Pathogenesis and phylogenetic analyses of canine distemper virus strain ZJ7 isolate from domestic dogs in China
- Bin Tan†1,
- Yong-Jun Wen†1,
- Feng-Xue Wang1,
- Shu-Qin Zhang1,
- Xiu-Dong Wang1,
- Jia-Xin Hu1,
- Xin-Chuan Shi1,
- Bo-Chao Yang1,
- Li-Zhi Chen1,
- Shi-Peng Cheng1 and
- Hua Wu1Email author
© Tan et al; licensee BioMed Central Ltd. 2011
Received: 9 September 2011
Accepted: 16 November 2011
Published: 16 November 2011
A new isolate of canine distemper virus (CDV), named ZJ7, was isolated from lung tissues of a dog suspected with CDV infection using MDCK cells. The ZJ7 isolate induced cytopathogenic effects of syncytia in MDCK cell after six passages. In order to evaluate pathogenesis of ZJ7 strain, three CDV sero-negative dogs were intranasally inoculated with its virus suspension. All infected dogs developed clinical signs of severe bloody diarrhea, conjunctivitis, ocular discharge, nasal discharge and coughing, fever and weight loss at 21 dpi, whereas the mock group infected with DMEM were normal. The results demonstrated that CDV-ZJ7 strain isolated by MDCK cell was virulent, and the nucleotide and amino acid sequences of strain ZJ7 had no change after isolation by MDCK cell when compared with the original virus from the fresh tissues. Molecular and phylogenetic analyses for the nucleocapsid (N), phosphoprotein (P) and receptor binding haemagglutinin (H) gene of the ZJ7 isolate clearly showed it is joins to the Asia 1 group cluster of CDV strains, the predominant genotype in China.
Canine distemper (CD) is an acute or subacute, highly contagious disease with signs of generalized infection including respiratory disease, foot pad hyperkeratosis, central nervous system disturbance or a combination of these symptoms . Its causative agent is a canine distemper virus (CDV) that is an enveloped virus particle with a diameter of 150 to 300 nm , belonging to the Morbillivirus of Paramyxoviridae family. CDV is actually a single-stranded negative-sense RNA virus (~15.7-kb RNA genome) and causes a highly infectious, systemic and fatal disease in the wild and domestic Canidae [3, 4]. The virus replicates primarily in lymphatic tissues of the respiratory tract. Temporary fever and the onset of lymphopenia appear after 3 to 6 days infection [5, 6]. Generally, an acute infection by CDV is associated with respiratory or gastrointestinal tract disease or both, and central nervous system . The genome of CDV encodes the following virion proteins: nucleocapsid (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin (H), and polymerase (L). H protein is responsible for viral attachment to host cell and may play a role in inducting the protective immunity as well . H protein is also one of the most variable morbillivirus proteins and thus has been commonly used to assess genetic changes between CDV isolates . Sequence analyses of CDV strains have been identified in diverse geographic areas and various animal species, indicating that H gene of CDV strains underwent a genetic drift related to the geographic locations of the circulating strains . Dogs infected with virulence CDV strains showed obviously clinical signs of canine distemper including conjunctivitis, ocular discharge, nasal discharge, depression, coughing, diarrhea, lymphopenia, high body temperature and body weight loss . All infected dogs were diagnosed with lymphopenia at 5 or 7 dpi, which is the most important clinical sign to reflect the immunosuppression  and may be affected by apoptosis . Lymphoid depletion started in the lymph nodes and thymus at 6 dpi without necrosis . However, the lymph node follicles of dogs that naturally infected with CDV have pathological findings from necrosis to lymphoid depletion .
An isolation of CDV strains from tissues by cell culture is difficult because the lipid-enveloped CDV is sensitive to the environment and easily inactive by heat and light . However, the field isolates of CDV have been reported to be successfully replicated in macrophages of dogs and ferrets [14, 15]. This attributed to many receptors on macrophages cell surface, such as the signaling lymphocyte activiation molecule (SLAM), which allows CDV strains entering the cells. Therefore, the CDV can be isolated by co-cultivation of lymphocytes from the suspected dogs and lymphocytes from mitogen-stimulated dogs . Kimoto focused on the Vero cell, modified and unmodified, to isolate the CDV strains . Lednicky et al. demonstrated an effective isolation of the wild-type CDV strains by MDCK, whose method is much earlier detecting the virus than others . It was known that the virulence for natural host could be lost when the CDV was adapted to the cell culture , and so the isolation of virulence CDV from the suspected dogs is more difficult . In this study, however, the virulence CDV had been isolated in MDCK cell from the infected and clinically sick dogs as early as three days after inoculation. This is may be because that MDCK cell is sensitive to the CDV filed strains and so the CDV strains can be replicated in vitro without selection and/or adaptation in the study. This method is an effective tool for the research of CD disease and development of CD vaccine candidate. In addition, the geographical lineage(s) of the current China CDV field strains have also been determined in this study.
Morphology and characteristics of new CDV isolate
The tolerance of CDV-ZJ7 strain for FUDR, ether, acid and heat
Test group (TCID50)
20% Ethylether, 4°C, 24 h
pH3.0, 37°C, 2 h
50°C, 30 min
Cytopathic effect (CPE) on MDCK cell and IFA detection of CDV antigen
Clinical features of the infected dogs
CDV detection by RT-PCR and virus recovery
CDV in the swabs from the infected dogs were detected by RT-PCR. The virus was still detected in conjunctival, nasal and swabs of one dog at 8 dpi and other dogs at 14 dpi. The virus was also re-isolated from the swabs of all infected dogs in MDCK cells, and no CDV were detected in the mock dogs By RT-PCR.
Sequencing and phylogenic analyses for N and P genes of CDV
Analyses for the amino acid sequences of H gene from wild-type CDV strains
In this study, a new ZJ7 isolate have been isolated from CV diseased dogs in Jilin province, a recent representative of CDV in China northeast, and the identification of the isolate have been confirmed by virulence investigation and molecular analysis. 100% identities of nucleotide and amino acid sequences of H and P genes have been determined between the ZJ7 strain isolated in MDCK cell and the original virus from infected fresh tissues (data not show). The results indicated that the CDV was genetically stable after isolation within a few passages and the MDCK cell was a suitable cell line to isolate CDV from fresh tissues. The ZJ7 strain isolated from several clinical cases were genetically distinct from the known vaccine strains, as previous studies of other CDV positive cases with history of CDV vaccination [19–22].
Many other studies have demonstrated the geographically distinct lineages of CDV strains by phylogenetic analysis of their H genes [19–23]. To phylogenetic analysis of ZJ7 isolate, we used the Onderstepoort (GenBank accession no. AF378705), the Snyder Hill (AF259552) and the TN (AF390348) strains as references of current China field isolates. A high degree of identity was detected among all studied China wild-type strains, which were separated from the vaccine strains from GenBank. The connected aspartic amide N glycosylation site potentially is a spotlight in H proteins between vaccine and wild strains of CDV. Usually, there are four (Onderstepoort strain) or seven (Convac strain) potential sites in the vaccine strains. However, Eight or nine sites have been detected in all wild CDV strains, of which 309 ~ 311 N-connected amide asparagine glycosylation sites are specific to CDV field strains . It was consistent with pathogenic analyses of CDV ZJ7 strain in this study, where eight potential N-connected amides asparagine glycosylation including 309 ~ 311 have been confirmed in ZJ7 isolate . Some studies believed that the variants from H protein glycosylation played a crucial role in the antigenic differences . In addition, the predicted amino acids of ZJ7 isolate lacked 3 amino acids SKP compared with the Onderstepoort vaccine strain (Figure 6), but it had difference in 9 amino acids from another China wild-tpye TN strain.
In this study, a CDV ZJ7 strain was successfully isolated from lung tissues of the dogs suspected with CDV infection using non-modified MDCK cells. The ZJ7 strain still induced pathogenic effects to the infected dogs after six passages, whereas the mock group infected with DMEM is normal. In addition, molecular and phylogenetic analyses of N, P, and H gene on ZJ7 isolate have clearly indicated its joins to the Asia 1 group cluster of CDV strains, the predominant genotype in China. In a word, this new CDV ZJ7 strain isolated by MDCK cell remains virulent, and its nucleotide and amino acid sequences are still conservative.
Materials and methods
Cell lines and culture
Cell line of MDCK (canine epithelial kidney cells) was purchased from American Type Culture Collection (ATCC) (Manassas, VA). The cells were grown routinely in polystyrene tissue culture flasks with filtercaps (Nalge Nunc International, Rochester, NY) at 37°C in a 5% humidified CO2 atmosphere. The culture medium was composed of Eagle's modified minimal essential medium with Earle's salts (EMEM-E) (Cellgro, Mediatech, Inc., Herndon, VA), 2.5 or 10% (as specified per application) heat-inactivated fetal bovine serum (HI-FBS, from Cellgro), antibiotics of 100 units/mL penicillin, 100 μg/mL streptomycin and 50 μg/mL gentamycin, 1 mM sodium pyruvate, 1X non-essential amino acid solution, 2 mM glutamine, and 0.075% (w/v) sodium bicarbonate.
Viruses and clinical specimens
The CDV3 and Onderstepoort vaccine strains used in this study were maintained in the Division of Zoonoses, Institute of Special Economic Animal and Plant Sciences, China. The organs of the infected dogs were obtained from an animal hospital in Jilin province of China at 2009. The suspect of CDV infection was initially found by the doctors on the basis of the clinical signs (fever, respiratory, enteric, and hyperkeratosis signs).
Virus isolation and titration
The virus isolation and titration were conducted as described previously . Briefly, the tissue samples from the infected dogs were collected from their necropsies and then stored at -80°C. Homogenates containing 0.2 g of lung samples and 2 mL of Dulbecco's modified Eagle's medium with antibiotics were sonicated and centrifuged. A monolayer of MDCK cell was infected with the homogenates in a 24-well culture plate for isolation. ZJ7 isolate was titrated in a 10-fold dilution using a 50% tissue culture infectious dose (TCID50) assay  in a 96-well culture plate.
Virus identification by electron microscope (EM) and immunofluorescence assay (IFA)
Before observing under EM, the MDCK cell infected with the virus isolates were harvested by freezing and thawing for three times, and a 1 mL harvested cell culture was centrifuged for 5 min at 800 ×g. The supernatant was then transferred into a new microtube and centrifuged for 10 min at 13, 400 ×g. A negative stain was prepared for transmission electron microscope observation. The observed virions were photographed and analyzed. As conventional methods , the isolated virus were tested by 5-fluoro deoxyuridine (FUDR), ethylether, acid and high temperature (Table 1). A 96 well microtitre plate (Costar, NY, USA) was seeded with MDCK cell in MEM with 10% NBCS, and cultivated at 37°C in 5% CO2 overnight until 70-80% confluence. The cultures were then inoculated with 20-fold diluted field CDV isolates at 3 passage. The uninfected cultures in left rows of the plate were taken as the negative controls. After 72 hrs incubation in 37°C, 5% CO2 atmosphere, the plate was fixed in 80% cold acetone/PBS, and then washed and incubated with mAbs at a 50-fold dilution. After washing with PBS, FITC-conjugated polyclonal antibody (Sigma, St. Louis, MO, USA) was added to the plate, and then followed by 1 h incubation in a 37°C humid box. After 3 times washes with PBS, a 50% glycerol in PBS was added to each well. Two infected wells were treated as a positive control to confirm viral growth. Fluorescence signal was observed using an fluorescence inverted microscope (Zeiss Axioskop-40, Germany).
Six 2-month-old female dogs, whose serology is negative to CDV, were purchased from Shifang Experiment Animal Corporation (Jiangshu, China) and raised in the isolated cages. All animal work and experimental procedures were conducted with an approval of Institutional Animal Care and Use Committee of Jinlin University, China.
Virus infection and sample collection
A CDV ZJ7 isolate, which has been directly isolated from lung tissues of a dog showing pathological changes consistent with canine distemper at necropsy, was passaged at least five times in MDCK cell before it was used to form a viral suspension at concentration of 1.2 × 106 TCID50 /1 mL. Under anesthesia with protocol (Institute of Special Economic Animal and Plant Sciences, Jilin, China), the viral suspension was dropped into the right conjunctiva and nostril of three dogs using a syringe without a needle. The infection was monitored daily by clinical and neurological examination until the dogs were euthanized with Nembutal (Solabio Pharmaceutical Co. Ltd., China) at the end of the experiment. The tissue samples were obtained all infected dogs. Clinical signs and rectal temperatures of the dogs were daily recorded. Nasal, tonsilar, conjunctival, rectal and vaginal swabs, which were used for reverse transcriptase (RT)-PCR and virus reisolation, were collected at 0 (before inoculation of virus), 5, 7, 9, 10, 12, 14, 19, 21, 23 and 28 dpi. All samples for isolation were stored at -80°C until being used.
Virus detection and recovery
A suspension containing 40 μl of nasal, tonsilar, conjunctival and vaginal swabs and antibiotics of 1000 units/mL penicillin and 1000 μg/mL streptomycin was inoculated into MDCK cell seeding in a 24-well culture plate. The cytopathogenic effect was observed by phase contrast microscopy. The presence of CDV was confirmed by reverse transcriptase-PCR with a specific CDV N gene primers: Upper: 5' GATAAAGCATGTCATTATAGTCCTAA 3' and Lower: 5'CTTGAGCTTTCGACCCTTC 3', and the expected fragment was 335 bp. Briefly, all RNA was extracted using the RNAeasy kit (QIAGEN). The extracted RNA was immediately used for RT-PCR or preserved at - 80°C before use. cDNA synthesis was performed with SuperScript II reverse transcriptase (Invitrogen) and oligodeoxy nucleotide primers by 10 mL of RNA sample and random primers as reverse transcription primer. The PCR amplification of cDNA was carried out in a 50 mL solution containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 3 mM MgCl2, 0.5 mM dNTP and 200 pmol of each primer accordingly. The internal gene sequences of primers, 5mL cDNA and 2.5U EXTaq DNA polymerase (Takara) were given in the Table 1. The PCR amplification cycle was optimized as follows: 94°C 45s, 52.2°C 45s and 72°C 45s, for 35 cycles with a final extension step at 72°C for 5 min.
Titration of VNA against CDV
For the neutralization assay, the antibody titre was measured in a 96-well culture microplate in Vero cells . Quadruplicate 0.05 mL serum was diluted 3-fold serially, and a 0.05 ml CDV solution with 10 TCID50 of the onderstepoort strain was added to each well, and then the plate was incubated for 90 min at 37°C. Subsequently, the Vero cells at concentration of 1.2 × 104 /0.05 mL were added to the serum-virus mixtures, and incubated for 7 days at 37°C, CO2. The plate was examined microscopically, and the titre was expressed as the highest dilution showing 50% inhibition of cytopathic effects. Serum from a dog was vaccinated with attenuated live CDV vaccine as positive control, while virus dilution without serum was used as negative control. The titre was calculated by the Reed and Munch method .
Phylogenetic analyses for nucleotide and amino acid sequences
Primers for RT-PCR and sequence analyses of CDV N, P and H genes
AACAA GGCTA GGGTT CAGAC CT
TTGTT GACTG ATGCA AGACT GGT
CGACC ACCCG TTCTA TC
GCGGA CTTAG GCTCT TGT
CTTAG GGCTC AGGTA GTCCA
ATTCA ATCGT CTGTA AGGGA
The authors would like to thank Wei-Jing Chen for the assistance during this work. This study was supported by the national science and technology supporting plan (2009BADB4B02) funded by the Ministry of Science and Technology of China.
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