Serological and Nucleocapsid Gene Based Molecular Characterization of Canine Distemper Virus (CDV) Isolated from Dogs of Southern Gujarat, India

Canine Distemper virus (CDV) is the fatal virus of canines and felines that affects dogs, foxes, wolves, lions and tigers (Guo et al., 2013). CDV comes under the genus Morbillivirus, subfamily, Paramyxovirinae, family Paramyxoviridae and in order of Mononegavirales (Murphy et al., 1999). It is a single-stranded, non-segmental, enveloped and negative-sense RNA virus having 150-300 nm size (Murphy et al., 1999). Genome of CDV is approximately 15.7 Kb having six structural proteins: haemagglutinin (H), large protein (L), phosphoprotein (P), nucleocapsid protein (N), fusion protein (F) and matrix protein (M) and one non-structural protein (C) which is produced by an alternative open reading frame in P gene (Lamb and Kolakofsky, 2001; Swati et al., 2015). CDV is multi-organ tropism virus which causes multiple organ damage and systemic disease. The virus primarily replicates in lymphatic system of respiratory tract and later on reaches to various organs including, ocular, brain, lymphoid organs, urinary bladder, respiratory system and gastro intestinal tract (GIT) (Appel, 1987). Even, it has more affinity to respiratory, central nervous system and GIT leads to severe damage and clinical manifestation (Patel and Heldensb, 2009).
       
It is highly immunosuppressive virus to the host that increases susceptibility to several secondary infections, which is the prime reason of death (Pawar et al., 2011; Agnihotri et al., 2018). CDV is deadliest in young puppies which cause sudden death within few days of infection. However, CDV vaccine is among the core vaccination for dogs, puppies that left unvaccinated are more prone to infection. CDV infection incidences are mostly seen in winter season. The disease is easily characterized by onset of clinical sings. Various laboratory techniques like rapid lateral flow assay (LFA), dot ELISA, virus isolation and characterization have been attempted in past for confirmative diagnosis making (Parthiban et al., 2000; Ramadass and Latha, 2001; Pawar et al., 2011; Agnihotri et al., 2018). Rising CDV infection seems to have increasing illegal commercial trading of pups (Elia et al., 2006), inappropriate vaccines (Kommonen et al., 1997) and unregulated vaccination.
       
Confirmative diagnosis is necessary to rule out different diseases as multiple clinical sings are overlapping with many diseases of dogs. Thus, it is required to do sensitive and molecular based diagnosis as it can detect even low amount of viral RNA in samples. Reverse transcriptase polymerase chain reaction  (RT-PCR) has been successfully applied on various samples for diagnosis of CDV in past (Frisk et al., 1999; Kim et al., 2001; Shin et al., 2004; Saito et al., 2006; Sachdeva, 2011; Swati et al., 2015; Swati et al., 2016; Agnihotri et al., 2017; Ashmi et al., 2017; Agnihotri et al., 2018). Mostly N gene based diagnosis was found more common because, it is highly conserved gene as compare to other. Nested/One step RT-PCR was employed to characterize the virus based on its N gene. Kim et al., (2001) have used N gene based molecular characterization. Perhaps, clinically at field condition demanding faster and point of care (POC) diagnosis at veterinary dispensary like, canine parvovirus detection can be faster by employing rapid diagnostic kit LFA and commercial dot ELISA (Desai et al., 2020). Thus, initially applying rapid POC based test are highly encouraged with subsequent molecular based diagnosis and virus isolation at last. Isolation of virus in different cell line was successfully attempted by Pawar et al., (2011); Sachdeva (2011) and Swati et al., (2015). Most of the seroprevalence study was reported from south India (Latha et al., 2007; Ashmi et al., 2017). Hence, attempts were made in the present study for diagnosis of CDV infection in dogs, its characterization and prevalence at southern Gujarat region of India.

Collection and preparation of samples
 
We analyzed 40 different samples collected from suspected dogs of CDV infection showing varying clinical signs viz. nasal discharge, respiratory distress, chorea etc. presented in Table 1. Samples were collected during August 2017 to August 2018 and processed at Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, Navsari Agricultural University, Navsari, Gujarat, India. All the samples were collected in sterile swabs and vials which were immediately transported with ice pack to the laboratory. Nasal and ocular swabs were processed by adding one ml of phosphate buffer saline (PBS) followed by vigorous vortex for 3-4 minutes and stored in -20°C until further use.
 

 
Immunochromatography based lateral flow test (LFA)
 
Immunochromatography based Lateral Flow test namely CDV Ag Test Kit (cat. no. RG1103DD) were procured from Bionote, Inc. (Republic of Korea) and used for rapid antigen detection of CDV. Briefly, swabs were merged into sample collection/diluent tube (supplied with kit) and stirred at least for 10 times. Swabs were discarded while squeezing against the wall of the tube. Then, supernatant was used by adding 4 drops into sample hole on the test device. Results were noted within 5-10 minutes.
 
Indirect enzyme linked immunosorbent assay based on IgG antibody detection (i-ELISA)
 
Indirect enzyme linked immunosorbent assay (i-ELISA) was employed to detect IgG antibodies against CDV in serum samples of dogs. Ingezim Moquillo IgG (cat. no. 15.CDG.K1, Inmunologia Y genetica aplicada, Spain) commercial ELISA kit was purchased. i-ELISA was performed according to the manufacturer’s protocol. Briefly, serum samples were diluted to 1:100 dilution in diluent supplied with kit. Hundred µl of positive, negative and each diluted serum samples were added to pre-coated ELISA plate (supplied with kit) and incubated for 10 minutes at room temperature (RT). Then after, four times plate was rinsed using 300 µl of wash buffer. After that, 100 µl of conjugate was added to each well and incubated for 10 minutes at RT. Plate was washed for four times and then 100 µl of substrate solution was added and kept for 5 minutes at RT. Finally, 100 µl of stop solution was added to each well and absorbance at 450 nm was noted for each sample within 5 minutes. Serum titres based on optical density (O.D) was calculated by following the kit instruction.
 
Viral RNA extraction
 
Total viral RNA from the samples like, nasal swab, ocular swab, pustular sample and cerebrospinal fluid were extracted by using QIAamp viral RNA mini kit (cat. no. 52906, Qiagen, Germany) according to manufacturer’s protocol. Briefly, samples were added to the viral lysis buffer and incubated for 5 minutes at RT. Ethanol was added and vortex for 1 minute. Then all material was transferred to the spin column (supplied with kit) and pure RNA was extracted by washing the column for two times by using supplied wash buffer. Finally, RNA was eluted in 60 µl of elution buffer and quality was checked by using Nanodrop 1000 (Thermo Scientific, USA) and stored in -40°C until further use.
 
One step reverse transcription polymerase chain reaction (One step RT-PCR) for initial screening
 
All samples were initially screened by employing one step RT-PCR using in house design N gene based primers (synthesized by Eurofins India, Bengaluru) (Table 2) and SuperScript III One-step RT-PCR kit (cat. no. 12574026, Invitrogen). Primers were design by retrieving published sequences of CDV N gene and aligned by CLUSTAL W. The most suitable and conserved sequence was selected (Accession No. KC667070.1) and primers were design by using Primer 3 online tool (Koressaar and Remm, 2007; Untergasser et al., 2012). One step RT-PCR reaction was made by adding Extracted 2 µl RNA to 12.5 µl of 2X reaction mix, 2 µl (20 pmole) each forward and reverse primer, 1 µl of SuperScript III RT enzyme and remaining nuclease free water was added to make 25 µl of volume. Reaction carried out in Thermal Cycler (Veriti, Applied Biosystems) by making initial cDNA synthesis on 55°C for 30 minutes, RT enzyme inactivation and pre-denaturation on 94°C for 2 minutes. Then after, forty cycles of 94°C for 15 seconds, 46°C for 30 seconds and 68°C for 30 seconds. Final extension was done on 68°C for 5 minutes. The amplified products were analyzed by 1.5% agarose (cat. no. 50004, SeaKem LE agarose, Lonza, USA) gel electrophoresis in 1× TBE buffer at constant 70 V for 1 hour and visualized by ethidium bromide and 100 bp ladder (cat. no. 239045, GelPilot 100 bp Plus ladder, Qiagen, Germany) as a DNA size marker under gel documentation system (Syngene, UK).
 

 
Nested one step RT-PCR
 
Nested One step RT-PCR was used to characterize N gene as reported earlier by Kim et al., (2001). Primers were described in Table 2. Nested one step RT-PCR was carried out according to Kim et al., (2001) and Pawar et al., (2011). Initially, partial N gene was amplified by outer primer by following one step RT-PCR method including, cDNA preparation and thermal cycler amplification condition as described earlier in article except annealing was done on 42°C. Amplified RT-PCR product visualized similarly as mention earlier elsewhere. Second round of RT-PCR was done for targeting internal control by using inner primer and taking earlier amplified product (RT-PCR product amplified by outer primer) as a template and following the same protocol as described earlier.
 
Restriction endonuclease (RE) analysis
 
Restriction endonuclease (RE) analysis was done by following Kim et al., (2001). Briefly, 50 µl of reaction having1 µl of AvaI enzyme, 5 µl of reaction buffer, 2 µl of outer primer RT-PCR amplified product and remaining nuclease free water added which is incubated at 37°C for 1 hour. Digested product was visualized on 1.5% agarose gel electrophoresis. Digested product expected to have 389 and 160 bp band on visualization.
 
Virus isolation
 
Virus isolation was done by using Madin Darby Canine Kidney (MDCK) cell line by following Sachdeva (2011) and Swati et al., (2015). MDCK cell line procured from National Centre for Cell Science (NCCS), Pune was used for virus isolation. Briefly, CDV suspected and CDV confirmed samples were inoculated at a volume of 1 ml into 25 cm² tissue culture flask (cat. no. 156340, Nunc EasY Flask 25 cm²) containing sub-confluent monolayer of MDCK in serum free plain DMEM media. The flask was incubated at 37°C for 1 hour for virus adsorption. Then after, cells either washed with Dulbecco’s Phosphate-Buffered Saline (DPBS) or directly supplied with DMEM maintenance-media containing non-essential amino acids (Hyclone), 2% serum (HiMedia), 1 X antibiotic-antimycotic solution (HiMedia). An uninoculated MDCK cell line flask was kept as a negative control. The flasks were incubated at 37°C with 5% CO₂ humidified incubator and examined daily for cytopathic effects (CPE) up to 7 days. At the end of the duration and every passage, cultural fluid was checked for CDV by one step RT-PCR.

Immunochromatography based lateral flow test
 
It is rapid, simple and easy to perform lateral flow test, representative photograph is depicted in Fig 1. Only two dogs were found positive by LFA test whereas 10 dogs were positive in CDV detection in one step RT-PCR. Though LFA is rapid in detection, it gives false negative results. It might be due to limit of detection and low device sensitivity along with secretion of virus might be low or interrupted which could be the reason of false negative results in nasal swabs, ocular swabs and CSF. Similarly An et al., (2008) reported slightly lower sensitivity of LFA for detection of CDV. On other hand Agnihotri et al., (2018) found LFA test more sensitive for CDV antigen detection than RT-PCR as they have used faecal samples for RT-PCR, whereas serum sample for LFA. Hence, selections of samples are also important in diagnosis of CDV infection.
 

 
Indirect enzyme linked immunosorbent assay based on IgG antibody detection
 
Indirect IgG based detection kit was used for antibodies detection from sera samples. Representative photograph of i-ELISA is presented in Fig 2. Fourteen dogs out of 18 samples were found positive for i-ELISA. As stray dogs were not vaccinated for CDV, all serum samples found positive for IgG antibody considered as confirmed case of infection. Usage of serum neutralization test is time and cost intensive (von Messling et al., 1999) therefore, ELISA prefer in low cost laboratory set. Established disease can be diagnosed by finding anti-CDV IgG through ELISA, as it is more accurate and sensitive. Generally, anti-CDV IgM detecting ELISA should prefer over IgG because of routine vaccination practices in pet dogs. Anti-CDV IgM detecting ELISA could detect active infection. However, vaccination is not practices in stray dogs in India. Therefore, anti-CDV IgG ELISA could be used. Even, von Messling et al., (1999) found comparable sensitivity of ELISA and RT-PCR for detection of CDV.
 

 
One step RT-PCR
 
One step RT-PCR was employed for initial screening by using in house designed N gene based primers followed by nested one step RT-PCR. Thirteen samples of 10 dogs were found positive by one step RT-PCR (Fig 3). One step RT-PCR is most reliable over LFA in antigen detection due to its high sensitivity. Our finding is in accordance with Pawar et al., (2011); Ashmi et al., (2017) and Agnihotri et al., (2018). Usage of One step RT-PCR over conventional RT-PCR is more reliable, easy, less time consuming and less tedious. Transitional period is much less in one step based RT-PCR kit whereas conventional method is much tedious. Kim et al., (2001) compared the one step and conventional RT-PCR for detection sensitivity and specificity, where they find similar results. Agnihotri et al., (2017) used additionally H gene for RT-PCR along with N gene for detection of CDV infection. However, Pawar et al., (2011) used H gene, Ashmi et al., (2017) used H and F gene for phylogeny construction and nucleotide characterization as F protein help in fusion between virus and infected cells and H protein is crucial for viral entry. As, different genotypes and strains are classified based on H gene sequences. Hence, any changes in these genes monitored for nucleotide variability which identify presence of circulating strain. Perhaps our study was in accordance with Kim et al., (2001) and Kapil et al., (2008) which emphasis on characterization of nucleocapsid gene. CDV was found in both ocular and nasal swabs. Ocular and nasal swabs both are ideal sample and must require at the time of sample collection for diagnosis of CDV. Although, more studies are required to find out CDV secretion pattern.
 
 

 
Nested one step RT-PCR and RE analysis
 
All positive samples were used for nested one step RT-PCR for N gene based characterization. Thirteen samples of 10 dogs found 549 bp by outer primer and 419 bp by inner primer (Fig 4) that were similar with the past studies of Kapil et al., (2008) and Pawar et al., (2011). On RE digestion by AvaI, characteristic fragments of 389 and 160 bp were seen (Fig 5). Our findings are in accordance with Kim et al., (2001) where they have found two fragments which showed the confirmed CDV isolate and characteristic of N gene remain same over the period of time.
 

 

 
Virus isolation
 
Positive samples were used for virus isolation in MDCK cell line. It took 24-72 hour for showing CPE (Fig 6). Characteristic CPE was seen like giant cell formation, syncitia formation and destruction and detachment of cells which are similar to Sachdeva, (2011). CPE formation was detected in 2^nd passage of samples. Initially CPE was not observed in three samples rather detachment of cells were observed within 24 hours. This might be due to higher titer of virus in inoculum samples at initial infection to MDCK cells. In subsequent passage, CPE observed in almost all positive samples. We had successfully grown CDV in MDCK cell line. Attempt of screening negative samples were carried out up to 3^rd passage, where all samples were found negative for CPE and one step RT-PCR. Swati et al., (2016) used dog lymphocyte by stimulating with Phytohaemagglutinin A (PHA) for CDV isolation then after used MDCK cell line but it has no difference in CPE as well as in infectivity titre with or without stimulation as reported earlier by Appel et al., (1992). Lednicky et al., (2004) reported variation in the formation of CPE in different cell lines. Generally, Vero cells expressing SLAM were efficient for the isolation of CDV and CPF formation. Same way, B95a cells were used for virus isolation (Pawar et al., 2011; Haritha, 2019), However, Sachdeva, (2011); Swati et al., (2016) and Keerthi, (2019) had successfully isolated virus in MDCK cell line and found characteristic CPE which were accordance to our findings.
 
Prevalence of CDV
 
Overall 14 (77.77%) dogs out of 18 found positive for CDV infection. As described in table 3, on age group wise percent positivity was found to be 71.42% (05/07) in 0 - ≤6 months, while 77.77% (07/09) in 6 - ≤12 months of age group, whereas, both the two samples were found positive in 12 months ≤ of group. Latha et al., (2007) found 70% of prevalence of CDV in suspected sample where they have found that dogs of age group of 1-5 years are more susceptible which is partially in accordance with our study where almost all age group of dogs infected with CDV. Similarly, Jozwik and Frymus, (2002) reported that 3-6 months of dogs are more susceptible. Generally, CDV infect almost all age group and unvaccinated dogs.
 

       
CDV is lethal virus for canines and felines, that’s why to prevent the disease, vaccination must be followed in susceptible population. Stray dogs are the major risk factor in transmission of disease to other dogs and wild animals. Interference of stray dogs with others is very critical in outbreaks as well as big threat in emergence of wild type new strain of virus. Therefore, vaccination should be the compulsory preventive measurement in enzootic area but in developing country like India, most of the stray dog population residing in rural area and free ranging, as free stray range dogs are not vaccinated against the prevailing disease, they are the major sources of infection (Ashmi et al., 2017). Further study on complete gene or genomic characterization required to find out viral strains presently circulating in our country which could help in designing of vaccine candidate.

We want to acknowledge Dr. V. B. Kharadi, Dean, College of Veterinary Science and Animal husbandry, Navsari Agricultural University, Navsari, for providing necessary funds to carry out work. We also want to extend our thanks to Mrs. Akruti Patel, Mrs. Amisha Chaudhari and Mrs. Akruti Naik, lab technicians of our department for work, support and help.