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.

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.

Canine morbillivirus (CDV): a review on current status, emergence and the diagnostics.

The increasing host range of canine morbillivirus (CDV) affecting important wildlife species such as Lions, Leopard, and Red Pandas has raised the concern. Canine distemper is a pathogen of dogs affecting the respiratory, gastrointestinal, and nervous systems. Seventeen lineages of CDV are reported, and the eighteenth lineage was proposed in 2019 from India. Marked genomic differences in the genome of wild-type virus and vaccine strain are also reported.The variations at the epitope level can be differentiated using specific monoclonal antibodies in neutralization tests. Keeping in mind the current status of the emergence of CDV, genetic and molecular study of circulating strains of the specific geographical region are the essential components of the disease control strategy. New target-based diagnostics and vaccines are in need to counter the effects of the emerging virus population. Control of CDV is necessary to save the endangered, vulnerable, and many other wildlife species to maintain balance in the ecological system. This review provides an overview on emergence reported in CDV, diagnostics developed till today, and a perspective on the disease control strategy, keeping wildlife in consideration.

Genetic analysis of two viroceptor genes of orf virus.

In the present study, we analyzed the chemokine-binding protein (CBP) and the GM-CSF/IL-2 inhibition factor (GIF) of orf virus (ORFV) isolates of sheep and goat origin from different geographical regions of India. Both are immunomodulatory proteins known for their unique strategy of establishing short-term immunity and re-infection in their host. The GIF gene is highly conserved, whereas the CBP gene is highly variable. Both the proteins have conserved potential N-glycosylation sites. The GIF protein contains the "WDPWV" motif responsible for receptor activation. In addition, the SUSHI/short consensus repeats (SCR) domain is reported for the first time in ORFV. Both proteins could potentially be used as immunotherapeutic agents in inflammatory diseases related to the overexpression of specific cytokines.

Circulation of orf viruses containing the NZ7-like vascular endothelial growth factor (VEGF-E) gene type in India.

Orf, a poxviral skin infection of small ruminants is caused by orf virus (ORFV) of the genus Parapoxvirus of the Poxviridae family. Vascular endothelial growth factor (VEGF) is an important virulence factor that is responsible for proliferative lesions in parapoxviral infections. VEGF gene shows high intra- and inter-species variability. Two variants of VEGF have been described globally in ORFV, viz. NZ2- and NZ7-like. In the present study, ORFV isolates of different geographic regions of India were analysed on the basis of the VEGF gene. Indian ORFV isolates showed 95.7-100 % nucleotide (nt) and 78.4-99.3 % amino acid (aa) identity with each other, except ORFV-Assam/LK/14 and ORFV-Meghalaya/03 which shared 85.1-88.35 % and 79.1-81.8 % identity, at nt and aa levels, respectively with other Indian ORFV isolates. All Indian ORFVs under the study demonstrated 83.5-99.1 % nt and 80.5-97.9 % aa identity with NZ7-like VEGF as compared to 41.2-44.8 % nt and 30.7-38.4 % aa identity with NZ2-like VEGF on comparison with global ORFV strains. Phylogenetic analysis based on the VEGF gene showed two clusters of ORFV in which the Indian ORFVs clustered with NZ7-like VEGF from global ORFV strains, mostly from China. Despite the considerable variation, VEGF protein from Indian ORFV strains showed conserved VEGF homology domain with eight cysteine residues. Homology modeling of Indian ORFV strains predicted the presence of extended Loop 3 similar to NZ7-like VEGF. Therefore, the present study showed the circulation of ORFV strains with comparatively less variable NZ7-like VEGF in India which implicates its importance in the epidemiology of ORFV infections in the country.

Poxviral E3L ortholog (Viral Interferon resistance gene) of orf viruses of sheep and goats indicates species-specific clustering with heterogeneity among parapoxviruses.

Orf is a contagious disease posing a serious threat to animal and human health. E3L is one of the evolutionarily acquired immunomodulatory proteins present in orf virus (ORFV) and is responsible for conferring resistance to interferons among poxviruses. Genetic analysis of ORFV isolates of different geographical regions including Indian subcontinent targeting viral interferon resistance (VIR) gene (a homolog of vaccinia virus E3L gene) revealed a high percentage of identity among themselves and other ORFV isolates at both nt and aa levels as compared to low identity among parapoxviruses (PPVs). Phylogenetic analysis showed species-specific clustering among PPVs along with sub-clusters based on host species of origin among ORFVs infecting sheep and goats. Conserved amino acids in N-terminal Z-DNA binding domain and C-terminal ds RNA binding domain of VIR proteins of PPVs corresponding to ORFV VIR positions namely N37, Y41, P57, and W59 (necessary for Z-DNA binding) and E116, F127, F141, and K160 (necessary for dsRNA binding) were found. Further, the predicted protein characteristics and homology model of VIR protein of ORFV showed high structural conservation among poxviruses. This study on E3L genetic analysis of ORFV isolates may provide a better understanding of the molecular epidemiology of circulating strains in India and neighboring countries. Also, E3L deleted or mutated ORFV may be an as vaccine candidate and/or compounds blocking E3L may prove as an effective method for treating broad spectrum poxviral infections, suggesting a wider application in control of poxvirus infections.

Genetic analysis of L1R myristoylated protein of Capripoxviruses reveals structural homogeneity among poxviruses.

Sheeppox virus (SPPV) and goatpox virus (GTPV) are members of the genus Capripoxvirus (CaPV) of the family Poxviridae. CaPVs are responsible for important contagious diseases of small ruminants that are enzootic to the Indian sub-continent, Central and Northern Africa and the Middle East. In the present study, the sequence and phylogenetic analysis of the L1R gene of sixteen CaPV isolates (seven SPPV and nine GTPV) from India were performed along with 3D homology modeling of the L1R protein. L1R is a myristoylated protein responsible for virion assembly and being present on intracellular mature virion (IMV) surface, it is also a potent target for eliciting neutralizing antibodies. Sequence analysis of CaPV L1R gene revealed an ORF of 738bp with >99% and >96% identity within species and between species, respectively, at both nucleotide as well as amino acid levels. Phylogenetic analysis displayed distinct clusters of members of genus Capripoxvirus, as GTPV, SPPV and LSDV. L1R at the protein level showed various species-specific signature residues that may be useful for future grouping or genotyping of CaPV members. CaPV L1R was predicted to possess myristoylation motif GAAASIQTTVNTLNEKI and a potential N-glycosylation site at amino acid residue 50 (Asn). Despite of different host specificity in poxviruses, comparative sequence analysis of L1R proteins revealed highly conserved nature with presence of myristoylation motif (GXXXS) and six cysteine residues forming three disulfide bonds among all poxviruses. The conserved and immunogenic nature of the CaPV L1R gene may prove to be a potential candidate/target for developing molecular diagnostics including recombinant protein based assays and prophylactics for the control of CaPV diseases in tropical countries like India.