Thermostable negative-marker foot-and-mouth disease virus serotype O induces protective immunity in guinea pigs.

Foot-and-mouth disease (FMD) is a contagious viral disease of high economic importance, caused by FMD virus (FMDV), a positive-sense single-stranded RNA virus, affecting cloven-hoofed animals. Preventive vaccination using inactivated virus is in practice to control the disease in many endemic countries. While the vaccination induces antibodies mainly to structural proteins, the presence of antibodies to the non-structural proteins (NSP) is suggestive of infection, a criterion for differentiation of infected from vaccinated animals (DIVA). Also, there is a growing demand for enhancing the stability of the FMD vaccine virus capsid antigen as the strength of the immune response is proportional to the amount of intact 146S particles in the vaccine. Considering the need for a DIVA compliant stable vaccine, here we report generation and rescue of a thermostable and negative marker virus FMDV serotype O (IND/R2/1975) containing a partial deletion in non-structural protein 3A, generated by reverse genetics approach. Immunization of guinea pigs with the inactivated thermostable-negative marker virus antigen induced 91% protective immune response. Additionally, a companion competitive ELISA (cELISA) targeting the deleted 3A region was developed, which showed 92.3% sensitivity and 97% specificity, at cut-off value of 36% percent inhibition. The novel thermostable-negative marker FMDV serotype O vaccine strain and the companion cELISA could be useful in FMDV serotype O enzootic countries to benefit the FMD control program. KEY POINTS: • Thermostable foot-and-mouth disease virus serotype O with partial deletion in 3A. • Inactivated thermostable marker vaccine induced 91% protection in guinea pigs. • Companion cELISA based on deleted region in 3A could potentially facilitate DIVA.

Long-term protective immunity to goatpox in goats after a single immunization with a live attenuated goatpox vaccine.

In this study, the duration of immunity following a single-dose vaccination using an attenuated live goatpox vaccine (GTPV/Uttarkashi/1978 strain) was evaluated in goatpox-seronegative goats for 52 months. Long-term immunity was evaluated by clinical protection upon virulent virus challenge and serum neutralization assay applied to serum samples. The rise in the level of GTPV-specific antibodies was found to reach a maximum at 21 days post-vaccination, and these antibodies were maintained for 1 to 2 years after immunization, with a steady decline. Upon virulent virus challenge at 12, 24, 42, and 52 months post-vaccination, protection in all the vaccinated animals was evident (100%), whereas, the control animals developed severe clinical disease. This is the first time that the long-term immunity of a live goatpox vaccine has been investigated up to 52 months after vaccination in goats by virulent virus challenge and demonstration of serum neutralization titres. This vaccine has immense potential for controlling and eradicating goatpox from an enzootic region.

A new blocking ELISA for detection of foot-and-mouth disease non-structural protein (NSP) antibodies in a broad host range.

Large-scale monitoring of foot-and-mouth disease (FMD) in livestock is imperative in an FMD control program. Detection of antibodies against non-structural proteins (NSP) of FMD virus (FMDV) is one of the best tools to estimate the prevalence of past infection; availability of such a well-validated test is therefore essential. Using a FMDV 3B protein-specific monoclonal antibody, we have developed a new NSP antibody blocking ELISA (10H9 bELISA) and validated it on large panels of sera from different susceptible species. The diagnostic sensitivity of the ELISA was 95% with a specificity of 98%, similar to the values found using a commercial kit (PrioCHECK FMD NS test). The 10H9 bELISA can be used in a broad range of FMD susceptible species making it a very useful tool in monitoring the foot-and-mouth disease control programs by detection of virus circulation in the vaccinated populations. KEY POINTS: • A new ELISA for detection of foot and mouth disease (FMD) antibodies. • Diagnostic sensitivity of 95% and specificity of 98%. • Tested with panels of validated sera from broad host range.

Functional characterization of recombinant major envelope protein (rB2L) of orf virus.

Orf, or contagious ecthyma, a highly contagious transboundary disease of sheep and goats, is caused by a double-stranded DNA virus (ORFV) belonging to the genus Parapoxvirus of the family Poxviridae. The ORFV genome encodes the major envelope proteins B2L and F1L, which have been found to be highly immunogenic and have multiple functional characteristics. In order to investigate the functional properties of the B2L protein, in this study, the B2L gene of ORFV strain 59/05, encoding recombinant mature B2L (aa 1M-D334), was produced as a fusion protein in Escherichia coli. The functional characteristics of purified rB2L fusion protein (~60 kDa) were evaluated in vivo and in vitro, showing that this protein had lipase and immunomodulatory activities. Immunization trials involving laboratory animals (mice, rabbits and guinea pigs) using either constant or graded doses of rB2L fusion protein with or without adjuvants (FCA, alum) as well as co-administration with candidate rErns-Ag protein of classical swine fever virus (CSFV) indicated that the rB2L protein is immunogenic and has immunomodulatory properties. This study shows the potential utility of the rB2L protein as a safe and novel adjuvant in veterinary vaccine formulations.

Comparison of different inactivation methods on the stability of Indian vaccine strains of foot and mouth disease virus.

In this study, the efficiency of binary ethyleneimine (BEI) in combination with formaldehyde (FA) and glutaraldehyde (GTA) in inactivating the Indian FMDV vaccine strains is compared. The acceptable safety of virus inactivation was faster and the inactivation rates were increased many-folds with combination of inactivants than BEI alone. FMDV A was inactivated rapidly than the other two serotypes with BEI + FA combination. Inactivation plots were linear for all the serotypes irrespective of inactivation process. Further, the integrity studies on 146S using serotype specific ELISA indicated no significant change in the antigenic mass of all the serotypes throughout the inactivation process. However, the loss of 146S antigen occurred in the subsequent steps of downstream processing. Further, the studies on intactness of viral RNA using real time PCR indicated the amplification of 1D gene sequences in all the preparations of timed samples irrespective of serotypes/inactivation process. Further, inactivated virus preparation (146S) was more stable at lower temperatures for all the serotypes/inactivation process. Among the combinations of inactivants, BEI + FA out performed compared to BEI + GTA and BEI in terms of inactivation rates, 146S yield and its storage stability, irrespective of the serotypes.

Synergistic effect of high-mobility group box-1 and lipopolysaccharide on cytokine induction in bovine peripheral blood mononuclear cells.

High-mobility group box 1 (HMGB1) is one of the potent endogenous adjuvants released by necrotic and activated innate immune cells. HMGB1 modulates innate and adaptive immune responses in humans and mice by mediating immune cells crosstalk. However, the immuno-modulatory effects of HMGB1 in the bovine immune system are not clearly known. In this study, the effect of bovine HMGB1 alone or in combination with LPS on the expression kinetics of cytokines upon in vitro stimulation of bovine peripheral blood mononuclear cells (PBMCs) was investigated by quantitative PCR assay. The biological activity of bovine HMGB1 expressed in this prokaryotic expression system was confirmed by its ability to induce nitric oxide secretion in RAW 264.7 cells. The present results indicate that HMGB1 induces a more delayed TNF-α response than does LPS in stimulated PBMCs. However, IFN-γ, IFN-ß and IL-12 mRNA transcription peaked at 6 hr post stimulation after both treatments. Further, HMGB1 and LPS heterocomplex up-regulated TNF-α, IFN-γ and IL-12 mRNA expression significantly than did individual TLR4 agonists. The heterocomplex also enhanced the expression of TLR4 on bovine PBMCs. In conclusion, the data indicate that HMGB1 and LPS act synergistically and enhance proinflammatory cytokines, thereby eliciting Th1 responses in bovine PBMCs. These results suggest that HMGB1 can act as an adjuvant in modulating the bovine immune system and thus lays a foundation for using HMGB1 as an adjuvant in various bovine vaccine preparations.

Immunological evaluation of mannosylated chitosan nanoparticles based foot and mouth disease virus DNA vaccine, pVAC FMDV VP1-OmpA in guinea pigs.

A DNA vaccine for foot and mouth disease (FMD) based on mannosylated chitosan nanoparticles was evaluated in guinea pigs. The DNA construct was comprised of FMD virus full length-VP1 gene and outer membrane protein A (Omp A) gene of Salmonella typhimurium as a Toll-like receptor (TLR)-ligand in pVAC vector. Groups of guinea pigs immunized either intramuscularly or intra-nasally were evaluated for induction of virus neutralizing antibodies, Th1(IgG2) and Th2 (IgG1) responses, lymphocyte proliferation, reactive nitrogen intermediate production, secretory IgA for naso-mucosal immune response and protection upon homotypic type O virulent FMD virus challenge. The results indicate the synergistic effect of OmpA on the immunogenic potential of FMD DNA vaccine construct delivered using mannosylated chitosan nano-particles by different routes of administration. These observations suggest the substantial improvement in all the immunological parameters with enhanced protection in guinea pigs.

Monoclonal antibody based solid phase competition ELISA to detect FMDV serotype A specific antibodies.

In Foot-and-mouth disease (FMD) enzootic countries, periodic vaccination is the key tool in controlling the disease incidence. Active seromonitoring of the vaccinated population is critical to assess the impact of vaccination. Virus neutralization test (VNT) and enzyme-linked immunosorbent assays (ELISA) are commonly used for antibody detection. Assays like liquid phase blocking ELISA (LPBE) or solid phase competition ELISA (SPCE) are preferred as they do not require handling of live FMDV and are routinely used for seromonitoring or for vaccine potency testing; however, false positives are high in LPBE. Here we report, a monoclonal antibody (mAb) based SPCE as a potential alternate assay for antibody titration. From a panel of 12 mAbs against FMDV serotype A, two mAbs were chosen for the development of SPCE. Based on a set of 453 sera, it was demonstrated that mAb 2C4G11, mAb 6E8D11and polyclonal antibody (pAb) based SPCE had a relative sensitivity of 86.1, 86.1 and 80.3 %; and specificity of 99.6, 99.1 and 99.1 %, respectively. The correlation, repeatability, and level of agreement of the assays were high demonstrating the potential use of mAb in large scale surveillance studies and regular vaccine potency testing.

In-process quality control in foot-and-mouth disease vaccine production by detection of viral non-structural proteins using chemiluminescence dot blot assay.

Foot-and-mouth disease (FMD) is a contagious viral disease of cloven-footed animals. Immunization with inactivated virus vaccine is effective to control the disease. Six-monthly vaccination regimen in endemic regions has proven to be effective. To enable the differentiation of infected animals from those vaccinated, non-structural proteins (NSPs) are excluded during vaccine production. While the antibodies to structural proteins (SPs) could be observed both in vaccinated and infected animals, NSP antibodies are detectable only in natural infection. Quality control assays that detect NSPs in vaccine antigen preparations, are thus vital in the FMD vaccine manufacturing process. In this study, we designed a chemiluminescence dot blot assay to detect the 3A and 3B NSPs of FMDV. It is sensitive enough to detect up to 20 ng of the NSP, and exhibited specificity as it does not react with the viral SPs. This cost-effective assay holds promise in quality control assessment in FMD vaccine manufacturing.

Effect of immunosuppression on pathogenesis of peste des petits ruminants (PPR) virus infection in goats.

In this study an attempt to address the effects of immunosuppression on pathogenesis of peste des petits ruminants (PPR) virus infection was undertaken. Cyclophosphamide and dexamethasone were used to immunosuppress the animals. The drug treated animals exhibited severe leukopaenia and lymphopaenia; one of the indicators of immunosuppression. Experimental peste des petits ruminants virus (PPRV) infection was then given to both drug-induced immunosuppressed and non-immunosuppressed goats and observed their effects. Findings indicated that, the immunosuppressed goats had a short period of viremia, more extensive and severe disease advancement and higher mortality rate than the non-immunosuppressed goats. PPRV antigen distribution in both ante-mortem and post-mortem materials was extensive and diffused in immunosuppressed animals than that of non-immunosuppressed. Some of the atypical organ(s)/tissues like liver, kidney, heart etc showed more antigen load than non-immunosuppressed group. Histopathological and immunohistochemical studies of tissues from the two groups showed that pathological changes in the non-immunosuppressed animals were confined only to gastrointestinal tract, whereas in the immunosuppressed animals histopathological changes and PPRV antigen distribution were more extensive and diffused. The present study indicated that immunosuppression increased the extent and severity of the pathological lesions associated with peste des petits ruminants virus infection.

Identification and phylogenetic analysis of orf viruses isolated from outbreaks in goats of Assam, a northeastern state of India.

Two outbreaks of orf virus (ORFV) (a parapoxvirus) infection in goats, which occurred in Golaghat and Kamrup districts of Assam, a northeastern part of India, were investigated. The disease was diagnosed by standard virological and molecular techniques. The entire protein-coding region of B2L gene of two isolates were cloned and sequenced. Phylogenetic analysis based on B2L amino acid sequences showed that the ORFVs identified in these outbreaks were closely related to each other and both were closer to ORFV-Shahjahanpur 82/04 isolate from north India. The present study revealed that the precise characterization of the genomic region (B2L gene) might provide evidence for the genetic variation and movement of circulating ORFV strains in India.

Seroprevalence of Peste des petits ruminants in cattle and buffaloes from Southern Peninsular India.

This study describes seroprevalence of Peste des petits ruminants (PPR) in cattle and buffaloes carried out during the period 2009-2010 using the randomly collected serum samples from different parts of Southern peninsular India. The report presents the results of PPR virus (PPRV)-specific antibodies in situations where either the subclinical or inapparent or non-lethal infection was there in cattle and buffaloes. A total of 2,548 serum samples [cattle = 1,158, buffaloes = 1,001, sheep = 303 and goat = 86] were collected and screened for PPRV antibodies by using a PPR monoclonal antibody-based competitive ELISA kit. Analysis of 2,159 serum samples indicates an overall 4.58% prevalence of PPRV antibody in cattle and buffaloes. The presence of PPRV-specific antibodies demonstrates that cattle and buffaloes are exposed to PPR infection naturally, and the transmission mode may be direct or indirect. Further, it implies the importance of bovines as subclinical hosts for the virus besides widespread presence of the disease in sheep and goats in the country.

Ribavirin as a curative and prophylactic agent against foot and mouth disease virus infection in C57BL/6 suckling and adult mice model.

Despite the availability of control measures for foot-and-mouth disease (FMD), the application of antiviral agents is imperative due to certain limitations in the prevention and control of FMD. This study pertains to systematic in vivo investigation of ribavirin as a prophylactic/curative agent, both in suckling and adult C57BL/6 mice against foot-and-mouth disease virus (FMDV) infection. In the adult mice, antiviral efficacy was assessed based on standard clinical score, body weight, and viral load. Only 13.33 to 33.33% of adult mice exhibited disease-specific symptoms following treatment and infection and vice versa, respectively, indicating the antiviral efficacy of the ribavirin. Further, the distribution of virus in different vital organs following ribavirin treatment and virus infection, and vice versa using SYBR green-based real-time PCR is reported. In the blood sample, the viral RNA was detected as early as two days post-infection and there was a significant reduction in virus titer (1000 to 10,000-folds) in the treatment groups compared to the infection control group. Animals receiving ribavirin had significantly lower organ virus titers at 2, 4, 6, 9, and 14 days post-challenge (dpc) than placebo-treated. In suckling mice, the treatment groups were 100% protected/cured compared to the control group. Thus, our data demonstrate that ribavirin may provide a feasible therapeutic approach to prevent as well as to treat FMDV infection. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13337-021-00746-8.

Protective effect of partially purified 35 kDa protein from silk worm (Bombyx mori) fecal matter against carbon tetrachloride induced hepatotoxicity and in vitro anti-viral properties.

CONTEXT: It has been found that many proteins from silkworm (Bombyx mori L.) fecal matter have been active against human immunodeficiency virus, Sendai virus, herpes simplex virus type-1, and nuclear polyhedrosis virus. OBJECTIVE: A partially purified 35 kDa protein from silkworm was screened for its hepatoprotective activity, and in vitro antioxidant, and antiviral properties against camelpox and goatpox viruses. MATERIALS AND METHODS: The study investigated the efficiency of the partially purified 35 kDa protein from silk worm fecal matter against CCl4-induced liver damage measured in terms of enzyme levels such as aspartate aminotransferase (AST), alanine amino transferase(ALT), alkaline phosphatase (ALP) and total bilirubin, which maintain liver integrity. In vitro antioxidant potential of this protein was determined based on its ability to scavenge 2, 2-diphenylpicrylhydrazyl (DPPH) and superoxide anions scavenging activity. Further, in vitro cytotoxic effect on Vero cells and antiviral activity against goatpox and camelpox viruses were also studied. RESULTS: The protein had significant hepatoprotection against CCl4-induced liver damage and scavenging of DPPH radical and superoxide anion activity. However, the protein did not inhibit the multiplication of either virus tested at its maximum non-toxic concentration (MNTC) in vitro. DISCUSSION AND CONCLUSION: The partially purified 35 kDa protein from silk worm Bombyx mori L fecal matter possessed protective effect against CCl4-induced oxidative stress in rat model. The protein was found to be ineffective against camelpox and goatpox viruses at its MNTC in vitro.

Comparative efficacy of conventional and taqman polymerase chain reaction assays in the detection of capripoxviruses from clinical samples.

Sheeppox and goatpox are economically important viral diseases of sheep and goats, respectively. Both diseases are reportable to the World Organization for Animal Health. To implement a control and eradication program for these diseases, a rapid and user-friendly diagnostic tool is imperative for screening. Therefore, in the present study, TaqMan quantitative polymerase chain reaction (qPCR) and conventional PCR assays targeting the DNA polymerase (DNA pol) gene were developed for the detection of Capripoxvirus DNA from clinical specimens of sheep and goats. The 2 assays used different primer sets. Conventional PCR yielded a specific product of 134 bp, whereas qPCR yielded a 180-bp product. The specificity of amplified DNA pol gene products was confirmed by their size and by sequence analysis. The 2 assays were specific for Sheeppox virus and Goatpox virus. However, in comparison to conventional PCR, the qPCR was more rapid, specific, and 100 times more sensitive, with a detection limit as low as 0.042 pg of purified DNA. The qPCR assay was more sensitive (84.05%) than conventional PCR (76.06%) when used on clinical samples (n = 71) from sheep and goats.

A polymerase chain reaction strategy for the diagnosis of camelpox.

Camelpox is a contagious viral skin disease that is mostly seen in young camels. The disease is caused by the Camelpox virus (CMLV). In the present study, a polymerase chain reaction (PCR) assay based on the C18L gene (encoding ankyrin repeat protein) and a duplex PCR based on the C18L and DNA polymerase (DNA pol) genes were developed. The former assay yields a specific amplicon of 243 bp of the C18L gene, whereas the duplex PCR yields 243- and 96-bp products of the C18L and DNA pol genes, respectively, in CMLV, and only a 96-bp product of the DNA pol gene in other orthopoxviruses. The limit of detection was as low as 0.4 ng of viral DNA. Both PCR assays were employed successfully for the direct detection and differentiation of CMLV from other orthopoxviruses, capripoxviruses, and parapoxviruses in both cell culture samples and clinical material. Furthermore, a highly sensitive SYBR Green dye-based, real-time PCR was optimized for quantitation of CMLV DNA. In the standard curve of the quantitative assay, the melting temperature of the specific amplicon at 77.6 degrees C with peak measured fluorescence in dissociation plot was observed with an efficiency of 102%. To the authors' knowledge, this is the first report to describe a C18L gene-based PCR for specific diagnosis of camelpox infection.

In vitro antiviral efficacy of pleconaril and ribavirin on foot-and-mouth disease virus replication.

Antiviral therapy is a promising strategy to control acute viral infections. FMDV causes an acute infection and the vaccination provides a protective immunity 7 days post immunization. If the infection is uncontained, then it affects the entire herd. In such circumstances, if antiviral drug is administered the infection can be checked in a herd. Ribavirin is known to cure persistently infected BHK21 cells with FMD virus. However, there have been no systematic studies on antiviral activity of ribavirin against FMDV at different time points with the application of ELISA, PCR or real-time PCR. Pleconaril is known to inhibit enteroviruses and rhinoviruses but has not been explored on FMDV. Hence, the present study evaluates the in vitro antiviral efficacy of pleconaril and ribavirin on FMDV replication. The maximum non-toxic concentrations (MNTC) of pleconaril and ribavirin for BHK21 cells respectively were 7.81 µg/50 µL and 15.62 µg/50 µL. Thus, drug concentrations below MNTC were tested for their antiviral activity against serial tenfold diluted FMDV O, A and Asia 1 serotypes. Pleconaril did not inhibit FMDV serotype O replication at 7.5 µg/50 µL based on CPE inhibition assay and this was further confirmed using sandwich ELISA, PCR/real-time PCR. On the other hand, ribavirin at 15.62 µg/50 µL inhibited the in vitro replication of FMDV O, A and Asia 1 and the inhibition was confirmed by serotype specific sandwich ELISA, PCR and real-time PCR assays. The inhibition was directly proportional to the concentration of ribavirin. Therefore, ribavirin could be explored for its in vivo efficacy as a potential therapeutic in the prevention of early spread of FMDV infection in a herd.

Evaluation of a recombinant major envelope protein (F1L) based indirect- ELISA for sero-diagnosis of orf in sheep and goats.

Orf or contagious ecthyma, is a highly contagious transboundary disease of sheep and goats. For sero-diagnosis of orf, recombinant antigen based assays are considered as alternatives to conventional approaches such as serum neutralization test (SNT) and counter-immuno-electrophoresis (CIE). A major envelope protein of orf virus (ORFV), F1L, is highly immunogenic and is a candidate for use in these assays. In this study, the F1L gene of the ORFV-59/05 strain encoding a recombinant mature F1L protein (1M-D302 aa) with a C- terminal truncation, was produced as a fusion protein (∼50 kDa) in Escherichia coli. The immunogenic potential of purified rF1L was confirmed by detecting specific anti-F1L antibody responses in sera collected from immunized rabbits and guinea pigs using ELISA and SNT. An indirect-ELISA based on rF1L was developed and optimized. In comparison to SNT by ROC analysis in the detection of ORFV specific antibodies, this new assay exhibited a diagnostic specificity of 94.04% and 92.53% with sheep and goat sera, respectively, while the sensitivity was 89.22% and 94.25%, for sheep and goat sera. No cross reactivity was noted with sera collected from small ruminants infected with other transboundary diseases (goatpox, sheeppox, peste des petits ruminants, foot-and-mouth disease and bluetongue). Furthermore, the rF1L-ELISA applied to screen the vaccinated/challenged goat sera resulted in better detection (30%) than by SNT (28%) in spite of lower levels of antibodies which could be due to predominant cell mediated immune response in vaccinated animals. This study highlighted the potential utility of rF1L protein as a safe and novel diagnostic reagent in comparison to live virus antigen, in the development of sero-diagnostic assay for surveillance of ORFV infection in sheep and goats.

Comparative sequence and structural analysis of Indian orf viruses based on major envelope immuno-dominant protein (F1L), an homologue of pox viral p35/H3 protein.

Orf virus (ORFV), a member of the genus Parapoxvirus in the family Poxviridae, is the cause of orf, a highly contagious zoonotic viral disease that affects mainly sheep and goats. In the present study, the sequence and phylogenetic analysis of Indian ORFV isolates (n = 15) from natural outbreaks in sheep and goats belonging to different geographical regions were analysed on the basis of F1L gene along with homology modelling of F1L protein. Multiple sequence alignments revealed highly conserved C-terminus and heterogeneity of N-terminus region of F1L among all orf viruses studied. Further, a comparative sequence alignment indicated conservation of various motifs such as glycosaminoglycan (GAG), Asp/Glu-any residue-Asp (D/ExD) and a Cx3C chemokine like motif among all poxviruses and unique motifs (proline rich region [PRR] and Lys-Gly-Asp [KGD]), in parapoxviruses including ORFV isolates irrespective of geography and host species. Phylogenetically, two major clusters were noticed which included Indian orf isolates along with foreign isolates. Structurally, ORFV F1L resembled the topology as exhibited by its homologue vaccinia virus H3 protein with mixed ß/α folds and ligand binding specificity in docking models. We noted that despite differences in host cell specificity and pathogencity, poxvirus proteins especially ORFV F1L protein and its homologues presumed to share similarities as they are highly conserved irrespective of species and countries of origin. Further, the study also indicated the possibilities of differentiation of ORFV strains based on N-terminal heterogeneity despite highly conserved C-terminal region with conserved motifs.

Sequence analysis of haemagglutinin gene of camelpox viruses shows deletion leading to frameshift: Circulation of diverse clusters among camelpox viruses.

Orthopoxviruses (OPVs) have broad host range infecting a variety of species along with gene-specific determinants. Several genes including haemagglutinin (HA) are used for differentiation of OPVs. Among poxviruses, OPVs are sole members encoding HA protein as part of extracellular enveloped virion membrane. Camelpox virus (CMLV) causes an important contagious disease affecting mainly young camels, endemic to Indian subcontinent, Africa and the Middle East. This study describes the sequence features and phylogenetic analysis of HA gene (homologue of VACV A56R) of Indian CMLV isolates. Comparative analysis of CMLV HA gene revealed conserved nature within CMLVs but considerable variability was observed between various species of OPVs. Most Indian CMLV isolates showed 99.5%-100% and 96.3%-100% identity, at nucleotide (nt) and amino acid (aa) levels respectively, among themselves and with CMLV-M96 strain. Importantly, Indian CMLV strains along with CMLV-M96 showed deletion of seven nucleotides resulting in frameshift mutation at C-terminus of HA protein. Phylogenetic analysis displayed distinct clustering among CMLVs which might point to the circulation of diverse CMLV strains in nature. Despite different host specificity of OPVs, comparative sequence analysis of HA protein showed highly conserved N-terminal Ig V-set functional domain with tandem repeats. Understanding of molecular diversity of CMLVs and structural domains of HA protein will help in the elucidation of molecular mechanisms for immune evasion and design of novel antivirals for OPVs.