Optimization of lateral flow assay for Canine morbillivirus detection and the application of the strip as sample substitute.

Canine distemper is an emerging disease, caused by the Canine morbillivirus (CDV) of the Paramyxoviridae family. The virus has evolved as a multi-host pathogen as it affects many wildlife animal species. The development of specific and sensitive diagnostic tests is the need for a control program. Several diagnostic tests are available for the detection of CDV antigen and antibody. Lateral flow assay (LFA) is the most promising point of care diagnostic test because of its specificity, easy use, and instant result. This study was designed to develop a lateral flow assay using the in-house developed monoclonal antibody (mAb) against the nucleocapsid protein (N) of the 'CDV/dog/bly/Ind/2018' isolate, which represents the circulating strains of India. The two mAbs included in the study showed high binding affinity in indirect ELISA and dot blot assay. Out of two, one mAb was selected due to its comparatively higher binding affinity in LFA format, and less non-specific binding to the biological matrix and buffer components. The limit of detection was found to be 106.5 TCID50/ml with the assay run time of 5 min. The fresh clinical samples collected on the spot were distinctly detected by the LFA, whereas the stored samples with a reduced titre of the virus showed inconsistent results. Moreover, the blood samples showed a clear distinction of positive and negative than the swab and tissue homogenates. The RNA extraction from the strip was successful with the some modifications in the Trizol RNA extraction method and the N and H gene fragments were amplified. Therefore, the study concludes that the LFA is suitable for CDV antigen detection in the field conditions and the strips can be used as the sample substitute for molecular study.

Molecular epidemiology of swinepox viruses circulating in India.

Swinepox is a sporadic virus disease of domestic and wild pigs that mainly occurs during the rainy season. Though the disease is known for a century, research on swinepox especially genetic characterization is scanty. Self-limiting nature of the disease, the non-availability of specific diagnostics as well as the resemblance of clinical signs with other pathogens are some of the issues in the slow progress in swinepox-related research. Recent whole genome sequencing data from the USA, India, and Germany enhanced our understanding of the biology of swinepox virus (SWPV). The objective of the present study is to investigate the molecular epidemiology of two swinepox outbreaks that occurred in 2015 and 2016 one each in Uttar Pradesh, and the Haryana states of India. The appearance of clinical signs in different swine breeds was recorded. The scab samples from infected pigs were collected, DNA extracted, host range genes of SWPV were PCR amplified, sequenced and analyzed for genetic and phylogenetic characterization. Desi (nondescript breed), Yorkshire White pigs, and Landrace cross were found to be infected with SWPV. Host range genes of SWPV analyzed from clinical samples showed very high nucleotide identity with each other. Phylogenetic analyses revealed that SWPVs circulating in India are distinct (Indian lineage) from the SWPV of the USA, Germany, and Russia (European-North American lineage). Our study affirms the existence of two distinct lineages of SWPV globally with differences in clinical lesions between breeds.

Development and characterization of mouse monoclonal antibodies to canine morbillivirus.

Canine morbillivirus is a highly contagious multi-host pathogen with high morbidity and mortality. Timely diagnosis is of utmost importance to effectively control such a dreadful disease. Monoclonal antibodies (mAbs) serve as a high throughput diagnostics and applied tools for research and development (R&D). In the present study, a total of six mouse monoclonal antibodies were developed. All the mAbs generated belonged to IgG class. Of the six mAbs, two of them, namely CD-2F8 and CD-3D8 were directed against the nucleocapsid protein of CDV as determined in western blotting. The reactivity of all the mAbs was checked in indirect-ELISA and cell-ELISA using different morbilliviruses. The mAbs could broadly be categorized as; CDV specific (CD-3D8 and CD-2F8), cross-reactive to PPR virus (CD-AB3 and CD-4D6) and cross-reactive to both PPR virus and measles virus (CD-5D10 and CD-6E5). The characterized mAbs were used for antigenic profiling of CDV, PPR virus and measles virus. Based on the reactivity pattern; a close antigenic relationship was found among CDV and PPR virus as compared to measles virus. A pair of CDV specific mAbs namely CD-2F8 and CD-3D8 were identified which did not cross-react with measles and PPR viruses and thus could be used for diagnostic applications.

Seasonal pattern in occurrence of rotavirus infection (RV) in diarrheic children, calves and piglets from Bareilly, India.

Rapid and reliable diagnosis for diarrhoeal disease is critically important for the differentiation of etiological agents and subsequent suitable treatment modalities. The objective of the study is to reveal the seasonal pattern in the occurrence of rotavirus in diarrheic children, calves and piglets from Bareilly, Uttar Pradesh, India. A total of 115 diarrhoeal samples were collected, out of which 51 were collected during post-monsoon/autumn (September 2018-November 2018) and 64 during the winter season (December 2018-February 2019). The samples were collected from children <5 years (n = 50), piglets <3 months (n = 35) and calves <6 months of age (n = 30). These samples were screened by ribonucleic acid-polyacrylamide gel electrophoresis (RNA-PAGE) and reverse transcriptase-polymerase chain reaction (RT-PCR) by targeting the VP6 gene of rotavirus A (RVA) and the two were compared. In RNA-PAGE 29.4% (5/17), 6.3% (1/16) and 0% (0/18) samples collected from children, calves and piglets, respectively were rotavirus positive during the autumn season while 45.5% (15/33), 21.4% (3/14) and 17.7% (3/17) samples in the winter season. In RT-PCR, 41.2% (7/17), 12.5% (2/16) and 0% (0/18) samples were rotavirus positive in the autumn season while 51.5% (17/33), 28.6% (4/14) and 29.4% (5/17) samples in winter season collected from children, calves and piglets, respectively. On statistical analysis, no significant difference between the season and number of positives in children and calves (p > 0.05) was observed, however in piglets significantly higher number of RVA positives were detected in the winter season than autumn (p < 0.01). The diagnostic test comparison of RNA-PAGE and RT-PCR showed no statistically significant difference in detecting the RVA positives (p > 0.05). Overall the percent positivity showed a seasonal pattern with higher positivity in winter as compared to autumn season.

Differentiation of bovine herpesvirus1 subtypes based on UL0.5 gene sequencing.

Infectious bovine rhinotracheitis/infectious pustular vulvovaginitis is one of the high economic importance diseases of cattle and caused by bovine herpesvirus1 (BoHV1). Based on the restriction endonuclease fingerprinting of viral DNA, the BoHV1 can be divided into three subtypes viz., BoHV1.1, 1.2a, and 1.2b. Since this method requires a pure viral DNA, it is time-consuming and labour intense. In the current study, the UL0.5 gene based PCR sequencing has been used for the subtyping of BoHV1. Out of five isolates, four had BoHV1-like signatures and one isolate had BoHV1.2-like signatures. Further, these viruses phylogenetically clustered under the respective subtypes. These results indicate that the UL 0.5 gene based PCR sequencing could be used as an alternate method of subtyping of BoHV1.