Characterization of Foot-and-Mouth Disease Virus Serotype O-Specific Single Domain Antibody Expressed in the pET Expression System.

Foot and mouth disease (FMD) is an extremely contagious disease of cloven-hoofed domesticated and wild animals, resulting in significant economic losses in many parts of the world. FMD virus (FMDV) serotype O is responsible for approximately 70% of global outbreaks. For detection of FMDV antigen or antibody, ELISAs are used worldwide and have several limitations, such as batch-to-batch variation in generating immunobiologicals, high production cost and ethical concerns over animal sacrifice. The use of single domain antibody (sdAb) or variable N-terminal domain of the heavy chain of heavy chain antibody (VHH) found naturally in camels has proven their effectiveness in diagnostics and therapeutics. In the present study, the anti-FMDV serotype O-specific VHH-C1 gene sequence (Accession no. KJ751546) was retrieved from the NCBI database. The gene was synthesized commercially in the pBluescript KS+ cloning vector and expressed in E. coli BL21 (DE3) cells using the pET303/CT-His expression system with a C-terminal 6X-His tag. The expressed sdAb, verified by SDS‒PAGE and western blotting, was purified by Ni-chelate chromatography and used as a coating antibody in double antibody sandwich (DAS) ELISA for FMDV detection and typing. The sdAb exhibited a high binding affinity for FMDV serotype O, without any cross-reactivity toward serotypes A and Asia-1. It exhibited better thermostability up to 85 °C than conventional rabbit polyclonal anti-FMDV sera. The potential of sdAbs thus produced without sacrificing lab animals could be explored for replacing polyclonal sera in DAS-ELISA as well as for developing biosensors or lateral flow devices for FMDV type O detection.

Positively charged residues at the five-fold symmetry axis of cell culture-adapted foot-and-mouth disease virus permit novel receptor interactions.

Field isolates of foot-and-mouth disease virus (FMDV) have a restricted cell tropism which is limited by the need for certain RGD-dependent integrin receptors. In contrast, cell culture-adapted viruses use heparan sulfate (HS) or other unidentified molecules as receptors to initiate infection. Here, we report several novel findings resulting from cell culture adaptation of FMDV. In cell culture, a virus with the capsid of the A/Turkey/2/2006 field isolate gained the ability to infect CHO and HS-deficient CHO cells as a result of a single glutamine (Q)-to-lysine (K) substitution at VP1-110 (VP1-(Q)110(K)). Using site-directed mutagenesis, the introduction of lysine at this same site also resulted in an acquired ability to infect CHO cells by type O and Asia-1 FMDV. However, this ability appeared to require a second positively charged residue at VP1-109. CHO cells express two RGD-binding integrins (α5ß1 and αvß5) that, although not used by FMDV, have the potential to be used as receptors; however, viruses with the VP1-(Q)110(K) substitution did not use these integrins. In contrast, the VP1-(Q)110(K) substitution appeared to result in enhanced interactions with αvß6, which allowed a virus with KGE in place of the normal RGD integrin-binding motif to use αvß6 as a receptor. Thus, our results confirmed the existence of nonintegrin, non-HS receptors for FMDV on CHO cells and revealed a novel, non-RGD-dependent use of αvß6 as a receptor. The introduction of lysine at VP1-110 may allow for cell culture adaptation of FMDV by design, which may prove useful for vaccine manufacture when cell culture adaptation proves intractable.

Co-infection of bluetongue virus serotypes 12 and 16 in sheep from Haryana, India.

World Organization for Animal Health has listed bluetongue (BT) under notifiable diseases. The BT is an arboviral infectious disease of domestic and wild ruminants caused by the bluetongue virus (BTV). Southern states of India had remained the point of attention for BT since first presence in 1964 in Maharashtra. Recently, northern states of India have also been reported positive for BTV in small ruminants. The present study reported the dual infection of BTV serotypes, BTV-12 and -16 in sheep population from Sirsa district of Haryana in the year 2016. After detection and serotyping with Seg-2 specific real time polymerase chain reaction (PCR), the Seg-2 and Seg-6 of BTV were PCR amplified and sequenced. On phylogenetic analysis it was detected to be clustered in nucleotype G and nucleotype B specific for BTV-12 and BTV-16, respectively. This was the first report of BTV-16 from Haryana. The results signified the co-infection of two different serotypes in an animal from a single outbreak.

Capsid proteins from field strains of foot-and-mouth disease virus confer a pathogenic phenotype in cattle on an attenuated, cell-culture-adapted virus.

Chimeric foot-and-mouth disease viruses (FMDVs) have been generated from plasmids containing full-length FMDV cDNAs and characterized. The parental virus cDNA was derived from the cell-culture-adapted O1Kaufbeuren B64 (O1K B64) strain. Chimeric viruses, containing capsid coding sequences derived from the O/UKG/34/2001 or A/Turkey 2/2006 field viruses, were constructed using the backbone from the O1K B64 cDNA, and viable viruses (O1K/O-UKG and O1K/A-Tur, respectively) were successfully rescued in each case. These viruses grew well in primary bovine thyroid cells but grew less efficiently in BHK cells than the rescued parental O1K B64 virus. The two chimeric viruses displayed the expected antigenicity in serotype-specific antigen ELISAs. Following inoculation of each virus into cattle, the rescued O1K B64 strain proved to be attenuated whereas, with each chimeric virus, typical clinical signs of foot-and-mouth disease were observed, which then spread to in-contact animals. Thus, the surface-exposed capsid proteins of the O1K B64 strain are responsible for its attenuation in cattle. Consequently, there is no evidence for any adaptation, acquired during cell culture, outside the capsid coding region within the O1K B64 strain that inhibits replication in cattle. These chimeric infectious cDNA plasmids provide a basis for the analysis of FMDV pathogenicity and characterization of receptor utilization in vivo.

Foot and Mouth Disease (FMD) incidence in cattle and buffaloes and its associated farm-level economic costs in endemic India.

The study investigated the important epidemiological parameters and farm-level economic costs of FMD incidence in cattle and buffaloes during 2013-14 to 2015-16 in various states of India. Multistage random sampling procedure was adopted for the primary survey and data was collected through face-to-face personal interview from 18,609 cattle and buffalo rearing farm households from 123 districts across twelve states and one Union Territory. Besides epidemiological parameters, different farm-level direct and indirect loss associated with FMD was assessed at disaggregated level (states) by employing deterministic mathematical models. Highest number of affected villages and disease incidence was observed in non- FMD control programme (FMD-CP) implemented Madhya Pradesh and Assam states, respectively whereas negligible incidence was in FMD-CP implemented Punjab state. The disease incidence was high during 2013-14 and declined during 2014-15 and 2015-16, respectively implied severe incidence scenario (2013-14) succeeded by moderate (2014-15) and mild (2015-16) scenarios. The crossbred and high productive animals were severely affected than local breeds whereas on sexwise and agewise comparison revealed higher incidence in females and adult animals. During severe incidence scenario, milk loss/animal ranged from USD 6.87-47.44, 18.42-125.88, 16.33-91.43, and 27.17-123.62; mortality loss/animal ranged from USD 32.61-804.27, 30.76-577.7, 65.36-502.2, and 188.04-413.7; distress sale loss/animal ranged from USD 3.22-188.63, 64.34-519.3, 214.47-341.8, and 209.11-450.3; and opportunity cost of labour/animal from USD 5.49-54.29, 5.49-67.78; 7.95-31.37 and 9.83-72.38 in indigenous cattle, crossbred cattle, local and improved buffalo, respectively. The estimated draught power loss/animal varied from USD 39.46-142.94 with least being in Madhya Pradesh and highest in Assam states whereas the median treatment cost/animal was USD 9.18 and USD 27.07 in indigenous cattle and upgraded buffaloes, respectively. The total farm-level economic loss projected due to FMD in cattle and buffaloes in India was USD 3159 million (INR 221,110 million), USD 270 million (INR 18,910 million) and USD 152 million (INR 10,610 million), respectively during the severe, moderate and mild incidence scenarios at 2015-16 constant prices. The loss varied across the states, and in severe incidence scenario, the country might lose USD 3.2 billion/year and hence, the bi-annual vaccination schedule need to be strictly implemented in all the states. Besides timely vaccination coverage, managing unabated animal movement, educating and motivating the farmers to vaccinate their animals might reduce the incidence and consequential losses to various stakeholders in endemic states like India.

Effect of TLR agonist on infections bronchitis virus replication and cytokine expression in embryonated chicken eggs.

Avian infectious bronchitis (IB) is an acute, highly infectious and contagious viral disease of chickens caused by avian infectious bronchitis virus (IBV) belonging to the genus Coronavirus and family Coronaviridae. It can affect all age groups of birds. The toll-like receptors (TLRs) are a major class of innate immune pattern recognition receptors that have a key role in immune response and defense against various infections.The TLRs are essential for initiation of innate immune responses and in the development of adaptive immune responses. An in ovo model was employed to study the antiviral activity of TLR ligands (Pam3CSK4, LPS and CpG ODN) on replication of IBV. It was hypothesized that optimum dose and specific timing of TLR ligands may reduce viral load of IBV in specific pathogen free (SPF) embryonated chicken eggs (ECEs). Further, the mechanism involved in the TLR-mediated antiviral response in chorioallantoic membrane (CAM) of ECEs was investigated. The ECEs of 9-11 days old were treated with different doses (high, intermediate and low) of TLR-2 (Pam3CSK4), TLR-4 (LPS) and TLR-21 (CpG ODN) ligands. In addition, to know the timing of TLR ligand treatment, six time intervals were analyzed viz. 36, 24 and 12 h prior to infection, time of infection (co-administration of TLR ligands and avian IBV) and 12 and 24 h post-IBV infection. For studying the relative expression of immuno-stimulatory genes (IFN-α, IFN-ß, IFN-γ, IL-1ß, iNOS and OAS) in CAM, TLR ligands were administered through intra-allantoicroute and CAM were collected at 4, 8 and 16 h post treatment. The results demonstrated that intermediate dose of all the three TLR ligands significantly reduced virus titers and used in the present study. However, the LPS reduced virus titer pre- and post-IBV infection but Pam3CSK4 and CpG ODN reduced only pre-IBV infection. Further analysis showed that TLR ligands induced IFN-γ, IL-1ß and IFN stimulated genes viz. iNOS and OAS genes in CAM. The present study pointed towards the novel opportunities for rational design of LPS as immuno-stimulatory agent in chickens with reference to IBV. It may be speculated that in ovo administration of these TLR ligands may enhance resistance against viral infection in neonatal chicken and may contribute towards the development of more effective and safer vaccines including in ovo vaccines.

Abatement of enteric methane production from lactating Murrah buffaloes (Bubalus bubalis) with improving production performance and immune status through dietary supplementation of composite feed additive.

Ruminant livestock production processes are the major sources of methane production in agriculture sector triggering global environmental pollution. Above 90% of world buffalo population present in Asian countries, India ranks first and contributes significantly to the environmental pollution by enteric methane emissions. In this study, we examined the effect of dietary composite feed additive supplementation on ruminal methane production, nutrient utilization, milk production and immune status of buffaloes (Bubalus bubalis). Eighteen lactating Murrah (Bubalus bubalis) buffaloes at early stage of lactation were divided into two groups of nine animals and fed a composite feed additive [consisted of (%, w/w) dried and ground leaves of Cordia dichotoma and Holoptelea integrifolia, 31.4 each; garlic oil, 0.6; sodium nitrate, 3.1; magnesium sulphate, 8.4; mustard oil, 12.6 and cottonseed oil, 12.5] which contained an ideal combinations of methane inhibitors, alternate hydrogen sinks and rumen stimulating agents to treatment (CFA) group animals along with basal feed of chaffed green sorghum (Sorghum bicolor) fodder, chaffed wheat straw and concentrate mixture for maintenance and milk production. The results showed a decrease (44.6%) in methane concentration in exhaled air of CFA group buffaloes with increase (p < 0.05) in digestibility of feed in comparison to control (CON). Total digestible nutrient (TDN) content of the ration fed to buffaloes of CFA group was significantly (p < 0.05) increased. The daily milk yield, 6% fat corrected milk (FCM) yield and immune response were also increased (p < 0.05) in CFA group. The study suggests that the supplementation of composite feed additive was effective to reduce enteric methane emissions and improvement in production performance and immune status of buffaloes.

Molecular analysis of genome segment-3 of bluetongue virus serotype 12 isolates from Haryana.

AIM: The present study was designed to characterize the genome segment 3 (Seg-3) of bluetongue virus (BTV) serotype 12 isolates from different outbreaks of Bluetongue disease in Haryana, India. MATERIALS AND METHODS: Blood and swab samples were collected from goat and sheep suspected to be suffering of BT from different outbreaks from Gurugram, Sirsa, Hisar, and Karnal districts of Haryana. The samples were grown in insect and mammalian cell lines. After preliminary identification, serotyping was done using BTV type-specific quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays. Sequencing was performed using terminal and walking internal primers specific for Seg-3 on ABI Capillary Sequencer 3130 using a "BigDye cycle sequencing kit." The obtained sequence data were analyzed with various bioinformatic tools. RESULTS: Real-time PCR results confirmed the samples to be positive for BTV-12. The Seg-3 of Indian isolates was most closely related to that of a south Indian isolate of BTV-12 from Andhra Pradesh (KC662614) with 97% nucleotide identity. CONCLUSIONS: The study confirmed the circulation of BTV-12 in Haryana, India. The variations shown in genome Seg-3 of BTV-12 isolates may have some significance and need to be further explored.

A comprehensive study on seroprevalence of bluetongue virus in Haryana state of India.

AIM: The aim of present study was to determine seroprevalence of bluetongue virus (BTV) in Haryana state of India. MATERIALS AND METHODS: A total of 803 serum samples, 408 of cattle and 395 of buffalo origin, respectively, were collected from different villages of Haryana. Sampling was done randomly to obtain unbiased results. The samples were evaluated by a competitive enzyme-linked immunosorbent assay for the presence of BTV antibodies. RESULTS: Overall seroprevalence of BTV antibody in cattle and buffaloes for all 21 districts of Haryana state was found to be 75.49% and 92.91%, respectively. The prevalence of BTV in different agroclimatic zones ranged between 72-77% and 90-94% for cattle and buffalo, respectively. In buffaloes, the BTV seroprevalence was comparatively higher than in cattle. CONCLUSION: The study showed that BTV is circulating in cattle and buffalo populations in the Northern part of India.

Phenotypic and functional characterization of T-cells and in vitro replication of FMDV serotypes in bovine lymphocytes.

Peripheral blood mononuclear cells (PBMCs) from unvaccinated and vaccinated bovine calves were employed to study the alteration of different T-cell subpopulations, functional competence and detection of foot-and-mouth disease virus (FMDV) at different hours post in vitro infection (hpi) with FMDV serotype either O or A or Asia1. The alteration in T-lymphocyte subpopulations was analyzed by flow cytometry analysis using T-cell subpopulation specific monoclonal antibodies at various hpi. All the three FMDV serotypes down-regulate boCD4(+) and boCD8(+) T-cells up to 48hpi whereas down-regulation of boWC1(+) T-cells was observed up to 48hpi with FMDV serotype O only from unvaccinated calves. In contrast, lymphocytes from vaccinated animals demonstrated significant up-regulation of boCD4(+), boCD8(+) and boWC1(+) T-cells following exposure to FMDV serotype either O or A or Asia1. The lymphoproliferative test for functional competence of PBMCs using PHA or inactivated FMDV serotype either O or A or Asia1 antigens, revealed that PHA driven lymphoproliferative response (LPR) was significantly suppressed at 24, 48 and 72hpi in unvaccinated animals. Homotypic as well as heterotypic FMDV antigen specific LPRs were suppressed in PBMCs of unvaccinated animals infected with FMDV serotype either O or A or Asia1 and stimulated with FMDV antigens. FMDV was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) and 3A-non-structural protein (NSP) expression in PBMCs infected in vitro with FMDV serotype either O or A or Asia1 at different hpi. Using RT-PCR, the amplified product revealed 1D gene specific product of FMDV serotype O (1301bp) or A (866bp) or Asia1 (914bp) in lymphocytes of both unvaccinated and vaccinated calves at different hpi with FMDV serotype either O or A or Asia1, respectively. Likewise, the 3A-NSP mAb based flow cytometry revealed significant increase in 3A-NSP expression in lymphocytes of unvaccinated and vaccinated calves at different hpi with FMDV serotype either O or A or Asia1. In conclusion, the present study provides significant evidence that FMDV infects PBMCs in vitro and leads to transient immuno-suppression of boCD4(+) and boCD8(+) T-cells.

Comparative analysis of the roles of simian immunodeficiency and bovine leukemia virus matrix proteins in Gag assembly in insect cells.

The role of the matrix (MA) domain of simian immunodeficiency virus (SIV) and bovine leukaemia virus (BLV) Gag in the assembly of virus-like particles (VLP) in insect cells has been investigated. Wild-type SIV and BLV Gag assembled to form discrete VLP structures typical of many retroviruses analysed by similar systems. When amino acids predicated by the three-dimensional structure to be at the interface of SIV MA monomers were deleted, VLP assembly was abolished consistent with a role for MA multimerization in assembly. When amino acids predicted to be in the analogous positions in BLV MA were mutated, however, VLP assembly was not affected. These data indicate that the models of assembly derived from one model retrovirus may not necessarily apply to more distantly related viruses despite the structural similarity present in equivalent Gag domains.