H Gene-based Molecular Characterization of Field Isolates of Canine Distemper Virus from Cases of Canine Gastroenteritis

In the recent past, there were increased incidences of gastroenteritis in canines leading to severe dehydration and death. There are multiple reported causes for canine gastroenteritis; canine distemper virus (CDV) is an important viral etiology among them (Castanheira et al., 2014). It is characterized by rapid onset of severe leucopenia and loss of lymphocyte proliferation ability resulting in immunosuppression in domestic dogs and in many other carnivores (Appel and Summers, 1995). CDV belongs to genus Morbillivirus within the subfamily Paramyxovirinae, family Paramyxoviridae, order Mononegalovirales (ICTV, 2011). The virus has a broad host range infecting different species of order carnivora such as dogs, wolves, foxes, jackals, ferrets, minks, skunks, weasels, badgers, raccoons, ursidae, civets, genets, lions, genets, linsangs and hyenas (Beineke et al., 2015). It is distributed all across the globe and reportedly has high morbidity and mortality (Appel and Summers, 1995). CDV is an enveloped virus that consists of single stranded, negative sense, non-segmented RNA (Murphy, 1999). The genome is 15.7 kb in size that code for six structural proteins namely; N, P, M, F, H and L each separated by untranslated regions (Griffin, 2001). The N protein involved in transcription, replication and encapsidation of viral RNA is highly conserved of all the structural proteins (Yoshida et al., 1998). The F and H proteins are two glycoproteins of the virus; F protein is involved in fusion of cells resulting in syncytia formation (Lamb et al., 2006), whereas H protein helps in attachment of virus to the host cell receptors (Von Messling et al., 2001).
       
From clinical cases, the virus can be isolated from peripheral blood leukocytes, cerebrospinal fluid and brain samples in B95a (Mori et al., 1994), Vero, MDCK (Muthiah et al., 2008) and MV 1 Lu cells (Lednicky et al., 2004). Further, the virus can be detected using serological tests like ELISA and VNT (Twark and Dodds, 2000), molecular tests like amplification of N, F or H gene by RT-PCR followed by DNA sequencing (Ashmi et al., 2017; Mira et al., 2018) and qRT-PCR (Elia et al., 2006; Scagliarini et al., 2007); along with other techniques like direct FAT, immunohistochemistry and in situ hybridisation (Maes et al., 2003). The prevalence of CDV was previously reported from China as higher in unvaccinated animals (Luo et al., 2017).
       
Based on the molecular evolutionary patterns, CDV is grouped into Asia-1, Asia-2, Asia-3, Asia-4, Asia-5/India-1, South America-1/Europe-1, South America-2, South America-3, European wild life, America-1, America-2, Africa-1, Africa-2, Arctic, Rockborn like lineages (Bhatt et al., 2019). In India, there were reports of isolation of CDV from ocular, nasal (Swati et al., 2015; Ashmi et al., 2017), brain tissue (Pawar et al., 2011) and blood (Aarthi et al., 2015); however, we couldn’t find any report of isolation of virus from enteric samples collected from clinical cases of gastroenteritis in India. The current study emphasizes on the isolation of enteric CDV and understanding its molecular evolutionary patterns against other isolates from different geographical regions based on partial H gene.

The study was conducted at Department of Veterinary Biotechnology, College of Veterinary Science, P.V. Narsimha Rao Telangana Veterinary University, Rajendranagar, Hyderabad during the year 2018-2019.
 
Cells and virus isolates
 
Epstein-Barr virus transformed marmoset B lymphoblastoid cell line (B95a), used for propagation of virus was procured from Translational Research Platform for Veterinary Biologicals (TRPVB), TANUVAS, Chennai, Tamil Nadu and were maintained with maintenance medium containing DMEM with 1% FBS and 1X antibiotic solution (HiMedia). The same media was also used for the purpose of infection. A total of 300 fecal/rectal swabs were collected from various clinics in and around Hyderabad, Telangana during the year 2019. Briefly, the sample was emulsified in 3 ml of 0.1 M PBS (pH 7.4) containing antibiotics (100 IU/mL Benzyl Penicillin, 100 μg/mL Streptomycin sulfate), centrifuged at 10,000 rpm for 10 min at 4^oC. The supernatant collected was filtered with 0.22 µ syringe filter and 100 μl of it was used for isolation of virus after confirming by real-time PCR.
 
RNA isolation and cDNA synthesis
 
The RNA of supernatant of fecal/rectal samples or the Nobivac DHPPi vaccine (positive control) was extracted using TRIZOL® reagent (Ref:15596018; Ambion®) as per the protocol described by the manufacturer. The Nobivac DHPPi vaccine consisting of Onderstepoort strain of CDV served as positive control. The quantity of the RNA was quantified using NanoDropTMLite Spectrophotometer (Thermo Fisher Scientific). The cDNA synthesis was carried out using 1 µg of RNA as template, random hexamers as primer from the PrimeScript™ 1st strand cDNA Synthesis kit (Cat No.6110A; TaKaRa) following the protocol described by the manufacturer.
 
Screening of CDV by real-time PCR
 
The samples were screened by real-time PCR using the cDNA as a template and primers described before (Elia et al., 2006) targeting the N gene. The reaction mix was prepared as described in the manufacturer’s guidelines using Power SYBR® Green PCR Master Mix (Takara). The reaction was set for 10 μl per well in MicroAmp® Fast 96-Well Reaction Plate (Applied Biosystems), sealed with ThermalSeal RTTM (Applied Biosystems) and the reaction was run on StepOnePlus Real-time PCR (Applied Biosystems) as per the following conditions: Initial denaturation of 95^oC for 30 sec; followed by 40 cycles of 95^oC for 5 sec, 48^oC for 60 sec, 60^oC for 60 sec; followed by melt curve. The samples showing amplification with a melting temperature similar to that of the positive control on the melt curve were considered positive for CDV. The positive samples were propagated for virus isolation in B95a cells as described above.
 
PCR amplification and sequencing
 
The PCR was performed targeting partial H gene of CDV with cDNA as template and primer pairs targeting the respective genes as described previously (Ashmi et al., 2017) using Emerald Amp® GT PCR Master Mix (TaKaRa) as per the protocol described by the manufacturer. Briefly, the PCR was carried out with an initial denaturation at 95oC for 10 min; 35 cycles each of denaturation at 94oC for 60 sec, primer annealing at 53^oC for 60 sec, extension at 72^oC for 40 sec; final extension at 72oC for 10 min. The positive PCR products were purified using XcelGen® PCR Purification Mini Kit as per the manufacturer’s instructions. After purification, the purity was tested on 1% agarose gel and quantified using NanoDrop TM Lite Spectrophotometer (Thermo Fisher Scientific). The purified PCR products were sequenced at the sequencing facility, Xcelris Labs Ltd, Gujarat using the same respective gene-specific primers used for amplification (Ashmi et al., 2017). The sequences obtained are aligned with the reference sequences from NCBI using MUSCLE from MEGA7 and the changes were compared with the peaks from the chromatogram using Chromas v.2.0 and the sequence editing if needed was performed in Lasergene software (DNASTAR Inc., Madison).
 
Multiple sequence alignment and phylogenetic analysis
 
After retrieving the sequences from the chromatogram, the sequences were aligned against the other isolates from the NCBI database using MUSCLE from MEGA7. The percentage nucleotide sequence identity of current CDV isolates with other isolates was compared using NCBI BLAST. Further, the amino acid sequences of the isolates was aligned with other available sequences to understand the variations in the protein sequence. The unrooted phylogenetic tree was generated using the maximum-likelihood method using Tamura-Nei model and bootstrap value of 1000 in MEGA7.

The incidence of canine gastroenteritis is increasing consistently in the recent past despite the routine vaccination, which is posing a potential challenge for veterinary clinicians all across the globe. CDV is one among the viral etiology that can cause gastroenteritis in canines. CDV was grouped into Asia-1, Asia-2, Asia-3, Asia-4, Asia-5/ India-1, South America-1/Europe-1, South America-2, South America-3, European wild life, America-1, America-2, Africa-1, Africa-2, Arctic and Rockborn like lineages (Bhatt et al., 2019). The virus is mainly shed from occulo-nasal discharges and is present mostly in peripheral blood (Pawar et al., 2011), hence the gastro-enteric infection was least explored. Moreover, for effective control of incidence of canine gastroenteritis, it is important to understand all possible causes. In the present study, the virus was isolated from the enteric samples from suspected animals during the year 2019, India. Further, the molecular relationship of the enteric virus was compared against other isolates from the world using phylogenetic analysis.
 
Screening of CDV by real-time PCR and isolation of virus
 
Real time PCR based screening/diagnosis is cost effective and time saving. There were reports on the use of real-time PCR for screening clinical samples for CDV using a pair of gene-specific primers targeting the N gene (Elia et al., 2006).  Following similar strategy, the 300 fecal samples/rectal swabs collected in the current study were preliminarily screened by real-time PCR. The Nobivac DHPPi vaccine was used as a positive control and the clinical isolates should have the melting temperature in the range of vaccine. The Ct value of less than 25 on the amplification plot was considered as positive for CDV. In the present study, we could screen 30 samples to be positive based on the amplification plot, which accounts for a morbidity of 10%.  All the 30 positive samples were propagated in B95a cells for virus isolation. Fig 1 show the cytopathic effect caused by the CDV in B95a cells in comparison to healthy cells. Eight isolates could be obtained upon passaging in B95a cell line and were named as F102/PVNRTVU/2019, F107/PVNRTVU/2019, F108/PVNRTVU/2019, F115/PVNRTVU/2019, F128/PVNRTVU/2019, F129/PVNRTVU/2019, F158/PVNRTVU/2019 and F200/PVNRTVU/2019.
 

 
Partial H gene of CDV is highly variable
 
The H protein is a glycoprotein of CDV that helps in attachment of virus to the host cell receptors (Von Messling et al., 2001). Previous reports suggest that the H gene is highly variable with variations of amino acid in the protein (Martella et al., 2006; Sarute et al., 2013). Hence, the partial H gene was targeted in the present study for understanding the molecular epidemiology of CDV. The partial H region was amplified from the cDNA of all the eight CDV isolates along with Nobivac DHPPi vaccine as a positive control using the primers described previously (Ashmi et al., 2017). The amplification with the amplicon size of 449 bp was seen in all the eight isolates along with positive control (Fig 2). All the positive PCR products of the isolates were purified, sequenced and submitted in the GenBank of NCBI database. The accession numbers for the same are: F102/PVNRTVU/2019 H gene (MN824437), F107/PVNRTVU/2019 H gene (MN824439), F108/PVNRTVU/2019 H gene (MN824440), F115/PVNRTVU/2019 H gene (MN824435), F128/PVNRTVU/2019 H gene (MN661141), F129/PVNRTVU/2019 H gene (MN824436), F158/PVNRTVU/2019 H gene (MN661142) and F200/PVNRTVU/2019 H gene (MN824438).
 

               
BLASTN analysis for the sequence of isolates for the respective genes show that they align with the sequences of the previously published database. The sequences of respective genes for the isolates were aligned with reference and vaccine sequences to check for nucleotide or amino acid variations. We report the nucleotide variation within H gene between the isolates from current study, Nobivac DHPPi vaccine and other isolates from the NCBI database. The H gene of CDV show the nucleotide identity of 93-95% among the eight isolates and 88-95% with other available sequences in the database. Further, the nucleotide sequence alignment revealed the presence of NdeI restriction site at 1571-1576 position of H gene of all the eight isolates in current study and reference isolates, which is absent in vaccine strains (Fig 3). Previously it was reported that the NdeI restriction site was found only in wild type isolates leaving out the vaccine strains (Hashimoto et al., 2001; Calderon et al., 2007). Based on this analysis we conclude that the circulating CDV during the year 2019 from India were of wildtype and not related to vaccine strains. The amino acid sequence alignment of partial H gene for isolates in current study with respective reference isolates show variations in the protein sequence (Table 1). The H gene of CDV isolates here revealed an amino acid identity of 90-91% among the eight isolates and 83-91% with other available sequences in the database. As previously reported the H gene region is highly variable with more number of amino acid variations (Martella et al., 2006; Sarute et al., 2013). Of all the reported amino acid variations in this study, we noticed that D530G was previously reported and was consistently reported in four isolates here (Bhatt et al., 2019).
 

 

 
Molecular epidemiology of CDV
 
It is important to understand the molecular epidemiology of the enteric virus that is posing a serious threat in canines. For the vaccination and control to be effective, it is noteworthy to know the evolutionary patterns of the circulating virus. The phylogenetic tree was reconstructed for partial H gene by maximum-likelihood method using Tamura-Nei model for the current isolates against other isolates distributed geographically that were collected from NCBI database. The phylogenetic analysis revealed that the circulating virus isolates during the year 2019 from India were grouped under three different lineages; F158/PVNRTVU/2019 H gene (MN661142) with South America-2, F115/PVNRTVU/2019 H gene (MN824435) with Asia-4 and other six isolates with Asia-5/India-1. All the eight isolates show distant relationship with vaccine strains (America-1) and other isolates from the NCBI database (Fig 4). However, the previous reports suggested that the Indian isolates were grouped either under European lineage (Swati et al., 2015; Ashmi et al., 2017) or Asia-5/India-1 (Bhatt et al., 2019). Our findings suggest some CDV strains circulating in India were evolving from South America-2 or Asia-4. More importantly, we noticed that the current isolates were distinctly separated from all the vaccine strains across the world including the Nobivac DHPPi vaccine used in this study.
 

We report the isolation of CDV from enteric samples from India and their molecular epidemiology. The phylogenetic analysis revealed that the circulating strains clearly differed from the vaccine strains indicating an immediate emphasis on development of vaccines against circulating strains of CDV and installing proper vaccination measures for effective control of CDV. Study conducted with a wider sample throughout the country can provide better insights on its molecular epidemiology.

The authors acknowledge, DBT (No.BT/ADV/Canine Health/TANUVAS), India for the financial support.

The work does not involve animal experimentation; hence, animal approval is not needed. The work has been performed in accordance with standard operating procedures adopted by the Departments of Veterinary Microbiology and Biotechnology, PVNR TVU, Hyderabad.