Indian Journal of Animal Research

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Research Article

Indian Journal of Animal Research, volume 55 issue 6 (june 2021) : 629-635

Isolation and Phylogenetic Analysis of the Canine Distemper Virus from a Naturally Infected Dog in China

Hongliang Zhang1,*, Peipei Meng2, Xiaoming Song2, Shuguang Li3, Ruimei Yang2, Chuanmei Zhang2, Hu Shan2, Yongjun Wen1,*
1Ministry of Agriculture Key Laboratory of Clinical Diagnosis and Treatment Technology in Animal Diseases, College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot-010 018, China
2Shandong Collaborative Innovation Center for Development of Veterinary Pharmaceuticals, College of Veterinary Medicine, Qingdao Agricultural University, Qingdao-266 109, China
3Shandong Lvdu Biotechnology Co., Ltd. Binzhou-256 600, China
Cite article:- Zhang Hongliang, Meng Peipei, Song Xiaoming, Li Shuguang, Yang Ruimei, Zhang Chuanmei, Shan Hu, Wen Yongjun (2020). Isolation and Phylogenetic Analysis of the Canine Distemper Virus from a Naturally Infected Dog in China . Indian Journal of Animal Research. 55(6): 629-635. doi: 10.18805/IJAR.B-1298.

ABSTRACT

Background: Canine distemper virus (CDV) can infect a variety of predators, such as Canidae, Mustelidae and Procyonidae family and cause high morbidity and mortality. Genetic/antigenic heterogeneity has been observed among various CDV strains, notably in the haemagglutinin (H) gene. Based on sequence analysis of the H gene, wild-type CDV strains can be divided into distinct genotypes. 

Methods: We used Vero/SLAM cells to isolate CDV strains from clinically naturally infected dog tissues. Here, the H gene sequence of the isolated strain was analyzed for the restriction enzyme polymorphism with RT-PCR-RFLP method and the method could be used to distinguish CDV vaccine strains from wild-type strains. Furthermore, the genetic and antigenic variation of the H gene was also analyzed.

Result: Our results indicated that Vero cells expressing canine SLAM allow the efficient isolation of CDV wild-type strains. The isolated CDV was named Shandong strain. Phylogenetic analysis identified that Shandong strain was CDV wild-type strain, the Asian genotype I. Analysis of the potential glycosylation site of the H gene and determination of the neutralization titer PD50 showed that there were immunogenic differences between the Shandong strain and the vaccine strains.

INTRODUCTION

The canine distemper virus (CDV) is a non-segmented, negative sense single-stranded RNA virus, closely related to measles and rinderpest virus, the other two members of the genus Morbillivirus of the Paramyxoviridae family. These viruses are similar in antigenic and molecular biological characteristics, especially almost identical in ultramicro morphology and structure (Bolt et al., 1997). Canine distemper is distributed worldwide and constitutes one of the most serious contagious diseases of warm-blooded animals. CDV is pathogenic for many species of carnivores including Canidae (such as dogs, foxes and the wolves), Mustelidae, Procyonidae and Felidae (such as tigers, lions, leopards, pandas) families (Adaszek et al., 2009). The positive rate of canine distemper virus in dogs is high and it is necessary to develop a mitigation strategy immediately and continuously to reduce the prevalence of CDV (Costa et al., 2019; Luo et al., 2017 ). The CDV hemagglutinin (H) gene is the most heterogenic and antigenic variable among all different strains of CDV, which could result in virulence and antigenic variations of the CDV (Martella et al., 2006). Hirama et al., (2003) reported that the CTL epitope(s) were localized in the H protein. Genetic and antigenic heterogeneity has been observed among the various CDV strains, notably in the H gene, which appears as a good target for epidemiological surveillance. One or two Nde I sites are present in wild-type strains H gene while absent in the vaccine strains (Mochizuki et al., 1999) and this allows to differentiate wild-type viruses from vaccine strains (DIVA). Herein, a strain of CDV was isolated through Vero cell culture from tissue suspensions obtained from a CDV-infected dog, which was named Shandong strain. The DNA fragment of H gene from Shandong strain was digested with Nde I and analyzed with RFLP. The phylogenetic analysis determined that Shandong strain clustered along with CDVs of the Asia I lineage.

MATERIALS AND METHODS

Clinical samples
 
In Dec. 2011, one dog at the Veterinary Teaching Hospital of Qingdao Agricultural University showed typical signs of canine distemper, such as fever, purulent ocular and nasal discharge, bronchitis, tonsillitis, gastroenteritis and neurological disturbance and the diagnosis was further confirmed by RT-PCR analysis. Tissue samples (Lung, liver, spleen and brain) were collected after euthanasia and necropsy. Samples for viral isolation were stored at -70° until use. Subsequent experimental research (Dec.2011-Jul.2013) was conducted in the Preventive Veterinary Laboratory of Qingdao Agricultural University.
 
Cell culture and virus isolation
 
Vero cells expressing canine signaling lymphocyte activation molecule (SLAM, the intrinsic receptor to CDV) were maintained in DMEM (GIBCO, USA), supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin and L-glutamine (2 mM) (Nakano et al., 2009). The supernatant of clinical samples was used for infection of cultured cells. At the same time, CPE control group was designed.
 
IFA detection of CDV
 
A 96 well microtitre plate was seeded with Vero cell in DMEM with 10% fetal bovine serum and cultivated at 37°C in 5% CO2 until 80% confluence. The cultures were then inoculated with CDV isolates. The uninfected cultures in left rows of the plate were taken as the negative controls. After 96 h incubation, the plate was fixed in 80% cold acetone/PBS and then washed and incubated with CDV McAb (targeted to CDV F protein) at a 10-fold dilution. After washing with PBS, FITC-goat anti-mouse IgG (Sigma, USA) was added to the plate and then followed by 1 h incubation in a 37°C humid box. Remove fluorescent antibody, washed five times, each time 10 min. Two infected wells were treated as a positive control to confirm viral growth. Fluorescence signal was observed using a fluorescence inverted microscope (OLYMPUS, Japan).
 
RT-PCR-RFLP analysis of CDV H gene
 
Total RNA was extracted from 250 μL infection of cultured cells with the TRIzol reagent (Invitrogen, USA) following the manufacturer’s instructions. CDV H gene was amplified using primers CDV-H-F/R designed in the conserved franking regions of the H gene (Table 1). PCR amplification was performed in a final volume of 25 μL and the target band (1921 bp) was purified with the EZ-10 Spin Column DNA Gel Extraction Kit (Sangon, Shanghai, China). In order to differentiate wild-type viruses from vaccine strains, the enzyme NdeI (TaKaRa, Dalian, China) was used to evaluate the restriction fragment length polymorphism (RFLP). Fragments from the wild-type strains could be digested to three fragments of 1282 bp, 345 bp and 294 bp, or two fragments of 1576 bp and 345bp, while fragments from the vaccine strains will not be digested.
 

Table 1: Primers used in this study.


 
Cloning, sequencing and phylogenetic tree construction
 
PCR products of the correct size (1921 bp) were cloned into the pMD18-T vector (TaKaRa, Dalian, China). Positive recombinant plasmids were sequenced in Beijing Liuhe Genomics Inc (Qingdao, China). MegAlign software was applied to analyze the nucleotide /amino acid sequence of CDV H gene of different wild-type and vaccine strains and the potential N-linked glycosylation sites of the H protein were predicted using the software NetNGlyc1.0. MegAlign and MEGA 5.0 software were used to build the phylogenetic tree based on CDV H gene sequences obtained in this study and published in GenBank of different genotypes of CDV strains.
 
Antigenicity analysis
 
The surface accessibility, hydrophilicity and antigenicity of H protein were also examined with protean software for Onderstepoort, Shandong, CDV3, 98-002 and other strains. The CDV3 antibody sera collected on the 21st day of SPF Beagle dogs were immunized with the CDV3 vaccine strain and the endpoint neutralization test (fixed virus dilution serum method) was used to determine the antibody neutralization behavior of Shandong strain and CDV3 strain. The Reed-Muench formula was used to calculate the neutralization titer PD50.

RESULTS AND DISCUSSION

Cytopathic effect of isolated shandong strain
 
Distemper-positive clinical sample filtrate was inoculated into vero cells. The cells became round and swelling, stretchy, showing cell fusion after 48h (Fig 1A). the cells aggregate significantly, round cells shrink and gradually fall off after 72h (Fig 1B). The mock inoculated control cells showed no lesions (Fig 1C).
 

Fig 1: The result of IFA detection of CDV (200×).


 
IFA detection of CDV
 
The presence of CDV isolates in vero cells has been confirmed by indirect immunofluorescence. The specific green signals of FITC were detected in the infected group (Fig 1D), whereas the mock group had no fluorescence signals (Fig 1E).
 
CDV H gene amplification and RFLP analysis
 
The 1921 bp fragment which contains the full-length H gene was amplified from the commercial canine distemper vaccine strains of Lederle and CDV3 strains, the reference wild-type strains and the isolated strain (Fig 2A). The reference wild-type and the isolated strains were digested to three fragments of 1282bp, 345bp and 294bp by NdeI while the vaccine strains were not digested (Fig 2B). This suggested that the isolated strain was a wild-type strain.
 

Fig 2: The result of CDV H gene amplification (A) and RFLP analysis (B).


 
Sequence and phylogenetic analysis of the H gene
 
The homology analysis results (Fig 3) of nucleotide and deduced amino acid sequence of the CDV H gene  showed that Shandong strain was most closely related with SD (09) 3 and HeB (09) strains, the nucleotide homology rates were 98.4% and 98.1% and the deduced amino acid sequences homology rates were 98.5% and 98.4%; The Shandong strain was furthest from the CDV3, Onderstepoort, Convac and Lederle vaccine strains, the rate of nucleotide homology was 91.0-91.1% and amino acid homology was 89.8%-91.0%. With phylogenetic analysis of the 46 CDV nucleotide sequences in the GenBank (Fig 4), it can be seen that the Shandong strain is wild-type and its genotype is similar as the majority of isolates from China belonging to the Asia-1 type. The H protein of Shandong strain has nine potential N-linked glycosylation sites, which is 2-3 sites more than the vaccine strains and 1-2 sites more than the Asia-2 type 98-002 and 97 Jindo strains. In addition, the 12 cysteine residues in all strains analyzed are conserved except the CDV3 vaccine strain, which has a 377C®377W mutation (Fig 5).
 

Fig 3: Homology analysis of nucleotide sequences (A) and deduced amino acid sequences (B) among CDV strains.


 

Fig 4: The phylogenetic relationship between CDV strains.


 

Fig 5: Alignment of deduced amino acid sequences of the CDV H genes of wild-type and vaccine strains.


 
CDV H gene cloning and sequencing was an effective tool for evolutionary relationship analysis and widely used in genotyping of canine distemper virus, variance analysis of viral isolates and vaccine strains, as well as the analysis of the interactions between the viruses and hosts. Phylogenetic and molecular evolutionary analyses of H gene sequence of CDV have revealed that the several CDV wild-type clusters are: Asia-1, Asia-2, Asia-3, Europe, Europe wild-life, America-1 and Arctic genotypes. Interestingly, Haritha et al., (2020) reported that the circulating CDV were grouped with three different lineages i.e., South America-2, Asia-4 and Asia-5/India-1 and distantly related with the vaccine strains. Zhao et al., (2010) reported that the popular CDV strains in China fox, raccoon and mink can be divided into Asia-1, Asia-3 and Arctic three genotypes. The complete H gene of 18 canine distemper virus (CDV) isolates from clinical samples from Henan Province, China, from 2012 to 2016 were isolated into two clusters within the Asian-1 genotype (Liu et al., 2019). Amino acid substitutions in the SLAM binding region of the CDV HA protein at position 530 (G/E to R/D/N) and 549 (Y to H) was proposed to be important to the spread of domestic dog-adapted CDV strains to other carnivores (Nikolin et al., 2012) and no substitution at amino acid 530 and 549 was found in the Shandong strain. All South American CDV strains showed high amino-acid divergence from vaccine strains (Panzera et al., 2012). This genetic variability may be a possible factor leading to the resurgence of distemper cases in vaccinated dog populations. The spread of CDV to novel host species may be associated with the potential N-linked glycosylation sites of the H gene (Pomeroy et al., 2008). Studies have shown that different strains of CDV H protein N-linked glycosylation sites as well as the 12 cysteine residues may affect viral antigenicity (Martella et al., 2006). In this study, the H protein of Shandong strain has nine potential N-linked glycosylation sites, which is 2-3 sites more than the vaccine strains and 1-2 sites more than the Asia-2 type 98-002 and 97 Jindo strains. The 12 cysteine residues in all strains analyzed are conserved except the CDV3 vaccine strain, which has a 377C®377W mutation.
 
Antigenicity analysis
 
The analysis of the surface accessibility, hydrophilicity and antigenicity of H protein of Onderstepoort, Shandong, CDV3, 98-002 and other strains by the Protean software suggested that there were significant differences in the domains of amino acid 230~250 and 326~350 and 375~410 between the Shandong strain and the other three strains, with the most significant differences observed with the Onderstepoort and CDV3 strains (Fig 6). The neutralization titer PD50 of the antibodies of Shandong and CDV3 strains determined by CDV3 vaccine strain antibodies were 10-1.8 and 10-2.1. The CDV wild-type strains are significantly distinct from attenuated vaccine strains originated in the 50-60s of last century (Old CDVs genotypes) (Needle et al., 2019). Combined with the results of PD50 neutralization titer, it can be analyzed that the mutation of the H gene antigenic site can cause differences in immunogenicity between Shandong strains of CDV wild-type strains and vaccine strains.
 

Fig 6: The antigenic index of H protein compared with Shandong strain.

CONCLUSION

The results suggested that Shandong strain was CDV wild-type strain, the Asian genotype I. Analysis of the potential glycosylation site of the H gene and determination of the neutralization titer PD50 showed that there were immunogenic differences between the Shandong strain and the vaccine strains.

ACKNOWLEDGEMENT

This work was supported by the National Key R & D Program for the 13th Five-Year Plan, China (No.2016 YFD0501004), Shandong Key Research and Development Program, China (No.2019GNC106074; 2017GRC5201) and the High-level Talents Introduction Project of Inner Mongolia Agricultural University (No.NDGCC2016-22).

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