Co-Circulation of the Rare CPV-2c with Unique Gln370Arg Substitution, New CPV-2b with Unique Thr440Ala Substitution, and New CPV-2a with High Prevalence and Variation in Heilongjiang Province, Northeast China

<div><p>To trace evolution of canine parvovirus-2 (CPV-2), a total of 201 stool samples were collected from dogs with diarrhea in Heilongjiang province of northeast China from May 2014 to April 2015. The presence of CPV-2 in the samples was determined by PCR amplification of the VP2 gene (568 bp) of CPV-2. The results revealed that 95 samples (47.26%) were positive for CPV-2, and they showed 98.8%–100% nucleotide identity and 97.6%–100% amino acid identity. Of 95 CPV-2-positive samples, types new2a (Ser297Ala), new2b (Ser297Ala), and 2c accounted for 64.21%, 21.05%, and 14.74%, respectively. The positive rate of CPV-2 and the distribution of the new2a, new2b and 2c types exhibited differences among regions, seasons, and ages. Immunized dogs accounted for 48.42% of 95 CPV-2-positive samples. Coinfections with canine coronavirus, canine kobuvirus, and canine bocavirus were identified. Phylogenetic analysis revealed that the identified new2a, new2b, and CPV-2c strains in our study exhibited a close relationship with most of the CPV-2 strains from China; type new2a strains exhibited high variability, forming three subgroups; type new2b and CPV-2c strains formed one group with reference strains from China. Of 95 CPV-2 strains, Tyr324Ile and Thr440Ala substitutions accounted for 100% and 64.21%, respectively; all type new2b strains exhibited the Thr440Ala substitution, while the unique Gln370Arg substitution was found in all type 2c strains. Recombination analysis using entire VP2 gene indicated possible recombination events between the identified CPV-2 strains and reference strains from China. Our data revealed the co-circulation of new CPV-2a, new CPV-2b, and rare CPV-2c, as well as potential recombination events among Chinese CPV-2 strains.</p></div

Phylogenetic analysis of CCoV strains on the basis of partial S gene sequences.

<p>(A) Phylogenetic tree generated using the nucleotide sequences of the partial S gene of CCoV-IIa. (B) Phylogenetic tree generated using the nucleotide sequences of the partial S gene of CCoV-I. (C) Phylogenetic tree generated using the nucleotide sequences of the partial S gene of CCoV-IIb. Red circles represent the CCoV strains identified in our study.</p

Phylogenetic analysis of CCoV strains using the nucleotide sequences of the partial M gene of CCoV.

<p>Red circles represent the CCoV strains identified in our study, and black diamonds represent the Chinese CCoV reference strains.</p

Characteristics of the CPV-2 positive dogs, the genotypes of CPV-2 strains, and amino acid substitution of the VP2 protein in northeast China.

<p><b><i>Note</i></b>. For breed, GM = Golden Malinois, LR = Labrador Retriever, RC = Rough Collie, CO = Caucasian Owtcharka, TM = Tibetan Mastiff, PD = Poodle, CH = Chihuahua, and JS = Japanese Spitz; For gender, F = female, and M = male; for age, M = month; for location, MDJ = Mudanjiang, HRB = Harbin, DQ = Daqing.</p><p>Characteristics of the CPV-2 positive dogs, the genotypes of CPV-2 strains, and amino acid substitution of the VP2 protein in northeast China.</p

Identification of the recombination event between the major parent strain MDJ-20 (KT156829) (green) and the minor parent strain G1 (KF482468) (blue), which led to the recombinant 08/09 strain (GU380305) (red).

<p>(A) Bootscan evidence for the recombination origin on the basis of pairwise distance, modelled with a window size 200, step size 20 and 100 bootstrap replicates. (B, C, and D) Fast neighbour-joining (NJ) tree (1000 replicates, Kimura two-parameter distance) constructed using the regions derived from minor parent strain (1–809, 1708–1755) (B), the recombination region (810–1707) (C), and non-recombinant region (D). <i>Note</i>. The potential recombination event was detected in Maxchi (<i>P</i> = 8.51E-04), SiSscan (<i>P</i> = 1.05E-02), and Chimaera (<i>P</i> = 7.25E-03) methods.</p

List of the entire VP2 gene for recombination analysis.

<p>List of the entire VP2 gene for recombination analysis.</p

Phylogenetic analysis of CPV-2 strains on the basis of the partial VP2 gene (507 bp).

<p>The red spot diagram represents CPV-2 strains identified in this study. The values in parentheses indicate the number of strains with identical sequences to that included in the tree. (1): KT074275 from Daqing; (2): KT074317 and KT074304 from Harbin; (6): KT074266, KT074270 from Mudanjiang; KT074276, KT074288, KT074293, and KT074306 from Harbin; (8): KT074262, KT074264, and KT074271 from Mudanjiang; KT074277, KT074282, KT074311, KT074314, and KT074315 from Harbin. (13): KT074340-KT074347 from Harbin; KT074348-KT074352 from Mudanjiang. (17): KT074320-KT074322 and KT074324-KT074334 from Harbin; KT074336-KT074338 from Daqing; (35): KT074267-KT074269 and KT074272 from Mudanjiang; KT074273 and KT074274 from Daqing; KT074278, KT074279, KT074280-KT074287, KT074289- KT074291, KT074294- KT074298, KT074300- KT074302, KT074305, KT074307- KT074310, KT074312, KT074313, KT074316, and KT074318 from Harbin.</p

Identification of the recombination event between the minor parent strain HRB-F8 (KT156836) (blue) and the major parent strain G1 (KF482468) (green), which led to the recombinant MDJ-20 strain (KT156829) (red).

<p>(A) Bootscan evidence for the recombination origin on the basis of pairwise distance, modelled with a window size 200, step size 20 and 100 bootstrap replicates. (B, C, and D) Fast neighbour-joining (NJ) tree (1000 replicates, Kimura two-parameter distance) constructed using the regions derived from minor parent strain (1–133, 558–1755) (B), the recombination region (134–557) (C), and non-recombinant region (D). <i>Note</i>. The potential recombination event was detected in Maxchi (<i>P</i> = 3.74E-02) and SiSscan (<i>P</i> = 1.42E-04) methods.</p