Baculovirus expression and purification of virion core and envelope proteins of goatpox virus to evaluate their diagnostic potential.

Goatpox and sheeppox are highly contagious and economically important viral diseases of small ruminants. Due to the risk they pose to animal health, livestock production, and international trade, capripoxviruses are a considerable threat to the livestock economy. In this study, we expressed two core proteins (A4L and A12L) and one extracellular enveloped virion protein (A33R) of goatpox virus in a baculovirus expression vector system and evaluated their use as diagnostic antigens in ELISA. Full-length A4L, A12L, and A33R genes of the GTPV Uttarkashi strain were amplified, cloned into the pFastBac HT A donor vector, and introduced into DH10Bac cells containing a baculovirus shuttle vector plasmid to generate recombinant bacmids. The recombinant baculoviruses were produced in Sf-21 cells by transfection, and proteins were expressed in TN5 insect cells. The recombinant proteins were analysed by SDS-PAGE and confirmed by western blot, with expected sizes of ~30 kDa, ~31 kDa, and ~32 kDa for A4L, A12L, and A33R, respectively. The recombinant proteins were purified, and the immunoreactivity of the purified proteins was confirmed by western blot using anti-GTPV serum. The antigenic specificity of the expressed proteins as diagnostic antigens was evaluated by testing their reactivity with infected, vaccinated, and negative GTPV/SPPV serum in indirect ELISA, and the A33R-based indirect ELISA was optimized. The diagnostic sensitivity and specificity of the A33R-based indirect ELISA were found to be of 89% and 94% for goats and 98% and 91%, for sheep, respectively. No cross-reactivity was observed with other related viruses. The recombinant-A33R-based indirect ELISA developed in the present study shows that it has potential for the detection of antibodies in GTPV and SPPV infected/vaccinated animals.

Chromium contamination in groundwater and Sobol sensitivity model based human health risk evaluation from leather tanning industrial region of South India.

The present investigation was conducted to find the possible chromium contamination in groundwater and the related health risks in a leather industrial region of south India using Sobol sensitivity modeling. Thirty-five groundwater samples were sampled from the field sites and were analyzed for pH, TDS (Total Dissolved Solids), EC (Electrical Conductivity), F- (Fluoride), NO3- (Nitrate) and Cr (Chromium). The concentration of nitrate varied from 3 to 81 mg/L with a mean of 48.6 mg/L. About 57% (n = 20) of the wells surpassed the drinkable limit (45 mg/L) for NO3- as per World Health Organization (WHO). The fluoride ion ranged from 0.1 to 2.7 mg/L with a mean of 1.5 mg/L. Around 51% (n = 18) of the samples crossed the recommended limit of WHO for F- (1.5 mg/L). The chromium varied from 0.01 to 0.19 mg/L in groundwater with a mean of 0.1 mg/L. About 66% (n = 23) of the samples overshoot the permissible limit of WHO standards (0.05 mg/L) for Cr. The spatial distribution map of chromium in the groundwater showed that 271.76 km2 area is under risk. Based on total hazard index (THI), 66%, 46%, and 43% of the groundwater samples surpassed the allowable limit (THI > 1) for children, women and men, correspondingly. Children pose severe health risks than women and men in this region. Using Sobol sensitivity indices, three different categories of risk effects were assessed: first order effect (FOE), total effect (TE) and second order effect (SOE). In the oral sensitivity model, concentration of Cr (Cw) in water and ingestion rate (IR) had the dominant role, whereas in the dermal model, skin surface area (SA) and contact fraction by skin (F) had vital role in addition to the concentration (Cw). Further, the outcome of this study insists the responsibilities of industrial, municipal and agricultural sectors to keep the environment pollution free and to ensure the supply of potable water to the people.

Evaluation of chromium in vegetables and groundwater aptness for crops from an industrial (leather tanning) sector of South India.

The main objective of the present study is evaluation of groundwater aptness for crops and chromium concentration in vegetables from an industrial (leather tanning) sector of South India using geospatial techniques. Seventy groundwater samples were collected from the open and tube wells during November 2017, February 2018, May 2018 and September 2018 to represent northeast (NE) monsoon (October-December), post-monsoon (winter) (January-February), pre-monsoon (summer) (March-May) and southwest (SW) monsoon (June-September) seasons, respectively. In addition, vegetables were also collected during the above-mentioned seasons from the market to assess the level of chromium content in them. All the groundwater samples were tested in the chemical laboratory using the American Public Health Association norms for various physicochemical parameters, viz. TDS, pH, sodium, potassium, calcium, magnesium, bicarbonate, chloride, sulfate, nitrate, fluoride and chromium. Northeast and southwest monsoon season samples mostly represented 'high to very high saline' and 'low alkaline' categories of irrigation water. However, post- and pre-monsoon samples represented 'high to very high saline' and 'low to medium alkaline' categories. 'High saline and low alkaline' water could be used for irrigation in all types of soil with less problem of exchangeable sodium. However, 'very high saline' water should not be applied for the crops having poor salt tolerance and soils having poor internal drainage. The concentration of chromium in groundwater and vegetables was within the permissible limits for human intake prescribed by the World Health Organization standards.

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.

Prokaryotic expression, purification and evaluation of goatpox virus ORF117 protein as a diagnostic antigen in indirect ELISA to detect goatpox.

Goatpox is an economically significant transboundary viral disease of goats that is caused by goatpox virus (GTPV). This study describes the prokaryotic expression of the GTPV ORF117 protein, a homologue of vaccinia virus A27L, and evaluation of its diagnostic potential in ELISA. The GTPV ORF117 gene was cloned into the pET32a vector to express recombinant ORF117 protein (rA27L) in E. coli BL21-CodonPlus (DE3)-RIPL. The bacterial expression of the protein was confirmed by western blot analysis using anti-GTPV polyclonal antibodies that detected rA27L, which is ~ 35 kDa in size. rA27L was affinity purified under native conditions and used to assess the antibody response in an optimized indirect ELISA. The purified antigen specifically reacted with anti-GTPV and anti-SPPV serum in ELISA. A preliminary screening of random and purposive serum samples (n = 520) from sheep and goats using this optimized ELISA gave a positivity rate of 19.4 % with a diagnostic specificity of 88.7% and diagnostic sensitivity of 98.5% when compared to the gold standard serum neutralization test. Our results suggest that the indirect ELISA based on the rA27L protein has potential for serosurveillance and seromonitoring of GTPV in goats.

Comparative efficacy of chemical stabilizers on the thermostabilization of a novel live attenuated buffalopox vaccine.

In the present investigation, the thermostability of a live attenuated buffalopox vaccine prepared with an indigenous baffalopox virus isolate (BPXV Vij/96) and freeze-dried under conventional lyophilizing conditions is described. Three different stabilizer combinations like LS (lactalbumin hydralysate + sucrose), LHT (lactalbumin hydralysate + Trehalose dihydrate) and TAA (Trehalose dihydrate + l- Alanine + l-Histidine) were used to prepare the vaccine. The study indicated that the LS stabilizer was found to be the stabilizer of choice followed by LHT and TAA for buffalopox vaccine at all temperatures studied. The presence of stabilizers has beneficial influence in preserving the keeping quality of the vaccine. Further, among the diluents used to reconstitute the freeze-dried buffalopox vaccine, double distilled water, 0.85% normal saline solution and phosphate buffer saline were the choice of diluents in that order. However, 1M MgSO4 did not perform well at higher temperatures. Investigation suggests for using LS as a stabilizer for freeze-drying and any of the three diluents except 1MgSO4 for reconstitution of buffalopox vaccine.

Loop-mediated isothermal amplification assay for rapid and sensitive detection of sheep pox and goat pox viruses in clinical samples.

A Loop-mediated isothermal amplification (LAMP) assay targeting the highly conserved DNA polymerase gene of capripox virus genome was developed and evaluated for rapid detection of sheep pox and goat pox viruses. The optimized LAMP assay is found specific and sensitive for amplification of target DNA with a diagnostic sensitivity and specificity of 96.6% and 100% respectively compared to quantitative PCR. The detection rate of LAMP, PCR and Q-PCR assays is found to be 81.5%, 67% and 83% respectively. This LAMP assay has the potential for rapid clinical diagnosis and surveillance of sheep pox and goat pox in field diagnostic laboratories.

Synthesis and regulation of chlorogenic acid in potato: Rerouting phenylpropanoid flux in HQT-silenced lines.

Chlorogenic acid (CGA) is the major phenolic sink in potato tubers and can constitute over 90% of total phenylpropanoids. The regulation of CGA biosynthesis in potato and the role of the CGA biosynthetic gene hydroxycinnamoyl CoA:quinate hydroxycinnamoyl transferase (HQT) was characterized. A sucrose induced accumulation of CGA correlated with the increased expression of phenylalanine ammonia-lyase (PAL) rather than HQT. Transient expression of the potato MYB transcription factor StAN1 (anthocyanin 1) in tobacco increased CGA. RNAi suppression of HQT resulted in over a 90% reduction in CGA and resulted in early flowering. The reduction in total phenolics and antioxidant capacity was less than the reduction in CGA, suggesting flux was rerouted into other phenylpropanoids. Network analysis showed distinct patterns in different organs, with anthocyanins and phenolic acids showing negative correlations in leaves and flowers and positive in tubers. Some flavonols increased in flowers, but not in leaves or tubers. Anthocyanins increased in flowers and showed a trend to increase in leaves, but not tubers. HQT suppression increased biosynthesis of caffeoyl polyamines, some of which are not previously reported in potato. Decreased PAL expression and enzyme activity was observed in HQT suppressed lines, suggesting the existence of a regulatory loop between CGA and PAL. Electrophysiology detected no effect of CGA suppression on potato psyllid feeding. Collectively, this research showed that CGA in potatoes is synthesized through HQT and HQT suppression altered phenotype and redirected phenylpropanoid flux.

Development and comparative evaluation of loop mediated isothermal amplification (LAMP) assay for simple visual detection of orf virus in sheep and goats.

A loop-mediated isothermal amplification (LAMP) assay targeting DNA Pol gene was optimized and evaluated for the rapid detection of orf virus in clinical samples. The LAMP assay was found to be specific and sensitive. The detection rate of LAMP (89.3%) was better than PCR (67.9%) and comparable to real-time PCR (91.1%) in clinical samples by gel electrophoresis and visual detection methods. This LAMP assay is simple and does not rely upon any special equipment and could be employed in clinical diagnosis and epidemiological survey of orf infection.

Evaluation of stability of live attenuated camelpox vaccine stabilized with different stabilizers and reconstituted with various diluents.

In this study, thermostability of a Vero cell attenuated live camelpox vaccine under conventional lyophilization conditions has been evaluated. Three stabilizers were used separately for freeze-drying the vaccine and the stability of the vaccine, both in freeze-dried and reconstituted forms at different temperatures was assessed. The study revealed that the camelpox vaccine lyophilized with TAA stabilizer found superior with a shelf life of 44 months, 227 days, 22 days and 20 days at 4, 25, 37 and 45 °C, respectively followed by LS stabilizer. In terms of half-life, TAA stabilizer proved better followed by LS and BUGS stabilizers at all temperatures except at 25 °C in which LS found relatively superior. Among the four diluents viz. 1x PBS (phosphate buffered saline, pH 7.4), 0.85% NaCl, distilled water and 1 M MgSO4, PBS was a better diluent followed by 0.85% NaCl. Both the diluents maintained the infectivity titer more than the minimum effective dose (3 log10TCID50 with a maximum titre of 6.53 log10TCID50 in both the diluents) for 60 h at 37 and 45 °C. However, 1 M MgSO4 found less suitable for camelpox vaccine dilution. The study indicates that the TAA and 1× PBS are the choice of stabilizer and diluent, respectively for camelpox vaccine.

"Candidatus Liberibacter solanacearum" titer over time in Bactericera cockerelli (Hemiptera: Triozidae) after acquisition from infected potato and tomato plants.

The potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae) is a serious pest of potato and other solanaceous crops. B. cockerelli has been associated with the bacterium "Candidatus Liberibacter solanacearum" (Lso), the causal agent of zebra chip, a new and economically important disease of potato in the United States, Mexico, Central America, and New Zealand. The biology of liberibacter transmission to potato and other host plants by the potato psyllid is largely unknown. The current study determined Lso acquisition by adult psyllids following different acquisition access periods (AAP) on potato and tomato, quantified Lso titer over time in postacquisition psyllids, determined Lso-acquisition rate in psyllids at each AAP on each source of inoculum, and determined influence of host plant Lso titer on Lso acquisition rates and postacquisition titer in psyllids over time. Results showed that Lso detection rates and titer increased over time in psyllids following AAPs of 8, 24, and 72 h on tomato and potato and Lso titer was highest when psyllids acquired Lso from tomato versus potato. Lso titer ranged from 200- to 400-fold higher in tomato leaves, petioles, and stems than those of potato. The increase of Lso titer in the insects reached a plateau after an average of 15 d following 24 and 72 h AAP on potato or tomato. At this 15-d plateau, Lso titer in postacquisition psyllids was comparable with that of infective psyllids from the Lso-infected laboratory colony. Lso-acquisition rate in psyllids fed on potato and tomato increased up to 5 and 20, 15 and 35, 35 and 75, and 80 and 100%, respectively, when the insects were allowed access to plants for 4, 8, 24, and 72 h, respectively.

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.

Effects of Temperature on 'Candidatus Liberibacter solanacearum' and Zebra Chip Potato Disease Symptom Development.

Temperature has been shown to have a significant effect on development of liberibacter species associated with citrus Huanglongbing disease. 'Candidatus Liberibacter africanus' and 'Ca. L. americanus' are both heat sensitive, whereas 'Ca. L. asiaticus' is heat tolerant. The recently described 'Ca. L. solanacearum' is associated with zebra chip (ZC), a newly emerging and economically important disease of potato worldwide. This psyllid-transmitted liberibacter species severely affects several other solanaceous crops and carrot. Experiments were conducted to evaluate effects of temperature on development of 'Ca. L. solanacearum' and ZC disease. Potato plants were inoculated with 'Ca. L. solanacearum' by briefly exposing them to liberibacter-infective potato psyllids at various temperatures under laboratory conditions. Following insect exposure, the plants were maintained at selected temperature regimes in growth chambers, monitored for ZC symptom development, and later tested for liberibacter by polymerase chain reaction to confirm infection. Results indicated that temperatures below 17°C appear to slow development of 'Ca. L. solanacearum' and ZC symptoms, whereas temperatures above 32°C are detrimental to this liberibacter. Compared to Huanglongbing liberibacters, 'Ca. L. solanacearum' appears heat sensitive. The sensitivity of this bacterium and its insect vector to temperature may partially explain incidence, severity, and distribution of ZC in affected regions.

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.

Vector transmission efficiency of liberibacter by Bactericera cockerelli (Hemiptera: Triozidae) in zebra chip potato disease: effects of psyllid life stage and inoculation access period.

Successful transmission of plant pathogens by insects depends on the vector inoculation efficiency and how rapidly the insect can effectively transmit the pathogen to the host plant. The potato psyllid, Bactericera cockerelli (Sulc), has recently been found to transmit "Candidatus Liberibacter solanacearum," a bacterium associated with zebra chip (ZC), an emerging and economically important disease of potato in several parts of the world. Currently, little is known about the epidemiology of ZC and its vector's inoculation capabilities. Studies were conducted in the field and laboratory to 1) assess transmission efficiency of potato psyllid nymphs and adults; 2) determine whether psyllid inoculation access period affects ZC incidence, severity, and potato yield; and 3) determine how fast the psyllid can transmit liberibacter to potato, leading to ZC development. Results showed that adult potato psyllids were highly efficient vectors of liberibacter that causes ZC and that nymphs were less efficient than adults at transmitting this bacterium. It was also determined that inoculation access period had little influence on overall ZC disease incidence, severity, and resulting yield loss. Moreover, results showed that exposure of a plant to 20 adult potato psyllids for a period as short as 1 h resulted in ZC symptom development. Furthermore, it was shown that a single adult potato psyllid was capable of inoculating liberibacter to potato within a period as short as 6 h, thereby inducing development of ZC. This information will help in developing effective management strategies for this serious potato disease.

Impact of precipitation disparity on groundwater fluctuation in a semi-arid region (Vellore district) of southern India using geospatial techniques.

In the present study, impact of precipitation disparity on groundwater level fluctuation was carried out in Vellore district, Tamil Nadu, India, using geospatial techniques. There are five rain gauge stations in the study area in which three rain gauge stations, namely Alangayam, Jolarpettai and Pernampet, receive more precipitation when compared with the average annual precipitation of Tamil Nadu state (920 mm). The other two stations, namely Madanur and Natrampalli, receive less than 920 mm of precipitation annually. The overall average annual precipitation of the study area is 913.6 mm. More than 100 mm precipitation is received in all the five rain gauge stations during southwest (SW) and northeast (NE) monsoon seasons. The maximum monthly precipitation is usually recorded during the month of November and the minimum precipitation is recorded during June. The post-monsoon precipitation is around 10.8 mm, which is almost negligible in the study area. The contribution of precipitation by various seasons is in the following sequence: Southwest monsoon > Northeast monsoon > Pre-monsoon > Post-monsoon. The spatial disparity study indicates that the intensity of average annual, pre-monsoon and post-monsoon precipitations increase towards west in the study area. The intensity of precipitation is more in the northern part during SW monsoon season, whereas the intensity is more in the southern part during NE monsoon season. The spatial disparity analysis of groundwater fluctuation shows that the depth of groundwater (below ground level) increases towards west during all the monsoon seasons. The minimum, mean and maximum depths of occurrence of groundwater in this region are, respectively, 1.6, 9.6 and 21.15 m. Declining trend in the regional groundwater level is observed from December to June because of less precipitation during non-monsoon season. However, the monsoon (both SW and NE monsoon) precipitation recharges the groundwater from June to December to reach the maximum in the month of December.

Association of "Candidatus Liberibacter solanacearum" with the psyllid, Trioza apicalis (Hemiptera: Triozidae) in Europe.

The psyllid Trioza apicalis Förster (Hemiptera: Triozidae) is a serious pest of carrots, Daucus carota L., in Europe. Carrots exhibiting symptoms of psyllid damage were observed in commercial fields in southern Finland in 2008. Symptoms in affected plants included leaf curling, yellow and purple discoloration of leaves, stunted growth of shoots and roots, and proliferation of secondary roots. Mechanisms by which T. apicalis induces symptoms in plants are not understood, and no plant pathogens have yet been associated with this insect. Given recent association of liberibacter with several crops affected by psyllids, an investigation on whether this bacterium is associated with T. apicalis was conducted. Polymerase chain reaction (PCR) primer pairs OA2/OI2c and LsoF/OI2c, specific for 16S rRNA gene from "Candidatus Liberibacter solanacearum," generated amplicons of 1,168 bp and 1,173 bp, respectively, from DNA extracted from field-collected psyllids (61 and 36.6%, respectively), laboratory-reared psyllids (70 and 33.3%, respectively), field-collected petioles from symptomatic carrots (80 and 55%, respectively), and laboratory-grown carrots (100% for both primer pairs). In contrast, no PCR products were detected in DNA extracted from insect-free plants. The DNA sequences of amplicons of the genes encoding liberibacter 16S rRNA from psyllids and carrots were identical. DNA of the 16S rRNA gene sequences determined from carrots and psyllids were 99.9% identical to analogous sequences of "Ca. L. solanacearum" amplified from several solanaceous crops and the psyllid Bactericera cockerelli (Sulc), a vector of this bacterium. This is the first report of a plant pathogen associated with T. apicalis and the second known psyllid species associated with "Ca. L. solanacearum".

Goat pox virus isolated from an outbreak at Akola, Maharashtra (India) phylogenetically related to Chinese strain.

In this study, we investigated a goat pox outbreak that occurred in an organized goat farm in a village named Yerenda near Akola, Maharashtra, India during 2007-2008. The outbreak involved in 175 goats including kids of local nondescript breeds with a morbidity, mortality, and case fatality rate, respectively, of 20%, 11.4%, and 60%. The goat pox virus (GTPV) antigen/nucleic acid in the clinical samples was detected by CIE and PCRs whereas virus-specific antibody was detected by using SNT and indirect ELISA. From classical clinical signs coupled with epidemiological details and various diagnostic assays, the causative agent of the outbreaks, GTPV was identified, successfully isolated in Vero cells and characterized. Further, sequence analysis of P32 envelope protein gene revealed that this isolate phylogenetically related closely to Chinese strain.

Isolation and identification of virulent peste des petits ruminants viruses from PPR outbreaks in India.

In this study, three outbreaks of peste des petits ruminants (PPR) in goats and sheep flocks with high morbidity and considerable mortality were recorded at Jhansi and Revati in Uttar Pradesh and Bhopal in Madhya Pradesh, India during 2003-2006. Clinical samples were collected from the affected flocks for laboratory investigation. The PPR virus (PPRV) antigen/nucleic acid in the infected tissues/swab materials was demonstrated by using sandwich enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction techniques, and the antibody to PPRV in serum samples was detected by competitive ELISA. The causative agent of the outbreaks, PPRV, was successfully isolated in Vero cells at first passage itself, and its identity was confirmed. The isolated PPR viruses belong to lineage IV based on phylogenetic analysis of partial fusion gene sequences and are closely related to other Asian or Indian PPRV isolates/strains.

Isolation and characterization of Indian isolates of camel pox virus.

In this study, we isolated and identified three camel pox viruses (CMLV) from two outbreaks of camel pox infection in camels associated with eruptions on cheeks, nostrils, limbs, scrotum, and sheath that occurred at different places of Bikaner district, Rajasthan (India). The scab specimens collected were subjected for virus isolation in Vero cell culture, and the isolated viruses were characterized by employing polymerase chain reaction (PCR) and sequencing. The causative agent was identified as CMLV, based on A-type inclusion, B5R and C18L genes-specific PCRs and partial sequencing of these genes, which clearly confirmed that the outbreaks were caused by CMLV and identity of CMLV isolates. Further, phylogenetic analysis of partial C18L gene sequences have showed that Indian CMLV are clustered together with other reported isolates/strains.