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.

Performance characteristics of virus neutralization test (VNT) and liquid phase blocking ELISA (LPBE) and their relationship in the cattle immunized with trivalent foot and mouth disease vaccine.

Virus neutralization test (VNT) and liquid phase blocking ELISA (LPBE) are accepted tests for screening and as in vitro alternativ to challenge in FMD vaccine potency testing. To replace VNT by LPBE for the screening of cattle, the optimized tests need to be first evaluated for their diagnostic performances. To replace it with LPBE in the absence of protection data, the interrelationship between VNT and LPBE have to be established to find out LPBE cut­off titer corresponding to the currently used VNT titers. Accordingly, VNT and LPBE were carried out using known negative (n = 306) and positive samples [Serotype O (n = 43), A (n = 14) and Asia1 (n = 11)], for the initial screening. The cut­off of < 1.5 log10 LPBE was comparable with that of < 1.2 log10 VNT titer for screening. LPBE was comparable to VNT in terms of specificity, sensitivity as shown by ROC curve and least varying (coefficient of variation 7.73% in LPBE vs 24.19% in VNT). Based on linear regression model using 471 bovine sera, the predicted LPBE titers corresponding to the currently used log 10 VNT titers of 1.65, 1.5 and 1.5, were 2.24, 1.87 and 2.00 for O, A and Asia1, respectively. These LPBE titers hence can be used as cut­off titers for classifying cattle as protected or not protected until correlation based on in vivo challenge between protection and antibody titer is established.

Short-term effect of foot-and-mouth disease (FMD) vaccination on the milk yield in the Deoni and crossbred cows.

Biannual vaccination of the cattle with inactivated foot-and-mouth disease (FMD) vaccine is the control strategy in endemic countries. Reduction in the milk yield is one of the main reasons for poor compliance of the cattle owners to FMD vaccination. As it can adversely affect the herd immunity, the present study aimed to quantify the losses in the milk yield post-FMD vaccination. Retrospective data on the milk yield (kg) recordings, days in milk, parity, and age at vaccination of the Deoni and crossbred cows were collected from 10 days before (-10) to 10 days after (+10) FMD vaccination (dpv). Days in milk were categorized into three stages of lactation for Deoni and crossbred cows. Age (month) was categorized into four classes. Least squares means of the milk yield were generated after adjusting for year, age, parity, and stage of lactation. Based on exploratory data analysis, the corrected milk yield records from -2 to +2 dpv for 5 years comprising 614 data points on Deoni cows (n=54) and 488 data points on crossbred cows (n=55) were used for the final analysis. Because of the correlated errors on the corrected milk yield, linear mixed model ANOVA was done by fitting dpv as fixed effect and cow as random effect, and the results revealed the effect of dpv was non-significant (P>0.05) in either breed. With respect to dpv 0, a marginal reduction of 90 g in the corrected milk yield in the Deoni cow was recorded on dpv 1, while the reduction was about 360 g on dpv 0 as compared dpv -1 in the crossbred cow. It was concluded that FMD vaccination caused a transient non-significant reduction in the milk yield in the Deoni and crossbred cows.

Efficient inhibition of foot-and-mouth disease virus replication in vitro by artificial microRNA targeting 3D polymerase.

Foot-and-mouth disease (FMD) is a devastating acute viral disease of livestock with cloven hooves. Among various therapeutic control measures, RNA interference (RNAi) is one of the methods being explored to inhibit FMD virus replication and spread. The RNAi is achieved by short hairpin RNAs or artificial microRNAs (amiRNAs). Utility of amiRNAs as antiviral, targeting conserved regions of the viral genome is gaining importance. However, delivery of miRNA in vivo is still a challenge. In this study, the efficacy of amiRNAs in preventing FMD virus replication in a permissive cell culture system was investigated, by generating stable cell lines expressing amiRNAs targeting three functional regions of the FMD virus (FMDV) genome (IRES, 3B3 and 3D). The results showed that amiRNA targeting 3D polymerase is relatively more efficient. However, expression of multiple microRNAs targeting the three regions did not exhibit additive effect. The data suggest that 3D specific miRNA is a potential valid strategy in developing novel antiviral measures against FMDV infection. Keywords: artificial microRNA; foot-and-mouth disease virus; virus inhibition.

Protective immune response to liposome adjuvanted high potency foot-and-mouth disease vaccine in Indian cattle.

BACKGROUND: Foot-and-mouth disease (FMD) vaccines applied for prophylactic use in endemic areas provide short-lived immunity requiring regular boosters. Indian FMD control program recommends twice a year vaccination. Development of high potency vaccines that provide better immune response can singificantly contribute to control programme by reducing the frequency of vaccination. The present study explores new adjuvants to enhance the protective efficacy of inactivated trivalent FMD vaccines. METHODOLOGY AND PRINCIPAL FINDINGS: VacciMax(®) is a novel adjuvant which uses a liposome-based oil emulsion platform. Cattle were immunized using VacciMax-A and VacciMax-B FMD vaccines and evaluated for protective efficacy. Similar groups of animals were also boosted after 6 months to study the effect of booster immunisation on protection against homologous challenge. Serum samples from immunized animals were tested by virus neutralization test (VNT) and liquid phase blocking ELISA (LPBE). After challenge, animals were screened for virus load by real-time PCR and reactivity in non-structural protein (3ABC) antibody detection ELISA to corroborate the protection data. A single dose of VacciMax-A formulation elicited higher percentage protection (63%) in VacciMax-A compared to 25% in VacciMax-B upon challenge at one-year post-vaccination. Upon boosting at 6 months also, VacciMax-A group showed higher levels of protection (100%) compared to VacciMax-B (86%), even though both the groups elicited comparable VNT titre (p=0.4964). The results also demonstrated that intramuscular route was preferrable over subcutaneous route of administration. CONCLUSION: The study demonstrates that immunization with VacciMax-A-IM adjuvanted FMD vaccine with high antigen payload under boosting regimen could effectively be used as potent vaccine to maintain herd immunity.