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. 2020 Feb 10;13(2):245–255. doi: 10.14202/vetworld.2020.245-255

Dynamic evolution of canine parvovirus in Thailand

N Inthong 1,2,3, S Kaewmongkol 3, N Meekhanon 3, K Sirinarumitr 2,4, T Sirinarumitr 2,5,✉
PMCID: PMC7096304  PMID: 32255965

Abstract

Background and Aim:

According to the previous study, the circulating canine parvovirus (CPV) in Thailand is 2a and 2b. Nowadays, CPV mutants, including CPV-2c, have been identified in many parts of the world. This study aimed to investigate the genetic diversity of the circulating CPV in Thailand.

Materials and Methods:

Eighty-five CPV-positive fecal samples were obtained from dogs with either acute hemorrhagic diarrhea or diarrhea. The complete VP2 gene of these samples was amplified using VP2 specific primers and polymerase chain reaction (PCR). The obtained full-length VP2 sequences were analyzed and a phylogenetic tree was constructed.

Results:

Sixty and 25 CPV-positive fecal samples were collected in 2010 and 2018, respectively. Thirty-four samples were new CPV-2a and 31 samples were new CPV-2b due to amino acids substitution at position 297 (Ser-Ala). In 2018, 5 new CPV-2a, 19 CPV-2c, and 1 feline panleukopenia virus (FPV) were found, but no new CPV-2b was detected. Moreover, most of the CPV in this study had amino acids mutations at positions 324 and 440. The phylogenetic construction demonstrated the close relationship between the current new CPV-2a with the previous CPV-2a reported from Thailand, China, Uruguay, Vietnam, Singapore, and India. Interestingly, the current new CPV-2b in this study was not closely related to the previous CPV-2b reported in Thailand. The CPV-2c in this study was closer to Asian CPV-2c and further from either European or South America CPV-2c. Interestingly, FPV was identified in a diarrhea dog.

Conclusion:

The evolution of CPV in Thailand is very dynamic. Thus, it is important to monitor for CPV mutants and especially the clinical signs relating to these mutants to conduct surveillance for the emergence of new highly pathogenic CPV in the future.

Keywords: canine parvoviruses, diversity, Thailand, VP2 gene

Introduction

Canine parvovirus (CPV) is one of the most common viruses in domestic dogs. It causes acute hemorrhagic gastroenteritis, leukopenia, nausea, diarrhea, and sometimes fatal myocarditis in young puppies [1]. CPV belongs to the family Parvoviridae, subfamily Parvovirinae, and genus Parvovirus. It is a non-enveloped, icosahedral, linearized, and single-stranded DNA virus. The genome of CPV is approximately 5.2 kb in length. The virus encodes two nonstructural proteins (NS1 and NS2) and three structural proteins (VP1, VP2, and VP3). The VP2 capsid protein is the main capsid protein and plays an important role in the determination of the antigenicity and host range of CPV [2]. CPV type 2 (CPV-2) was first identified in the USA in 1978 and was found to have spread worldwide in domestic and wild canid populations [3]. CPV is genetically close to feline panleukopenia virus (FPV); however, CPV has at least seven amino acid differences from FPV which determine the canine or feline host range, such as amino acid positions 80 (Lys-Arg), 93 (Lys-Asn), 103 (Val-Ala), 232 (Val-Ile), 323 (Asp-Asn), 564 (Asn-Ser), and 568 (Ala-Gly) [4].

The original CPV-2 was replaced worldwide by CPV-2a and CPV-2b in 1985. CPV-2a and CPV-2b have some nucleotide changes at the VP2 gene compared to the original CPV-2. There are six amino acid differences at residues 87 (Met-Leu), 101 (Ile-Thr), 300 (Ala-Gly), 305 (Asp-Tyr), 375 (Asn-Asp), and 555 (Val-Ile) between CPV-2 and CPV-2a and six amino acid differences at residues 87 (Met-Leu), 101 (Ile-Thr), 300 (Ala-Gly), 305 (Asp-Tyr), 375 (Asn-Asp), and 426 (Asn-Asp) between CPV-2 and CPV-2b. The differences between CPV-2a and CPV-2b are the substitution of two amino acids in the VP2 capsid protein, namely, Asn-426 in 2a (Asp-426 in 2b) and Ile-555 in 2a (Val-555 in 2b). Recently, the emergence of new CPV-2a and CPV-2b has been reported having an amino acid mutation at position 297 (Ser-Ala). Moreover, the new CPV-2a has a mutation at amino acid position 555 that changes isoleucine back to valine [4-8]. CPV-2c is a new CPV strain that has a glutamate substitution at the 426 residues of the VP2 protein [9-11]. Recently, CPV-2c has been detected in Argentina [12], Australia [13], Italy [10], Laos [14], Spain [6], Taiwan [15], and Uruguay [16]. According to the information above, CPV has a high mutation rate and has been dynamically evolving in many parts of the world. There is a growing concern about the severity and effectiveness of vaccine regarding the new mutant or genotype of CPV.

Molecular surveillance may be used as a tool for the detection of the new mutant, prediction of disease severity and providing the important data for the development of the better vaccine or the better diagnostic test in the future. Moreover, the knowledge of the current genotype of the CPV in Thailand is limited. This study aimed to investigate the current genotype of CPV circulating in Thailand and to determine the existence of CPV-2c and other CPV strains.

Materials and Methods

Ethical approval

This study was approved by the Animal Ethics Committee of the Faculty of Veterinary Medicine, Kasetsart University, Thailand (ACKU62-VET-007).

Samples

Eighty-five fecal samples were used in this study based on a positive result for CPV according to routine polymerase chain reaction (PCR) testing. In 2010 and 2018, 60 and 25 positive samples, respectively, were collected (Table-1). These fecal samples were collected from dogs that displayed either acute hemorrhagic diarrhea or diarrhea and nausea at the Veterinary Teaching Hospital, Kasetsart University, Rattanatibeth Referral Animal Hospital, Bangkok, Thailand, and the Amphawa Pet Hospital, Samut Songkhram, Thailand. These dogs were either vaccinated or unvaccinated and were aged from 1 month to 5 years. The fecal samples were stored at −80°C until used for DNA extraction.

Table-1.

Age of parvovirus-infected dogs, vaccination, year of sample collection, GenBank accession number, genotype of CPV, and amino acid at important position (NR=no report).

No. Sample Amino acid position CPV type Previous vaccination Age GenBank accession number Year of collection
80 87 93 103 232 267 297 300 305 323 324 370 426 440 555 564 568
1 FPV-VT-2020 K M N V V F S A D D Y Q N T V N A FPV NR 1 years MN270937 2018
2 CPV-VT-1 R L N A I Y A G Y N I Q N A V S G New 2a No NR KP715658 2010
3 CPV-VT-7 R L N A I Y A G Y N I Q N A V S G New 2a No 3 M KP715659 2010
4 CPV-VT-13 R L N A I F A G Y N I Q N T V S G New 2a No 3 M KP715660 2010
5 CPV-VT-14 R L N A I Y A G Y N I Q N A V S G New 2a No 2 M KP715661 2010
6 CPV-VT-30 R L N A I F A G Y N I Q N T V S G New 2a Yes 3 M KP715662 2010
7 CPV-VT-37 R L N A I F A G Y N I Q N A V S G New 2a Yes 2 M KP715663 2010
8 CPV-VT-39 R L N A I Y A G Y N I Q N A V S G New 2a No 5 M KP715664 2010
9 CPV-VT-40 R L N A I Y A G Y N I Q N A V S G New 2a No 2 M KP715665 2010
10 CPV-VT-42 R L N A I Y A G Y N I Q N A V S G New 2a No 1 M KP715666 2010
11 CPV-VT-45 R L N A I Y A G Y N I Q N A V S G New 2a Yes 2 M KP715667 2010
12 CPV-VT-51 R L N A I Y A G Y N I Q N A V S G New 2a Yes 6 M KP715668 2010
13 CPV-VT-58 R L N A I Y A G Y N I Q N A V S G New 2a No 2 M KP715669 2010
14 CPV-VT-61 R L N A I Y A G Y N I Q N A V S G New 2a No 3 M KP715670 2010
15 CPV-VT-62 R L N A I Y A G Y N I Q N A V S G New 2a No >3 M KP715671 2010
16 CPV-VT-71 R L N A I Y A G Y N I Q N A V S G New 2a Yes 5 M KP715672 2010
17 CPV-VT-81 R L N A I Y A G Y N I Q N A V S G New 2a No 4 M KP715673 2010
18 CPV-VT-82 R L N A I Y A G Y N I Q N A V S G New 2a No 2 M KP715674 2010
19 CPV-VT-83 R L N A I Y A G Y N I Q N A V S G New 2a No 3 M KP715675 2010
20 CPV-VT-87 R L N A I Y A G Y N I Q N A V S G New 2a No 2 M KP715676 2010
21 CPV-VT-88 R L N A I Y A G Y N I Q N A V S G New 2a NR 3 M KP715677 2010
22 CPV-VT-89 R L N A I Y A G Y N I Q N A V S G New 2a No 1.5 M KP715678 2010
23 CPV-VT-92 R L N A I Y A G Y N I Q N A V S G New 2a No 3 M KP715679 2010
24 CPV-VT-93 R L N A I Y A G Y N I Q N A V S G New 2a No 3 M KP715680 2010
25 CPV-VT-97 R L N A I Y A G Y N I Q N A V S G New 2a No 3 M KP715681 2010
26 CPV-VT-103 R L N A I Y A G Y N I Q N A V S G New 2a No >3 M KP715682 2010
27 CPV-VT-109 R L N A I Y A G Y N I Q N A V S G New 2a NR NR KP715683 2010
28 CPV-VT-115 R L N A I Y A G Y N I Q N A V S G New 2a NR NR KP715684 2010
29 CPV-VT-138 R L N A I Y A G Y N I Q N A V S G New 2a NR 3 M KP715685 2010
30 CPV-VT-139 R L N A I Y A G Y N I Q N A V S G New 2a NR 3.5 M KP715686 2010
31 CPV-VT-0561 R L N A I Y A G Y N I Q N A V S G New 2a NR NR MN270938 2018
32 CPV-VT-1377 R L N A I Y A G Y N I Q N A V S G New 2a NR NR MN270939 2018
33 CPV-VT-2097 R L N A I Y A G Y N I Q N A V S G New 2a NR 2 M MN270940 2018
34 CPV-VT-2098 R L N A I Y A G Y N I Q N A V S G New 2a NR 2 M MN270941 2018
35 CPV-VT-2961 R L N A I Y A G Y N I Q N A V S G New 2a NR 1 M MN270942 2018
36 CPV-VT-3 R L N A I Y A G Y N I Q D T V S G New 2b No 2 years KP715687 2010
37 CPV-VT-12 R L N A I Y A G Y N I Q D T V S G New 2b Yes 7 M KP715688 2010
38 CPV-VT-18 R L N A I Y A G Y N I Q D T V S G New 2b No 2 M KP715689 2010
39 CPV-VT-28 R L N A I Y A G Y N I Q D T V S G New 2b Yes 3 M KP715690 2010
40 CPV-VT-43 R L N A I Y A G Y N I Q D T V S G New 2b NR 1 years KP715691 2010
41 CPV-VT-49 R L N A I Y A G Y N I Q D T V S G New 2b Yes 4 M KP715692 2010
42 CPV-VT-53 R L N A I Y A G Y N I Q D A V S G New 2b No 2 M KP715693 2010
43 CPV-VT-54 R L N A I Y A G Y N I Q D T V S G New 2b No 2 M KP715694 2010
44 CPV-VT-56 R L N A I Y A G Y N Y Q D T V S G New 2b No 5 M KP715695 2010
45 CPV-VT-65 R L N A I Y A G Y N I Q D T V S G New 2b No NR KP715696 2010
46 CPV-VT-66 R L N A I Y A G Y N I Q D T V S G New 2b No 2 years KP715697 2010
47 CPV-VT-68 R L N A I Y A G Y N I Q D T V S G New 2b No 4 M KP715698 2010
48 CPV-VT-74 R L N A I Y A G Y N I Q D T V S G New 2b No NR KP715699 2010
49 CPV-VT-75 R L N A I Y A G Y N I Q D T V S G New 2b No 2 M KP715700 2010
50 CPV-VT-80 R L N A I Y A G Y N I Q D T V S G New 2b No 4 M KP715701 2010
51 CPV-VT-84 R L N A I Y A G Y N I Q D T V S G New 2b No 4 M KP715702 2010
52 CPV-VT-86 R L N A I Y A G Y N I Q D T V S G New 2b No 5 M KP715703 2010
53 CPV-VT-90 R L N A I Y A G Y N I Q D T V S G New 2b Yes 6 M KP715704 2010
54 CPV-VT-99 R L N A I Y A G Y N I Q D T V S G New 2b No 5 M KP715705 2010
55 CPV-VT-101 R L N A I Y A G Y N I Q D T V S G New 2b No 1 M KP715706 2010
56 CPV-VT-106 R L N A I Y A G Y N I Q D T V S G New 2b NR NR KP715707 2010
57 CPV-VT-108 R L N A I Y A G Y N I Q D A V S G New 2b NR NR KP715708 2010
58 CPV-VT-114 R L N A I Y A G Y N I Q D T V S G New 2b NR NR KP715709 2010
59 CPV-VT-120 R L N A I Y A G Y N I Q D T V S G New 2b No 3 M KP715710 2010
60 CPV-VT-121 R L N A I Y A G Y N I Q D T V S G New 2b No 2 M KP715711 2010
61 CPV-VT-123 R L N A I Y A G Y N I Q D T V S G New 2b No 8 M KP715712 2010
62 CPV-VT-129 R L N A I Y A G Y N I Q D T V S G New 2b No 1.5 years KP715713 2010
63 CPV-VT-135 R L N A I Y A G Y N I Q D T V S G New 2b No NR KP715714 2010
64 CPV-VT-142 R L N A I Y A G Y N I Q D T V S G New 2b NR 1 M KP715715 2010
65 CPV-VT-143 R L N A I Y A G Y N I Q D T V S G New 2b No 4 M KP715716 2010
66 CPV-VT-148 R L N A I Y A G Y N I Q D A V S G New 2b NR 2 M KP715717 2010
67 CPV-VT-0861 R L N A I Y A G Y N I R E T V S G 2c NR 1 M MN270943 2018
68 CPV-VT-0937 R L N A I Y A G Y N I R E T V S G 2c NR NR MN270944 2018
69 CPV-VT-1161 R L N A I Y A G Y N I R E T V S G 2c NR 11 M MN270945 2018
70 CPV-VT-1261 R L N A I Y A G Y N I R E T V S G 2c NR 6 M MN270946 2018
71 CPV-VT-1361 R L N A I Y A G Y N I R E T V S G 2c NR 4 M MN270947 2018
72 CPV-VT-1373 R L N A I Y A G Y N I R E T V S G 2c NR NR MN270948 2018
73 CPV-VT-1374 R L N A I Y A G Y N I R E T V S G 2c NR NR MN270949 2018
74 CPV-VT-1375 R L N A I Y A G Y N I R E T V S G 2c NR NR MN270950 2018
75 CPV-VT-1383 R L N A I Y A G Y N I R E T V S G 2c NR NR MN270951 2018
76 CPV-VT-1661 R L N A I Y A G Y N I R E T V S G 2c NR 1 years MN270952 2018
77 CPV-VT-2016 R L N A I Y A G Y N I R E T V S G 2c NR 2 M MN270953 2018
78 CPV-VT-2018 R L N A I Y A G Y N I R E T V S G 2c NR 3 M MN270954 2018
79 CPV-VT-2019 R L N A I Y A G Y N I R E T V S G 2c NR 3 years MN270955 2018
80 CPV-VT-2021 R L N A I Y A G Y N I R E T V S G 2c NR 7 M MN270956 2018
81 CPV-VT-2388 R L N A I Y A G Y N I R E T V S G 2c NR 3 M MN270957 2018
82 CPV-VT-2389 R L N A I Y A G Y N I R E T V S G 2c NR 5 M MN270958 2018
83 CPV-VT-2391 R L N A I Y A G Y N I R E T V S G 2c NR 2.5 years MN270959 2018
84 CPV-VT-2461 R L N A I Y A G Y N I R E T V S G 2c NR 3 M MN270960 2018
85 CPV-VT-3761 R L N A I Y A G Y N I R E T V S G 2c NR 21 days MN270961 2018
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CPV=Canine parvovirus, FPV=Feline panleukopenia virus

DNA extraction and PCR

DNA was extracted from the fecal samples using the acid guanidinium thiocyanate-phenol-chloroform extraction method. A set of primers was designed to amplify the whole VP2 gene: F (5’-ATG AGT GAT GGA GCA GTT CA) and R (5’-TTA ATA TAA TTT TCT AGG TGC TAG TTG). The PCR mixture (25 µl) was composed of 1× buffer (20 mM Tris-HCl [pH 8.4], 50 mM KCl2), 0.2 mM dNTPs, 2.5 mM MgCl2, 100 pmol of each of the forward and reverse primers, 1 unit Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA), and 2.5 µl of DNA template to give a total volume of 25 µl. After an initial denaturing at 94°C for 5 min, the amplification was performed using 35 cycles at 94°C for 40 s, annealing at 50°C for 40 s, and extension at 72°C for 90 s, and a final extension at 72°C for 10 min. The expected PCR products were 1755 bps in size. The PCR products were analyzed using 1% agarose gel electrophoresis at 100 V for 30 min and visualized under ultraviolet illumination. The PCR products were purified using an UltraClean®15 DNA Purification Kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA) and cloned into plasmid pGEM-T Easy (Promega Corporation, Madison, WI, USA). The sequences of the cloned full-length VP2 were determined at First BASE Laboratories Sdn Bhd, Selangor, Malaysia.

Analysis and phylogenetic construction of full-length VP2 gene of CPVs

The nucleotide sequences were translated into amino acid and multiple alignments of the amino acid sequences using the Bioedit biological sequence alignment editor computer package (version 7.1.3; Ibis Biosciences, Carlsbad, CA, USA). The phylogenetic analysis was constructed from the amino acid sequences of all 85 samples in this study and other full-length VP2 sequences obtained from the GenBank database (Table-2) using the MEGA program (version 7.0, The Biodesign Institute, Tempe, AZ, USA) with the neighbor-joining method and running 1000 replicates in the bootstrap. Bayesian phylogenetic analysis was also performed for more extensive amino acids analysis to analyze the selective pressures on certain amino acids using mixed model analysis. The phylogenetic tree was created by MrBayes version 3.2.6 (https://nbisweden.github.io/MrBayes/download.html) [17]. The tree was viewed using FigTree software version 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/).

Table-2.

GenBank accession numbers of CPV used in phylogenetic tree construction.

Order Origin GenBank accession number Year of collection Genetic type
1 China MF467224 2015 2a
2 China FJ435343 2008 2a
3 China FJ435345 2008 2a
4 China GU380304 2009 2a
5 China GU569936 2008 2a
6 France DQ026002 - 2a
7 India KX469433 2015 2a
8 Italy FJ005259 2008 2a
9 Singapore KY083098 2014 2a
10 Thailand GQ379047 2009 2a
11 Thailand GQ379045 2009 2a
12 Thailand GQ379046 2009 2a
13 Uruguay KM457139 2011 2a
14 Uruguay JF906788 2010 2a
15 Vietnam LC214970 2013 2a
16 China GU569937 2002 2b
17 China KF482468 2009 2b
18 China GU569938 2002 2b
19 China GU569944 2002 2b
20 China JQ743891 2010 2b
21 Italy FJ005263 2005 2b
22 Thailand FJ869123 2008 2b
23 Thailand FJ869124 2008 2b
24 Vietnam AB120724 2013 2b
25 USA JX475261 2010 2b
26 Argentina KM236569 2013 2c
27 China KT162005 2014 2c
28 China KT162016 2014 2c
29 China KP260509 2014 2c
30 China KY937641 2016 2c
31 Croatia KP859576 2014 2c
32 Ecuador KF149984 2012 2c
33 Germany FJ005202 1997 2c
34 Germany FJ005204 1999 2c
35 Greece GQ865518 2008 2c
36 Indonesia LC216905 2013 2c
37 Italy HQ025913 2010 2c
38 Singapore KY083092 2014 2c
39 Uruguay KM457112 2008 2c
40 - M74849 - 2b
41 - M24003 - 2a
42 - M38245 - 2
43 - FJ405225 - FPV
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CPV=Canine parvovirus, FPV=Feline panleukopenia virus

Results

Thirty-nine and nine samples out of 85 positive samples were from non-vaccinated and vaccinated dogs, respectively. The other 37 positive samples did not have a history of vaccination. The youngest CPV-infected dog was aged 21 days, and the oldest was 3 years. The youngest age of positive vaccinated dogs was 2 months; however, the individual histories of booster vaccination for these positive, vaccinated dogs were not available (Table-1).

For the 2010 data, the amino acid sequences analysis revealed that 29 samples were new CPV-2a and 31 samples were new CPV-2b due to amino acids substitution at position 297 (Ser-Ala) (Table-1). The number of positive samples for new CPV-2a and new CPV-2b was nearly equal in 2010. For 2018, 5 new CPV-2a, 19 CPV-2c, and 1 FPV were found, but CPV-2b was not detected (Table-3). In this study, most of the CPV had amino acid substitution at positions 324 and 440. Eighty-three CPV had 324 (Tyr-Ile) substitution due to a T-to-A transversion at nucleotide 970 and an A-to-T transversion at nucleotide 971 with the exception of 1 sample of new CPV-2b in 2010 (CPV-VT 56) (Figure-1). Thirty-two out of 34 new CPV-2a and three out of 31 new CPV-2b (CPV-VT 53, CPV-VT 108, and CPV-VT 148) had 440 (Thr-Ala) substitution due to an A-to-G transition at nucleotide 1318 (Table-1). The new CPV-2a in this study was closely related to CPV-2a from Thailand in 2009 (GQ379045, GQ379046, and GQ379047), and from Uruguay in 2010 and 2011 (JF906788 and KM457139), China in 2015 (MF467224), India in 2015 (KX469433), Singapore in 2014 (KY083098), and Vietnam in 2013 (LC214970) (Figures-2 and 3) due to similar amino acids at positions 267 (Tyr), 324 (Ile), and 440 (Ala). However, two new CPV-2a (CPV-VT 13 and 30) were closely related to new CPV-2a from China in 2008 (FJ435343, FJ435345, GU380304, and GU569936) (Figures-2 and 3) due to similar amino acids at positions 267 (Phe), 324 (Ile), and 440 (Thr). All CPVs in this study had 267 (Phe-Tyr) due to an A-to-T transversion at nucleotide 800 substitution except for the two new CPV-2a samples (CPV-VT 13 and 30) that did not have 440 (Thr-Ala) substitution and one new CPV-2a (CPV-VT 37) that had substitution at position 440. All CPV-2a samples had valine at position 555.

Table-3.

Number of parvoviruses found in each year.

Year Type of parvoviruses
FPV CPV2a CPV2b CPV2c Total
2010 0 29 31 0 60
2018 1 5 0 19 25
Total 1 34 31 19 85
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CPV=Canine parvovirus, FPV=Feline panleukopenia virus

Figure-1.

Figure-1

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Amino acid comparison of VP2 gene of CPV-2a: CPV-VT13 (T440), CPV-VT93 (A440), CPV-VT2097, CPV-2b: CPV-VT56 (324Y), CPV-VT90 (440T), and CPV-VT108 (440A), CPV-2c: CPV-VT2391, FPV: FPV-VT2020 in this study, with reference strains FPV (FJ405225), CPV-2 (M38245), CPV-2a (M24003) and CPV-2b (M74849) and other isolates from other parts of the world. CPV=Canine parvovirus, FPV=Feline panleukopenia virus.

Figure-2.

Figure-2

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Phylogenetic tree constructed from 80 amino acid sequences of the VP2 gene of canine parvovirus (CPV) and feline panleukopenia virus (FPV) in this study and other CPV and FPV sequences obtained from GenBank database using the neighbor-joining method and bootstrap analysis performed with 1000 trials. Drawn using MEGA version. ■=Samples collected in 2018 and ▲=Samples collected in 2010.

Figure-3.

Figure-3

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Phylogenetic tree constructed from 80 amino acids of the VP2 gene of canine parvovirus (CPV) and feline panleukopenia virus (FPV) in this study and other CPV and FPV sequences obtained from GenBank database using MrBayes version 3.2.6 with Markov chain Monte Carlo, with 5 million generations. Node values (in percentages) indicate posterior clade probabilities. Vertical bars indicate clades of CPV in each group, A=CPV-2c in Europe and South America, B=new CPV-2b in this study, C=CPV-2c in this study, and Asia, D=new CPV-2b that has alanine at amino acid position 440, F=new CPV-2a in this study.

In contrast to new CPV-2a in this study, all new CPV-2b in this study was distanced from CPV-2b reported previously in Thailand in 2008 (FJ869123 and FJ869124) and Europe in 2005 (FJ005263) (Figures-2 and 3) because of the amino substitution at position 324 (Tyr-Ile). However, three new CPV-2b (CPV-VT 53, CPV-VT 108, and CPV-VT 148) were closely related to the new CPV-2b from China in 2010 (JQ743891) and were also closely related to a cluster of new CPV-2a. These three CPV-2b distanced from the other new CPV-2b in this study (Figures-2 and 3) due to amino substitution at positions 324 (Tyr-Ile) and 440 (Thr-Ala). Nineteen CPV-2c in this study had amino acids substitution at positions 267 (Phe-Tyr), 324 (Tyr-Ile), and 370 (Gln-Arg) due to an A-to-G transition at nucleotide 1109 and a G-to-T transversion at nucleotide 1110. CPV-2c in this study was closely related to CPV-2c from Asia including China in 2014 and 2016 (KP260509, KT162005, KT162016, and KY937641), Indonesia in 2013 (LC216905), and Singapore in 2014 (KY083092) due to these three amino acids substitution (Figures-2 and 3), but distanced from the CPV-2c in Europe and South America. Interestingly, one dog with clinical signs of diarrhea was positive for FPV. The six amino acids (at 80, 103, 232, 323, 564, and 568) out of the seven amino acids which determine the canine or feline host range were similar to the reference FPV; however, there was an amino acid substitution at position 93 (Lys-Asn). According to this amino acid substitution, FPV in this study was in the different clades compared to the reference FPV (Figures-2 and 3).

Discussion

The number of samples positive for new CPV-2a and 2b circulating in central Thailand in 2010 was approximately equal. This result contrasted with a study in Thailand (data collected between 2003 and 2008) in which the predominant genotype in central Thailand was CPV-2a [11]. Interestingly, the majority CPV genotype in 2018 was CPV-2c, but new CPV-2b was not found. This study showed that CPV-2c had become the predominant genotype during the year 2018 in Thailand. Besides of t he diminishing of CPV-2b, there was only one amino acid difference at position 426 between CPV-2b and CPV-2c but there were two amino acids differences at positions 426 and 440 between CPV-2a and CPV-2c. Thus, the emergence of CPV-2c in Thailand might have derived from the new CPV-2b (2010) that had a mutation at position 426 (Asp-Glu).

From the previous study in Thailand (2003-2009), both CPV-2a and CPV-2b had alanine at amino acid position 297 [11]. According to this finding, CPV-2a and CPV-2b (2003-2009) were new CPV-2a and new CPV-2b. However, most of the CPV in this study had additional amino acid substitution at position 324 (Tyr-Ile), except for 1 CPV-2b (CPV-VT 56). Thus, the new CPV-2b (2010) was different from the new CPV-2b in Thailand (2003-2009) and Europe (2005). The amino acid substitution at residue 324 (Tyr-Ile) was also reported in Brazil [18], China [19], Hungary [20], India [21,22], Italy [23], Nigeria [24], South Korea [25], Taiwan [1,15], and Uruguay [26]. According to the above, amino acid position 324 has been shown to undergo a strong positive selection in the parvovirus of all carnivores [27]. Amino acid position 324 was also adjacent to the critical amino acid position 323 that has been known to control canine cell infection. The changes in the region of the capsid surface around VP2 residue 300, within a raised region on the shoulder of the three-fold spike of the capsid, have been shown to influence the binding of the virus to the canine TfR [28-30]. This change may influence the interactions with their various host receptors and may be expected to result in an increased host range.

Moreover, most of the current new CPV-2a had alanine at amino acid position 440; however, most of the new CPV-2b still had threonine. Amino acid substitution at position 440 (Thr-Ala) was also reported in the previous studies in China [19], India [21,22], Nigeria [24,31], Pakistan [32], South Africa [33], South Korea [25], and Uruguay [26]. Amino acid position 440 is also important because it is located at the top of the three-fold spike, the main antigenic site of the virus [34,35]. Interestingly, this mutation was not detected in a previous study in Thailand [11]. In the current study, two new CPV-2a (CPV-VT13 and 30) had phenylalanine and threonine at amino acid positions 267 and 440 similar to CPV-2a in 2003-2004 as reported in the previous study in Thailand, but these two strains had isoleucine at amino acid position 324 as seen in CPV-2a in 2008-2009 and new CPV-2a in this study [11]. However, most of the current new CPV-2a in the current study had tyrosine and alanine at positions 267 and 440, as seen in CPV-2a in 2008-2009, respectively. A CPV-2a (CPV-VT37) had phenylalanine and isoleucine at positions 267 and 324 similar to CPV-2a in 2003-2004 but had alanine at amino acid position 440 as seen in CPV-2a in 2008-2009 and new CPV-2a in this study. CPV-VT13, 30, and 37 might represent the transition evolution of the original new CPV-2a to the current new CPV-2a. CPV-2b (CPV-VT56) had similar amino acids at amino acid positions 267, 324, 426, and 440 (phenylalanine, tyrosine, aspartic acid, and threonine, respectively), as reported in the previous study in Thailand [11]. However, new CPV-2b in this study had isoleucine at amino acid 324. Thus, CPV-VT56 might also represent the transition evolution of the original new CPV-2b in the current new CPV-2b.

The CPV-2c in this study was similar to CPV-2c in Asia, such as in China [36], Laos [14], and Taiwan [15,37] due to amino acid substitution at amino acid positions 267 (Tyr), 324 (Ile), and 370 (Arg). CPV-2c in this study differs from CPV-2c in Europe and South America at three amino acid positions (Phe267Tyr, Tyr324Ile, and Gln370Arg). The amino acid substitution at position 440 (Thr-Ala) has been found in Argentina, but this change was not found in this study [12]. Interestingly, FPV was found in a dog that had clinical signs of diarrhea. This FPV sample had an amino acid substitution at position 93 (Lys-Asn). The amino acid at position 93 is important because it is one of the amino acids that determine the canine host range [38]. FPV infection in dogs has also been reported in Pakistan [39]. These findings demonstrated that parvoviruses in Thailand have been dynamically evolving as those in the other part of the world [40,41]. This mutation rate is as high as seen in RNA viruses [41]. The rapid mutation of CPV has resulted in growing concern about the effectiveness of vaccines regarding the new mutant or genotype of CPV. Molecular surveillance of CPV is crucial for the prediction of disease severity and may be important for the development of more effective vaccines or diagnostic tests in the future.

Conclusion

Two genotypes of CPV (new CPV-2a and CPV-2c) are circulating in Central Thailand and the predominant circulating genotype of CPV has been changed from CPV-2a in the past to CPV-2c at present. Currently, CPV-2b has not been found in Central Thailand. The current new CPV-2a circulating in Thailand has amino acid substitutions at positions 324 (Tyr-Ile) and 440 (Thr-Ala). FPV was found in a dog that had acute diarrhea; however, the importance of this finding remains to be determined. Our results provided additional information on the dynamic evolution of CPV in Thailand, which is following the same evolutionary trend observed in the others part of the world.

Authors’ Contributions

NI and TS designed the experiment and made DNA extraction, PCR, multiple alignment, and phylogenetic study. NM, SK, KS, and TS were involved in scientific discussion and provided suggestions for the overall work. All authors read and approved the final manuscript.

Acknowledgments

This research was supported by grants from the Kasetsart University Research and Development Institute, Bangkok, Thailand (Grant no. 21.53), and the Center of Excellence on Agricultural Biotechnology, Science and Technology Postgraduate Education and Research Development Office, Office of Higher Education Commission, Ministry of Education, Science, Research and Innovation (AG-BIO/PERDO-CHE), Bangkok, Thailand (Grant no. AG-BIO/59-002-015). We would like to thank the Faculty of Veterinary Technology, Kasetsart University, for providing technical support, the Veterinary Teaching Hospital, Faculty of Veterinary Medicine, Kasetsart University, Rattanatibeth Referral Animal Hospital, Bangkok, Thailand, and the Amphawa Pet Hospital, Samut Songkhram, Thailand, for providing specimens.

Competing Interests

The authors declare that they have no competing interests.

Publisher’s Note

Veterinary World remains neutral with regard to jurisdictional claims in published institutional affiliation.

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