/33.3 66.7
6 103.0 72 ± 4 3 ± 1 / 100/0 100
6 102.0 72 ± 4 3.5 ± 1 / 100/0 100
6 101.0 / 4 ± 1 / 100/0 100
6 C / / / / /
No., the number of animals. C, Control. ID, Inoculated doses (TCID50). MTA, Mean time loss of appetite (hours). MTD, Mean time to show watery diarrhea (days). MTH, Mean time to death after showing clinical signs (hours). M/M, Morbidity/mortality (%). RI, Recovery rate after MEV-SD1 infection (%)./, no found.
MEV type 3, and the antigenic differences between FPV/MEV-1 and MEV-2 were resulted from a mutation of VP2 amino acid residue 300 Ala to Val (Parrish et al., 1984). In the late 1970s, CPV-2 rapidly spread worldwide and initially killed thousands of unprotected dogs, and was 96
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Fig. 3. Histopathologic appearance of tissue of the experimental animals. (A) H & E-stained intestinal tissue taken on day 2 p.i. from a dead mink infected oral gavage with MEV-SD1, characterized by inflammatory cells infiltration of the intestinal lamina propria and intestinal vilus breakage. (B) H & E-stained intestina tissue from a mink inoculated oral gavage with 0.9% NaCl solution. (C) H & E-stained spleen tissue taken on day 2 p.i. from a dead mink infected oral gavage with MEV-SD1, presenting lymphocytopenia and necrosis. (D) H & E-stained spleen tissue from a mink inoculated oral gavage with 0.9% NaCl solution. Original magnification was × 200 for all images. HE stain.
Competing interests statement
to FPV but had a natural mutation of VP2 residue A300P, classified as FPV-like. The A300P mutation likely contributed to the adaptation of BFPV to blue foxes (Chen et al., 2011). RDPV from the raccoon dog and MCPV from the masked civet were antigenically similar to CPV-2a, but had a change at G300S, classified as CPV-2a-like (Chen et al., 2011). V300I were found in MEV Shandong4 (GU392245) and MEV LYT-2 (FJ712221). The substitution of residue V300A in VP2 protein differentiates the isolates from the reference MEVs, and A300 is a characteristic of FPV. The mutation might affect the pathogenicity of the parvoviruses in mink. Mutation of residue 232 of MEV VP2 didn’t decrease the released viral genome copies and did not significantly affect capsid assembly and viral genome packaging (Mao et al., 2016). Furthermore, phylogenetic analysis based on VP2 genes indicated that the isolates located in the same branch of all the reference strains of FPV. The NS1 genes of the six isolates and the three reference FPVs formed one unique evolutionary branch. So the study demonstrated that the isolates were FPV. Although host factors, such as age, immune status, and genotype, play a significant role in determining the outcome of parvovirus infection, the variability in disease severity is indisputably regulated by viral determinants (Jing et al., 2010). Experimental infection showed that FPV infection caused a high morbidity and mortality in mink, and the inoculation dose had an effect on pathogenicity of MEVSD1 in mink. In China, dogs and/or cats were usually fed in many mink farms. Mink may therefore act as an intermediate host providing parvoviruses the possibility of transmitting between divergent hosts (Wang et al., 2012). The etiology and epidemiological surveillance of the parvovirus should be strengthened for mink industry.
The authors declare that they have no competing financial interests. Acknowledgements We thank the staff of College of Veterinary Medicine, Shandong Agricultural University, in Taian, Shandong Province, China. And we also thank the staff of Fur Animal Disease Inspection Institute, Weifang, Shandong, China. This study was funded by Shandong Modern Agricultural Technology & Industry System (SDAIT-21-07). References Appel, M.J., Scott, F.W., Carmichael, L.E., 1979. Isolation and immunisation studies of a canine parco-like virus from dogs with haemorrhagic enteritis. Vet. Rec. 105, 156–159. Buonavoglia, C., Martella, V., Pratelli, A., Tempesta, M., Cavalli, A., Buonavoglia, D., Bozzo, G., Elia, G., Decaro, N., Carmichael, L., 2001. Evidence for evolution of canine parvovirus type 2 in Italy. J. Gen. Virol. 82 (Pt 12), 3021–3025. Carlson, J., Rushlow, K., Maxwell, I., Maxwell, F., Winston, S., Hahn, W., 1985. Cloning and sequence of DNA encoding structural proteins of the autonomous parvovirus feline panleukopenia virus. J. Virol. 55, 574–582. Chang, S.F., Sgro, J.Y., Parrish, C.R., 1992. Multiple amino acids in the capsid structure of canine parvovirus coordinately determine the canine host range and specific antigenic and hemagglutination properties. J. Virol. 66, 6858–6867. Chen, X.Y., Xie, Z.J., Zhao, Z.P., Jiang, S.J., Zhao, H.K., Zhu, Y.L., Zhang, X.X., 2011. Genetic diversity of parvovirus isolates from dogs and wild animals in China. J. Wildl. Dis. 47, 1036–1039. Christensen, J., Tattersall, P., 2002. Parvovirus initiator protein NS1 and RPA coordinate replication fork progression in a reconstituted DNA replication system. J. Virol. 76, 6518–6531. Govindasamy, L., Hueffer, K., Parrish, C.R., Agbandje-Mckenna, M., 2003. Structures of host range-controlling regions of the capsids of canine and feline parvoviruses and mutants. J. Virol. 77, 12211–12221.
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carnivores. J. Wildl. Dis. 37, 594–607. Stucker, K.M., Pagan, I., Cifuente, J.O., Kaelber, J.T., Lillie, T.D., Hafenstein, S., Holmes, E.C., Parrish, C.R., 2012. The role of evolutionary intermediates in the host adaptation of canine parvovirus. J. Virol. 86, 1514–1521. Sun, J.Z., Wang, J., Yuan, D., Wang, S., Li, Z., Yi, B., Mao, Y., Hou, Q., Liu, W., 2013. Cellular microRNA miR-181b inhibits replication of mink enteritis virus by repression of non-structural protein 1 translation. PLoS One 8, e81515. Truyen, U., Gruenberg, A., Chang, S.F., Obermaier, B., Veijalainen, P., Parrish, C.R., 1995. Evolution of the feline-subgroup parvoviruses and the control of canine host range in vivo. J. Virol. 69, 4702–4710. Truyen, U., Evermann, J.F., Vieler, E., Parrish, C.R., 1996. Evolution of canine parvovirus involved loss and gain of feline host range. Virology 215, 186–189. Uttenthal, A., Larsen, S., Lund, E., Bloom, M.E., Storgård, T., Alexandersen, S., 1990. Analysis of experimental mink enteritis virus infection in mink In situ hybridization, serology, and histopathology. J. Virol. 64, 2768–2779. Wang, J., Cheng, S., Yi, L., Cheng, Y., Yang, S., Xu, H., Zhao, H., Yan, X., Wu, H., 2012. Evidence for natural recombination between mink enteritis virus and canine parvovirus. Virol. J. 9, 252. Wills, C.G., 1952. Notes on infectious enteritis of mink and its relationship to feline enteritis. Can. J. Comp. Med. Vet. Sci. 16, 419–420. Yuan, W., Parrish, C.R., 2000. Comparison of two single-chain antibodies that neutralize canine parvovirus: analysis of an antibody-combining site and mechanisms of neutralization. Virology 269, 471–480. Yuan, D., Wang, J., Li, Z., Mao, Y., Sun, J.Z., Xi, J., Wang, S., Hou, Q., Yi, B., Liu, W., 2014. Establishment of a rescue system for an autonomous Parvovirus mink enteritis virus. Virus Res. 183, 1–5.
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Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Original Research
Molecular characterization of feline panleukopenia virus isolated from mink and its pathogenesis in mink
MARK
Diao Fei-feia,b,1, Zhao Yong-fenga,b,1, Wang Jian-lia,b, Wei Xue-huaa,b, Cui Kaic, Liu Chuan-yia,b, ⁎ Guo Shou-yua,b, Shijin Jianga,b, Xie Zhi-jinga,b, a b c
Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Taian, Shandong, 271018, China College of Veterinary Medicine, Shandong Agricultural University, Taian, Shandong, 271018, China College of Animal Science and Veterinary Medicine, Qingdao Agricultural University, Qingdao, Shandong, 266109, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Feline panleukopenia virus Mink Animal experiment
Six feline panleukopenia viruses (FPV) were detected in the intestinal samples from the 176 mink collected in China during 2015 to 2016, named MEV-SD1, MEV-SD2, MEV-SD3, MEV-SD4, MEV-SD5 and MEV-SD6. The VP2 genes of the isolates shared 98.9%–100% identity with the reference sequences. The substitution of residue V300A in VP2 protein differentiates the isolates from the reference MEVs, and A300 is a characteristic of FPV. Furthermore, phylogenetic analysis of VP2 genes indicated that the six isolates were clustered into the same branch of all the reference FPVs. The NS1 genes of the isolates shared 98.2%–100% identity with the reference sequences. The NS1 genes of the six isolates and the three reference FPVs formed one unique evolutionary branch. To clarify the pathogenicity of the isolates, animal experiments were performed on healthy mink, using MEV-SD1. As a result, the morbidity of the inoculated animals was 100% and the mortality was as high as 38.9%. It was implied that the FPV infection caused a high morbidity and mortality in mink and the inoculation dose had an effect on pathogenicity of MEV-SD1 in mink.
1. Introduction Feline panleukopenia virus (FPV), mink enteritis virus (MEV) and canine parvovirus (CPV) are very closely related viruses, showing a genome identity of more than 98% (Mcmaster et al., 1981), and belong to the Protoparvovirus genus within the Parvovirinae subfamily of the Parvoviridae family of single-stranded DNA viruses. FPV-induced disease in cats has been known since the 1920s (Steinel et al., 2000) and can infect many species within the order Carnivora, including large and small cats, mink, raccoons and foxes (Steinel et al., 2001). MEV was first reported in southern Canada (Wills, 1952), and causes intestinal enteritis, myocarditis and lymphopenia in mink, especially in neonates and young mink (Uttenthal et al., 1990; Yuan et al., 2014). Whereas CPV-2 and its associated disease in dogs were recognized in the late 1970s and spread globally within a few months (Appel et al., 1979). CPV has undergone a series of evolutionary selections in nature which have resulted in the global distribution of new virus variants, including CPV-2a, CPV-2b and CPV-2c (Parrish et al., 1999; Buonavoglia et al., 2001 Buonavoglia et al., 2001). The viruses show different biological characteristics such as the pH dependence of haemagglutination (HA)
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and host cell specificity in vitro and in vivo. CPV can replicate in canine and feline cell lines, however, FPV and MEV do not replicate or replicate only poorly in canine cell lines such as A72 canine fibroma, MDCK and Cf2Th (Horiuchi et al., 1994). FPV, CPV and MEV are linear, single-stranded DNA viruses with an average genome size of approximately 5000 bp, containing two large open reading frames (ORFs), one in the 5′ half and the other in the 3′ half of the genome, with encoding two nonstructural proteins (NS1 and NS2) and two capsid proteins (VP1 and VP2), respectively (Kariatsumari et al., 1991; Langeveld et al., 1995; Christensen and Tattersall, 2002). Several amino acid residues in VP2 protein influence the antigenicity and host range of both CPV-2 and FPV and have subsequent effects on the viral surface structure (Chang et al., 1992; Truyen et al., 1995, 1996; Langeveld et al., 1995; Parrish, 1999; Buonavoglia et al., 2001; Govindasamy et al., 2003). The amino acids N93, A103, and N323 are critical for CPV-2 replication in dogs, whereas K80, N564, and A568 are critical for FPV replication in cats. Amino acid differences at positions 87, 300, and 305 of VP2 protein distinguish CPV-2a and CPV-2b from CPV-2. The feline host range of the new antigenic types CPV-2a and CPV-2b is most likely determined by the
Corresponding author at: College of Veterinary Medicine, Shandong Agricultural University, Taian, Shandong, 271018, China. E-mail address: [email protected] (X. Zhi-jing). The authors contributed equally to this work.
http://dx.doi.org/10.1016/j.vetmic.2017.05.017 Received 8 March 2017; Received in revised form 10 May 2017; Accepted 20 May 2017 0378-1135/ © 2017 Elsevier B.V. All rights reserved.
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were compiled and edited using the Lasergene sequence analysis software package (DNASTAR, Madison, WI, USA). The deduced amino acid sequences were also compared using DNASTAR software. Multiple sequence alignment was carried out by using CLUSTAL W. Phylogenetic trees were constructed using MEGA6.0 software by the neighborjoining method and the maximum composite likelihood model was used to calculate distances between sequences. Bootstrap values were calculated on 1000 replicates of the alignments.
amino acid changes M87L, A300G, and D305Y. The substitution of N426D differentiates CPV-2b from CPV-2a. In 2000, a new mutant with VP2 D426E, a strategic residue for the antigenicity of CPV, was reported in Italy and was designated CPV-2c (Buonavoglia et al., 2001). MEV vaccines have been used with some success to prevent further spread of the viral disease with significant decreases in morbidity and mortality (Sun et al., 2013). However, due to the genetic variability of MEV, these vaccines are becoming increasingly inadequate (Sun et al., 2013). The objectives of the study were to clarify molecular characterization of the parvoviruses isolated from the mink in China, and whether experimental oral gavage infection of the mink results in clinical signs and leads to virus shedding.
2.4. Mink pathogenesis experiments To determine the pathogenicity of the isolates, animal experiments were performed on 42 healthy mink (2 months of age) which were negative for parvovirus antigen and anti-parvovirus antibody. Fortytwo mink were divided into 7 groups on average. MEV-SD1 was titrated by 50% tissue culture infectious dose (TCID50) assay (Yuan and Parrish, 2000) in CRFK cells. The mink in group 1 to 6 were lightly anesthetized with ketamine chloride (Ketalar, Parke-Davis) and were inoculated via oral gavage with 106.0 TCID50, 105.0TCID50, 104.0 TCID50, 103.0 TCID50, 102.0 TCID50 and 101.0 TCID50, using MEV-SD1. Six mink in group 7 were inoculated via oral gavage with 0.9% NaCl solution, serving as the unchallenged group. The animals were housed individually and fed twice daily on a commercial meat-based diet, and water was freely available at all times. The animal experiments were performed in accordance with regulatory standards and guidelines approved by the Shandong Agricultural University’s Animal Care and Use Committee, and the approved NO. is SDAUA-2015-002. From postinfection (p.i.) onwards, clinical signs of the mink were monitored and scored daily for 15 days or until the inoculated mink died from MEV-SD1 infection. To determine virus shedding, rectal swabs were collected from the animals for 15 days and were confirmed by PCR as above. The tissue samples were collected from the mink either killed by MEV-SD1 infection or euthanized on days 15 after MEVSD1 inoculation, including cerebrum, cerebellum, tonsil, retropharyngeal lymph node, thymus, lung, myocardium, bone marrow, liver, spleen, kidney, bladder, mesenteric lymph node, ileum, jejunum, colon and rectum. The samples were rapidly immersed in 10% neutral formalin buffer to prevent autolysis, and then processed into paraffin, sectioned at 4 um using the microtome Leica RM2235 (Leica Microsystems Ltd.), and stained with hematoxylin and eosin (HE) for the detection of histological lesions by light microscopy. Serum samples were collected on day 15 p.i. for serological testing using HI (Yuan and Parrish, 2000). The LD50 of MEV-SD1 in mink was titrated using Reed and Muench.
2. Material and methods 2.1. Samples During 2015–2016, the intestinal samples from 176 mink affected by enteritis at the age of 90–110 days were collected from seven mink farms throughout an east coast of China. The 176 mink were all vaccinated at the age of 50–60 days, using licensed inactivated cellderived MEV vaccine in China. The samples were transported on ice to the laboratory and stored frozen at −20 °C until further use. The study was approved by the Shandong Agricultural University’s Animal Care and Use Committee and also complied with the European Union (EU) Animal Welfare legislation. 2.2. Virus isolation DNA was extracted from the 176 intestinal samples by using the EasyPure Genomic DNA Kit (TransGen Biotech, China). The samples were tested by PCR using the primers for FPV-like parvovirus, VP2-P1: 5′- CTTTGCCTCAATCTGAAGGAG-3′, VP2-P2: 5′- GAATTGGATTCCAAGTATGAG-3′. The PCR conditions were available upon request. The 6 of the 176 intestinal samples were positive for VP2 gene by PCR and were homogenized in phosphate-buffered saline solution supplemented with 2000 unit/ml penicillin and 2000 mg/ml streptomycin, immediately centrifuged at 5000 × g for 5 min to precipitate debris. Subsequently, the intestinal suspensions were filtered through a 0.22μm Millipore filter (Millipore, Bedford, MA, U.S.A.) for virus isolation in Crandell feline kidney (CRFK) cells. The cells were cultured at 37 °C in a humidified 5% CO2 incubator in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal calf serum. A typical cytopathic effect of parvovirus infection was observed in the cell monolayer on day 4 postinoculation.
3. Results 2.3. Nucleotide sequencing and phylogenetic analysis 3.1. Six parvoviruses isolated from the mink DNA was extracted from the 6 intestinal samples by using the EasyPure Genomic DNA Kit (TransGen Biotech, China). PCR was used to amplify a 1755 bp segment of VP2 gene using primer pair VP2-F 5′ATGAGTGATGGAGCAGTTCAACCAG-3′ and VP2-R 5′-TTAATATAATTTTCTAGGTGCTAG-3′, and a 2007 bp segment of NS1 gene using primer pair NSI-F 5′-ATGTCTGGCAACCAGTATACTGAG-3′ and NS1-R 5′-TTAATCCAAGTCGTCTCGAAAATC-3′. PCR conditions used were available upon request. The PCR products were extracted from agarose gels, using a GenScript QuickClean gel extraction kit (GenScript, Piscataway, NJ, USA), and sequencing was performed in Sangon Biological (Shanghai) Co., Ltd (Shanghai, China). The sequences of the VP2 and NS1 genes of the 6 isolates were submitted to GenBank, and were assigned GenBank accession number individually, accession numbers KY094112 to KY094117 and KY094120 to KY094125. BLAST analyses (http://blast.ncbi.nlm.nih.gov/Blast.cgi) were used on each sequence to identify related reference viruses. To investigate more precisely the genotype and genetic origin, the DNA sequences
In the study, six parvoviruses were isolated from the mink exhibiting diarrhea disease, named MEV-SD1, MEV-SD2, MEV-SD3, MEV-SD4, MEV-SD5 and MEV-SD6, respectively. 3.2. Molecular characterization of the six parvoviruses The VP2 genes of the 6 isolates showed 99.8%–100% identity at the nucleotide level, which shared 98.9%–100% identity with the reference strains. Phylogenetic analysis of VP2 genes revealed that the isolates and the reference were clustered into 4 branches (Fig. 1). The isolates were clustered into one evolutionary branch and shared the identical branch with the reference FPVs. To identify the molecular characteristics of the isolates in detail, the deduced amino acid sequences of the VP2 proteins of the isolates were analyzed and aligned using DNASTAR software, and had 99.3%–100% similarity, which shared 98.1%-100% similarity with the reference strains. The mutations were shown in Table 1. The NS1 genes of the six isolates shared 99.9%–100% at the 93
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Fig. 1. Phylogenetic tree of the VP2 gene sequences of the 37 parvoviruses. The tree was constructed with MEGA 6.0 using neighbor-joining method. The origin of the reference sequences was also stated in Table 1.
weak, and developed diarrhea. By the fourth day after inoculation, the clinical signs were observed in the thirty-six inoculated mink. Following the appearance of diarrhea, the feces contained large quantities of mucus intestinal casts were seen frequently in the droppings. Thereafter, the feces consisted mostly of yellowish green watery fluid, even bloody fluid. Some of the animals died after inoculation. The survived animals were severely dehydrated and debilitated, but resumed eating and achieved complete clinical recovery. The serum samples that collected from the survived mink on day 15 p.i. were antibody-positive for MEV-SD1, HI titre 1:128-1:512. The control mink
nucleotide level, which showed 98.2%–100% identity with the reference strains. The NS1 proteins of the six isolates shared 99.6%–100% similarity, which had 98.1%–100% similarity with the reference strains. Phylogenetic analysis of NS1 genes indicated that the 6 NS1 genes and the three reference FPV NS1 genes formed one unique evolutionary branch (Fig. 2). 3.3. Pathogenesis of MEV-SD1 in mink Twenty hours p.i., some of the inoculated mink were anorectic and 94
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Table 1 Amino acid sequence variations in the VP2 proteins of FPV and the reference viruses.
lymphocytopenia and necrosis in spleen, lymphocytopenia and reticulocytosis in mesenteric lymph nodes, intestinal vilus breakageand inflammatory cells infiltration of the intestinal lamina propria (Fig. 3).
showed no clinic signs, virus shedding and seroconversion. The major differences in clinical symptoms of the mink infected with the six different virus titers were shown in Table 2. The mean time to loss of appetite (hours), the mean time to show watery diarrhea (days) and the mean time to death after showing clinical signs (hours) were shorter and shorter with the increase of the inoculation doses. Virus shedding from the inoculated mink was confirmed by PCR from on days 1–13 p.i. The morbidity rate of the inoculated animals was 100%, the mortality rate 38.9%. The LD50 of MEV-SD1 in mink was 104.75 TCID50. Histologic lesions were found in the mink that died from infection, including
4. Discussion The diseases caused by FPV were known to occur only in cats or raccoons until the mid-1940s, when a similar disease with a high mortality was observed in mink, named MEV, which was classified into three antigenic types, MEV type 1 (MEV-1), MEV type 2 (MEV-2) and 95
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Fig. 2. Phylogenetic tree of the NS1 gene sequences of the 32 parvoviruses. The tree was constructed with MEGA 6.0 using neighbor-joining method.
antigenically and genetically very close to FPV (Appel et al., 1979; Parrish et al., 1999; Buonavoglia et al., 2001). FPV and CPV are classified as host-range variants of FPV. In the study, six parvoviruses were isolated from the mink suffering from enteritis in China. The rest of the 176 sample were negative for parvovirus, but positive for Escherichia coli or salmonella, which also cause diarrhea disease. Residues 80, 93, 103, 323, 564 and 568 of VP2 protein affect the antigenic properties of parvoviruses isolated from carnivores (Parrish, 1999), and were conserved in the isolates. Four mutations in VP2 protein at residues 87, 300, and 305 have been shown previously to alter host range, TfR binding, and antigenic structure in parvoviruses (Stucker et al., 2012), whereas residues 87, 101 and 305 were also conserved in the isolates. Residue 300 in VP2 protein exhibits extensive amino acid variation among the carnivore parvoviruses and the VP2 300 loop region (299, 300 and 301 residues) of the threefold spike is a key determinant of host range via control of TfR binding (Jing et al., 2010; Wang et al., 2012). The capsid regions are also highly antigenic, and serves as the target of many neutralizing antibodies (Carlson et al., 1985), A holds the position in CPV-2, G in CPV-2a and CPV-2b, D in CPV-2c, and V in RRPV. BFPV from a blue fox was antigenically similar
Table 2 Clinical symptoms among mink pathogenesis experiments of MEV-SD1. Groups No. ID MTA MTD MTH M/M RI
6 106.0 22 ± 4 1.5 ± 0.5 24 ± 7 100/100 0
6 105.0 22 ± 4 1.5 ± 0.5 24 ± 7 100/100 0
6 104.0 26 ± 6 2 ± 0.5 36 ± 5 100/33.3 66.7
6 103.0 72 ± 4 3 ± 1 / 100/0 100
6 102.0 72 ± 4 3.5 ± 1 / 100/0 100
6 101.0 / 4 ± 1 / 100/0 100
6 C / / / / /
No., the number of animals. C, Control. ID, Inoculated doses (TCID50). MTA, Mean time loss of appetite (hours). MTD, Mean time to show watery diarrhea (days). MTH, Mean time to death after showing clinical signs (hours). M/M, Morbidity/mortality (%). RI, Recovery rate after MEV-SD1 infection (%)./, no found.
MEV type 3, and the antigenic differences between FPV/MEV-1 and MEV-2 were resulted from a mutation of VP2 amino acid residue 300 Ala to Val (Parrish et al., 1984). In the late 1970s, CPV-2 rapidly spread worldwide and initially killed thousands of unprotected dogs, and was 96
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Fig. 3. Histopathologic appearance of tissue of the experimental animals. (A) H & E-stained intestinal tissue taken on day 2 p.i. from a dead mink infected oral gavage with MEV-SD1, characterized by inflammatory cells infiltration of the intestinal lamina propria and intestinal vilus breakage. (B) H & E-stained intestina tissue from a mink inoculated oral gavage with 0.9% NaCl solution. (C) H & E-stained spleen tissue taken on day 2 p.i. from a dead mink infected oral gavage with MEV-SD1, presenting lymphocytopenia and necrosis. (D) H & E-stained spleen tissue from a mink inoculated oral gavage with 0.9% NaCl solution. Original magnification was × 200 for all images. HE stain.
Competing interests statement
to FPV but had a natural mutation of VP2 residue A300P, classified as FPV-like. The A300P mutation likely contributed to the adaptation of BFPV to blue foxes (Chen et al., 2011). RDPV from the raccoon dog and MCPV from the masked civet were antigenically similar to CPV-2a, but had a change at G300S, classified as CPV-2a-like (Chen et al., 2011). V300I were found in MEV Shandong4 (GU392245) and MEV LYT-2 (FJ712221). The substitution of residue V300A in VP2 protein differentiates the isolates from the reference MEVs, and A300 is a characteristic of FPV. The mutation might affect the pathogenicity of the parvoviruses in mink. Mutation of residue 232 of MEV VP2 didn’t decrease the released viral genome copies and did not significantly affect capsid assembly and viral genome packaging (Mao et al., 2016). Furthermore, phylogenetic analysis based on VP2 genes indicated that the isolates located in the same branch of all the reference strains of FPV. The NS1 genes of the six isolates and the three reference FPVs formed one unique evolutionary branch. So the study demonstrated that the isolates were FPV. Although host factors, such as age, immune status, and genotype, play a significant role in determining the outcome of parvovirus infection, the variability in disease severity is indisputably regulated by viral determinants (Jing et al., 2010). Experimental infection showed that FPV infection caused a high morbidity and mortality in mink, and the inoculation dose had an effect on pathogenicity of MEVSD1 in mink. In China, dogs and/or cats were usually fed in many mink farms. Mink may therefore act as an intermediate host providing parvoviruses the possibility of transmitting between divergent hosts (Wang et al., 2012). The etiology and epidemiological surveillance of the parvovirus should be strengthened for mink industry.
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