Manimuthu Prabhu

TaqMan real-time PCR assay based on DNA polymerase gene for rapid detection of Orf infection.

Both conventional and real time PCR (rt-PCR) assays based on the amplification of a 103bp fragment from the DNA polymerase (DNA pol) gene (conserved, non-structural) of Orf virus (ORFV) were developed for detection and semi-quantitation of ORFV DNA from infected cell culture and clinical samples. The latter technique was based on TaqMan chemistry. The rt-PCR assay was specific and sensitive as it could detect as low as 3.5fg or 15 copies of ORFV genomic DNA. Both intra- (0.38-1.0%) and inter-assay (0.53-2.87%) variabilities of rt-PCR were within the acceptable range meaning the high efficiency and reproducibility of the assay. The rt-PCR was applied successfully to detect ORFV DNA from suspected clinical samples. Further, the assay has shown a relative diagnostic sensitivity and specificity of 100% and 93.5%, respectively, when compared to B2L gene based semi-nested PCR implying a wide potential of this rt-PCR for rapid field diagnosis of Orf in sheep and goats.

Camelpox: epidemiology, diagnosis and control measures

Camelpox is an economically important contagious skin disease of camelids caused by camelpox virus (CMLV) and is characterized by mild local skin infection and less common severe systemic infections. The disease is confined to camel-rearing belts particularly in developing countries and causes economic impact due to considerable loss in terms of morbidity, mortality, loss of weight and reduction in milk yield. The virus has gained attention from researchers due to its recent emergence with close genetic relatedness to variola virus, the causative agent of smallpox, and carrying genes responsible for host immune evasion mechanisms. CMLV was earlier thought to be a zoonotic agent but so far little evidence has been documented from Somalia. Although the disease can be diagnosed based on clinical signs, the similar confounding skin lesions necessitate identification of infection by molecular biology based diagnostic techniques, namely restriction enzyme analysis of the virus genome and specific genes, genus- and species-specific diagnostic PCRs including real-time quantitative PCR, and sequence and phylogenetic analysis for diagnosis and differentiation of CMLV. The entire genome sequence of CMLV is known and it contains more than 211 putative genes, which code for different proteins with host range, immunomodulation, virulence and other functions. Both inactivated and live-attenuated vaccines are available in some countries. However, live vaccines are preferred as they provide long-lasting immunity. Considering the virus spreads through contaminated environments, an improved diagnostic and control method would be of immense value to curtail the infection in the field. Alternative therapeutics such as antiviral agents is an area that needs to be explored. This article discusses the epidemiology and biology of the disease, novel diagnostic approaches and control measures.

TaqMan hydrolysis probe based real time PCR for detection and quantitation of camelpox virus in skin scabs

The study describes the development of TaqMan hydrolysis probe based real time PCR (rt-PCR) assay targeting the ankyrin repeat protein (C18L) gene sequences for the detection and quantitation of camelpox virus (CMLV) nucleic acid and its comparison with established conventional and SYBR green rt-PCR assays. The assay was specific with an efficiency of 99.4%. The analytical sensitivity was 4 × 10¹ and 0.35 in terms of copy number and picogram of virus genomic DNA, respectively. The assay was linear with an acceptable intra (0.9-2.83% and 0.9-2.3%) and inter-assay (0.46-2.3% and 0.9-3.3) variations, when standard plasmid DNA and genomic DNA from purified CMLV respectively were tested. The assay was rapid, specific and sensitive as that of SYBR green and 1000 times more sensitive than the conventional PCR. It is suitable for the detection of CMLV nucleic acid directly from clinical samples. Further, the assay was evaluated using cell culture adapted CMLV isolates (n=11) and clinical samples (n=23) from camels and humans suspected of camelpox. This is an improved technique over conventional and SYBR green rt-PCR methods for the detection and quantitation of CMLV from skin scabs.

Comparative efficacy of live replicating sheeppox vaccine strains in Ovines.

In the present study, two sheeppox vaccines made from strains [sheeppox virus-Srinagar (SPPV-Srin) and Ranipet (SPPV-R)] indigenous to India and adapted to Vero cells were compared in terms of their safety, potency, efficacy and antigenic value with the commercial in-use Roumanian Fanar (SPPV-RF) vaccine, a foreign strain adapted in primary lamb testes cells. The safety test indicated that the SPPV (Sri and RF) vaccines were safe while SPPV-R was not completely attenuated and caused excessive adverse reactions at the passage level tested. The immunized animals showed DTH reaction and resisted virulent SPPV challenge, while control animals developed disease. Specific virus could be detected in the controls and animals immunized with lower dilutions of vaccines after challenge but not in any of the sheep immunized with 1 and 100 doses of each vaccine. All vaccines were found potent and the PD(50) was highest for SPPV (Srin and R) followed by RF. The immunized animals were seroconverted following vaccination with sustained antibody responses after challenge. In conclusion, indigenous SPPV-Srin vaccine was found to be as efficacious as SPPV-R and SPPV-RF vaccines. Thus, there is potential benefit in replacing the currently used commercial vaccine SPPV-RF with indigenous SPPV-Srin vaccine for use in India.

Real time PCR: A rapid tool for potency estimation of live attenuated camelpox and buffalopox vaccines

In the present study, SYBR Green and TaqMan real time PCRs (rt-PCR) based on the C18L gene (encodes ankyrin repeat protein) of camelpox (CMLV) and buffalopox viruses (BPXV) were, respectively employed for potency evaluation of live attenuated camelpox and buffalopox vaccines. Cells infected with the respective vaccine viruses were harvested at critical time points and subjected to respective PCRs. The critical time points of harvests for CMLV and BPXV respectively, were 36 and 30 h post infection and were respectively determined based on maximum slopes of (-3.324) and (-3.321) standard curves. On evaluation of eight batches of camelpox and seven batches of buffalopox vaccines, the results indicated that the titres estimated by respective rt-PCRs were well comparable to the conventional TCID(50) method. The rt-PCR assays were found relatively more sensitive, specific and rapid than end point dilution assay. Thus, they could be used as additional tools for estimation of live CMLV and BPXV particles in camelpox and buffalopox vaccines.