Infection and protection responses of deletion mutants of non-structural proteins of foot-and-mouth disease virus serotype Asia1 in guinea pigs.

The development of a negative marker vaccine against the foot-and-mouth disease virus (FMDV) will enhance the capabilities to differentiate vaccinated from infected animals and move forward in the progressive control pathway for the control of FMD. Here, we report the development of mutant FMDV of Asia1 with partial deletion of non-structural proteins 3A and 3B and characterization of their infectivity and protection response in the guinea pig model. The deleted FMDV Asia1/IND/63/1972 mutants, pAsiaΔ3A and pAsiaΔ3A3B1 were constructed from the full-length infectious clone pAsiaWT, the viable virus was rescued, and the genetic stability of the mutants was confirmed by 20 monolayer passages in BHK21 cells. The mutant Asia1 viruses showed comparable growth pattern and infectivity with that of AsiaWT in the cell culture. However, the AsiaΔ3A3B1 virus showed smaller plaque and lower virus titer with reduced infectivity in the suckling mice. In guinea pigs, the AsiaΔ3A3B1 virus failed to induce the disease, whereas the AsiaΔ3A virus induced typical secondary lesions of FMD. Vaccination with inactivated Asia1 mutant viruses induced neutralizing antibody response that was significantly lower than that of the parent virus on day 28 post-vaccination (dpv) in guinea pigs (P < 0.05). Furthermore, challenging the vaccinated guinea pigs with the homologous vaccine strain of FMDV Asia1 conferred complete protection. It is concluded that the mutant AsiaΔ3A3B1 virus has the potential to replace the wild-type virus for use as a negative marker vaccine after assessing the vaccine worth attributes in suspension cell and protective efficacy study in cattle.Key points• Deletion mutant viruses of FMDV Asia1, developed by PCR-mediated mutagenesis of NSP 3A and 3B1, were genetically stable.• The growth kinetics and antigenic relatedness of the mutant viruses were comparable with that of the wild-type virus.• Vaccination of guinea pigs with the deletion mutant viruses conferred complete protection upon challenge with the homologous virus.

RT-LAMP assay: an alternative approach for profiling of bovine heat shock protein 70 gene in PBMC cultured model.

The purpose of this study is to develop a novel Reverse Transcriptase Loop-mediated isothermal amplification (RT-LAMP) based assay for in vitro profiling of heat shock protein 70 (Hsp70) in bovine peripheral blood mononuclear cell (PBMC) culture model utilizing the absorbance level of magnesium pyrophosphate-a by-product of LAMP reaction. A set of bovine Hsp70 specific RT-LAMP primers were designed to detect the differential absorbance level of magnesium pyrophosphate by-product which signifies the degree of Hsp70 amplification from cDNA of thermally induced cultured cells at different recovery periods. The study revealed significant (P < 0.05) correlation between absorbance level and the fold change of Hsp70 transcripts at different kinetic intervals of heat stress recovery in bovine PBMC cell culture models. RT-LAMP based absorbance assay can be used as an indicator to measure the degree of bovine Hsp70 transcripts produced during thermal stress and can be used as an alternative to the traditional Real time PCR assay. Developed RT-LAMP assay can be used as a cost-effective method for profiling of bovine HSP70 gene.

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.

Comparative expression profile of NOD1/2 and certain acute inflammatory cytokines in thermal-stressed cell culture model of native and crossbred cattle.

Thermotolerance depends mainly on the health and immune status of the animals. The variation in the immune status of the animals may alter the level of tolerance of animals exposed to heat or cold stress. The present study was conducted to investigate the expression profile of two important nucleotide binding and oligomerization domain receptors (NLRs) (NOD1 and NOD2) and their central signalling molecule RIP2 gene during in vitro thermal-stressed bovine peripheral blood mononuclear cells (PBMCs) of native (Sahiwal) and crossbred (Sahiwal X HF) cattle. We also examined the differential expression profile of certain acute inflammatory cytokines in in vitro thermal-stressed PBMC culture among native and its crossbred counterparts. Results revealed that the expression profile of NOD1/2 positively correlates with the thermal stress, signalling molecule and cytokines. Present findings also highlighted that the expression patterns during thermal stress were comparatively superior among indigenous compared to crossbred cattle which may add references regarding the better immune adaptability of Zebu cattle.

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.

Differential effect of thermal stress on HSP70 expression, nitric oxide production and cell proliferation among native and crossbred dairy cattle.

In a tropical country like India, thermal stress is one of the major factors which significantly affects the productivity of dairy cattle. The present study was aimed to identify the effect of heat and cold stress on cell viability, mitogen stimulation indices, nitric oxide production and HSP70 expression in Sahiwal and Holstein crossbred (Frieswal) population in India. The results indicated that the Sahiwal breed can better withstand the effect of heat and cold stress significantly (P<0.05) when compared to the crossbred cattle due to the higher survivability of the Peripheral Blood Mononuclear Cells (PBMCs) and Phytohemagglutinin (PHA-P) mitogen based stimulation indices. The study also revealed the significant differences (P<0.05) in the level of nitric oxide (µM) production amongst the pre and post thermal stressed samples of Sahiwal and Frieswal crossbred samples. Further, the expression of HSP70 was significantly (P<0.05) higher in Sahiwal compared to Frieswal immediately after heat/cold shock to 6h of recovery as indirect ELISA analysis showed gradual rise in the Hsp70 protein concentration (ng/ml) immediately after heat and cold stress (0h) and reached the peak at 6h of recovery. Western blot and immune fluorescent assay results were also corroborated with the findings of indirect ELISA. In Sahiwal cattle the mRNA expression of HSP70 and its protein concentration were higher (P<0.05) during peak summer (44°C) and winter (10°C) as compared to Frieswal cattle. This investigation supports the earlier information on the higher adaptability of indigenous cattle breeds to hot and humid conditions compared to the crossbreds of temperate cattle breeds.

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.

Characterization of monoclonal antibodies targeting SARS-CoV-2 spike glycoprotein: Reactivity against Delta and Omicron BA.1 variants.

The cross-species transmissibility of SARS-CoV-2 infection has necessitated development of specific reagents for detecting infection in various animal species. The spike glycoprotein of SARS-CoV-2, which is involved in viral entry, is a highly immunogenic protein. To develop assays targeting this protein, we generated eight monoclonal antibodies (mAbs) against the S1 and seven against the S1/S2 protein (ectodomain) of SARS CoV-2. Based on neutralization capability and reactivity profile observed in ELISA, the mAbs generated against the S1/S2 antigen exhibited a broader spectrum of epitope specificity than those produced against the S1 domain alone. The full-length ectodomain induced antibodies that could neutralize the two most important variants of the virus encountered during the pandemic, namely Delta and Omicron. The availability of these reagents could greatly enhance the development of precise diagnostics for detecting COVID-19 infections in various host species and contribute to the advancement of mAb-based therapeutics.

Effect of physical form of the therapeutic diet on the behaviour of crossbred calves experimentally infected with foot-and-mouth disease virus.

The oral lesions associated with foot-and-mouth disease (FMD) negatively affect animal behavior, which can adversely impact animal production and welfare. Physical form of a therapeutic diet (TD) can improve the feed intake and wellbeing during the acute phase of FMD. Accordingly, we tested the effect of two physical forms of a previously developed TD on the behavior of calves experimentally infected with FMD virus (FMDV). Crossbred Holstein Friesian male calves of 10-12 months (n = 12) were experimentally infected with a virulent strain of FMDV and were offered a TD enriched with 19% CP and 2.9 Mcal ME/kg for 11 days post-FMDV infection. One group received the TD in mash form (TDM) while the other (n = 6/group) received it in cooked form (TDC). A group of four calves served as uninfected control and were fed TDM. The time spent by the calves on certain behaviours was recorded in a pre-set form from 06:00-18:00 h for 10 days from day 2-11 post-FMDV infection. The data was divided into two sessions. Session 1 (06:00-13:00 h) represented after the offering of TD, while session 2 (13:01-18:00 h) represented the data after offering green fodder. Based on exploratory data analysis, data recorded from day 2-7 post-FMDV infection was included in the final analysis. Linear mixed model was used by fitting treatment, day and their interaction as fixed effects while calf as random effect. Orthogonal contrast was applied by comparing the infected TDM with other two groups. The results revealed that the cooked form of TD improved the ingestion time, resting time, sleeping time and licking time from day 2-7 post-FMDV infection as compared with the infected TDM group. Ingestive behaviour was better in the infected TDC than that of TDM group (p < 0.01). The sleeping time was significantly high in the infected groups as compared to the uninfected TDM group (p < 0.01) till day 6 post-FMDV infection. Daily activities such as licking, standing and resting differed significantly between the infected TDM and TDC groups in session 1, but not in session 2. Urination and defecation did not differ significantly between the infected TDM and TDC groups. It was concluded that cooked form of TD remediated the effects of infection with FMDV as evidenced by improvement in the behaviour of the calves.

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.

The complete genome sequence of Indian sheeppox vaccine virus and comparative analysis with other capripoxviruses.

Sheeppox virus (SPPV) is responsible for a significant economic loss to sheep husbandry in enzootic regions of Africa, the Middle East, and Asia including the Indian subcontinent. In this study, we present the complete genome sequence of SPPV vaccine strain SPPV-Srin38/00 from India determined by next-generation sequencing (NGS) using Illumina technology. The attenuated Srinagar vaccine strain of SPPV (SPPV-Srin38/00) was developed by serial passaging the virus initially in lamb testes (LT) cells followed by Vero cell line. The SPPV-Srin38/00 virus has a genome size of 150, 103 bp, which encodes for 147 functional putative genes and consists of a central coding region flanked by two identical 2353 bp inverted terminal repeats (ITRs). Comparative phylogenetic analysis based on complete genome sequences of Capripoxviruses formed three distinct groups each for SPPV, GTPV, and LSDV with clustering of SPPV-Srin38/00 strain with SPPV-A strain. Nine ORFs of SPPV-Srin38/00 namely SPPV-Srin_002/SPPV-Srin_155, SPPV-Srin_004/SPPV-Srin_153, SPPV-Srin_009, SPPV-Srin_013, SPPV-Srin_026, SPPV-Srin_132, and SPPV-Srin_136 were found to be fragmented as compared to LSDV, whereas only one ORF (such as SPPV-Srin_136) was found to be fragmented as compared to GTPV. SPPV genomes, including the SPPV-Srin38/00 strain, shared 99.78-99.98% intraspecies nucleotide identity, indicating that SPPV strains have extremely low genetic diversity. The strain shared 96.80-97.08% and 97.11-97.61% nt identity with GTPV and LSDV strains, respectively. Its ORFs 016, 021, 022, 130 and 138 are the least identical ORFs among three species of the genus Capripoxvirus with 72.5-93% aa identity to GTPV and LSDV strains and may be potentially used for differentiation of CaPV species. This study may contribute to a better understanding of the epidemiology and evolution of capripoxviruses as well as the development of specific detection methods, better expression vectors, and vaccines with improved safety and efficacy.

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.

Sero-epidemiological study of peste des petits ruminants in sheep and goats in India between 2003 and 2009.

This study describes the serosurveillance of peste des petits ruminants (PPR) in sheep and goats that was carried out between 2003 and 2009 using serum samples from animals suspected of PPR that were submitted to the Rinderpest and Allied Disease Laboratory (Division of Virology of the Indian Veterinary Research Institute [IVRI]). A total of 2,197 serum samples from sheep and 2,687 from goats were screened for PPR virus (PPRV) antibody using a monoclonal antibody-based competitive enzyme-linked immunosorbent assay developed at IVRI. Screening of the 4,884 serum samples showed that the prevalence of PPRV antibody in sheep and goats was 41.01% (95% confidence interval [CI]: 31.86 to 50.16) and 46.11% (95% CI: 37.18 to 55.04), respectively, with an overall prevalence of 43.56% (95% CI: 36.78 to 50.34) during the period. This indicates increased and widespread infection with the virus in India compared with earlier reports, which is attributed to the variations in sheep and goat husbandry practices in different regions, the agro-climatic conditions, the topography of different states, the socio-economic status of individual farmers and the migration of livestock in India.

Partial genetic characterization of viruses isolated from pox-like infection in cattle and buffaloes: evidence of buffalo pox virus circulation in Indian cows.

Characterization of field isolates of viruses associated with pox-like outbreaks involving both cows (cattle) and buffaloes was carried out. PCR and electron microcopy of representative virus isolates from these animals, initially identified them as orthopoxviruses (OPXVs). Sequence and phylogenetic analyses of A-type inclusion and haemagglutinin (HA) genes of these isolates revealed a closer relationship with other OPXVs. Sequencing of the HA gene of these isolates revealed sequence identity of 96.2-99.8 and 94.6-98.7% at the nucleotide and deduced amino acid level, respectively, with VACVs, particularly with VACV-MVA, a vaccine strain. Further, C18L (ankyrin repeat protein)-gene-based BPXV-specific PCR confirmed them as BPXV. It is apparent from this study that pox-like outbreaks in cows and buffaloes in India are caused, in most cases, by BPXV. Considering the zoonotic implications of buffalopox, such outbreaks involving both buffaloes and cows in a mixed flock may pose a significant public health threat. Transmissibility of BPXV between different species including cows, buffaloes and human beings implies the potential reemergence of the virus in the subcontinent, similar to vaccinia-like outbreaks witnessed recently in other countries.

Comparative efficacy of peste des petits ruminants (PPR) vaccines.

Peste des petits ruminants (PPR) is a highly contagious, economically important viral disease of sheep and goats with high morbidity and mortality rates. In order to control the disease effectively, highly sensitive diagnostic tests coupled with potent vaccines are important pre-requisites. At present, there are three live attenuated PPR vaccines available in India including Sungri 96, Arasur 87 and Coimbatore 97. Indian Veterinary Research Institute (IVRI) Mukteswar developed the PPR Sungri 96 (isolate of goat origin) vaccine; while Tamil Nadu Veterinary and Animal Sciences University (TANUVAS) developed the Arasur 87 (isolate of sheep origin) and Coimbatore 97 (isolate of goat origin). In this study, the potency of these vaccines including a fourth vaccine from Institute of Animal Health and Veterinary Biologicals, Bangalore (IAH&VB) were tested as per the office International des Epizooties (OIE) guidelines by challenge studies in sheep and goats and their efficacies were evaluated using PPR C-ELISA. Potency tests of these vaccines in sheep and goats revealed that three of the vaccines were potent; however, the IAH &VB vaccine was comparatively less potent. The three vaccines could presumably be used for mass vaccination of both sheep and goats while contemplating PPR control program.

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.

Sequence analysis of morbillivirus CD150 receptor-Signaling Lymphocyte Activation Molecule (SLAM) of different animal species.

Signaling Lymphocyte Activation Molecule-SLAM (CD150) molecule has been reported as a putative receptor for most morbilliviruses for their respective host species. In this study, we determined the complete nucleotide sequence of the gene coding for the morbillivirus receptor-SLAM from the four species, namely, goat (Capra hircus), sheep (Ovis aries), Indian cattle (Bos indicus), and buffalo (Bubalus bubalis). The nucleotide (nt) open reading frame sequence of SLAM gene in all the four species studied was 1017 nucleotides in length encoding a polypeptide of 339 amino acids (aa), similar to Bos taurus, but different from canine, human, marmoset, and mouse SLAM, which were 1029, 1008, 1011, and 1032 nts, respectively, in length, and coding for 343, 336, 337, and 344 aa, respectively. Sequence analysis revealed 96.3-98.5% and 92.9-96.8% identities among the four species at the nt and aa level, respectively. Sequence diversity at aa level between various species revealed that the critical functional region of SLAM protein among different species is relatively conserved, thereby facilitating this molecule to act as a receptor for morbillivirus. Phylogenetic relationship based on the aa sequences of SLAM protein revealed that caprine, ovine, cattle, and buffalo fall under a defined cluster but caprine SLAM is more closely related to ovine, followed by bovine.