Emergence of foot-and-mouth disease virus serotype Asia1 group IX in India.

Foot-and-mouth disease virus (FMDV) serotype Asia1 is prevalent in India and is responsible for a minor proportion of FMD outbreaks. Globally, serotype Asia1 is grouped into nine different groups (GI-IX) based on genetic analysis. In India, only Asia1/G-III and Asia1/G-VIII have been documented so far. Phylogenetic analysis of recent serotype Asia1 isolates from India revealed the emergence of Asia1/G-IX. The Asia1/G-IX lineage shares recent common ancestry with Asia1/G-VIII dating to 2016. The root state posterior probabilities of Asia1/G-VIII are inclusive and there may have been either an incursion of the virus from Bangladesh, where it was first identified, or in situ evolution of the virus within India, which is an intriguing possibility.

Construction of an infectious cDNA clone of foot-and-mouth disease virus type O 1 BFS 1860 and its use in the preparation of candidate vaccine.

Foot-and-mouth disease virus (FMDV) serotype O is the most predominant among the endemic serotypes in India. A stable,full-length cDNA clone of FMDV type O 1 BFS 1860 preceded by a bacteriophage T7 polymerase promoter was assembled in a plasmid vector pGEM R- - 7Zf(-). An 8.2 kb PCR product was amplified from the cDNA clone and a full-length RNA was generated from it by in vitro transcription.Transfection of BHK-21 cells with the in vitro transcripts resulted in the production of infectious recombinant FMDV particles as evidenced by cytopathic effects (CPE). Further, characterization of the recombinant virus by immunofluorescence, microneutralization test (MNT), antigen ELISA,RT-PCR, plaque assay and electron microscopy revealed similarity to the parental strain. The immunogenicity of an oil-adjuvant vaccine prepared using the inactivated recombinant virus was tested in guinea pigs and cattle. Neutralizing antibodies were produced in both vaccinated guinea pigs and cattle. Vaccinated animals were protected on challenge. The results demonstrated that the recombinant virus was as stable and effective as the parental strain for the preparation of inactivated vaccine, suggesting the potential application of this strategy to make genetically engineered FMDV vaccines.

Genetic analysis of foot-and-mouth disease virus serotype A of Indian origin and detection of positive selection and recombination in leader protease-and capsid-coding regions.

The leader protease (L pro) and capsid-coding sequences (P1) constitute approximately 3 kb of the foot-and-mouth disease virus (FMDV). We studied the phylogenetic relationship of 46 FMDV serotype A isolates of Indian origin collected during the period 1968-2005 and also eight vaccine strains using the neighbour-joining tree and Bayesian tree methods. The viruses were categorized under three major groups -Asian, Euro-South American and European. The Indian isolates formed a distinct genetic group among the Asian isolates. The Indian isolates were further classi?ed into different genetic subgroups (<5% divergence).Post-1995 isolates were divided into two subgroups while a few isolates which originated in the year 2005 from Andhra Pradesh formed a separate group. These isolates were closely related to the isolates of the 1970s. The FMDV isolates seem to undergo reverse mutation or convergent evolution wherein sequences identical to the ancestors are present in the isolates in circulation. The eight vaccine strains included in the study were not related to each other and belonged to different genetic groups. Recombination was detected in the L pro region in one isolate (A IND 20/82) and in the VP1 coding 1D region in another isolate (A RAJ 21/96). Positive selection was identi?ed at aa positions 23 in the L pro (P < 0.05; 0.046*) and at aa 171 in the capsid protein VP1 (P < 0.01; 0.003**).

Experimental transmission of foot-and-mouth disease among Indian buffalo (Bubalus bubalis) and from buffalo to cattle.

Indian buffalo and cattle were infected experimentally with a serotype O strain of foot-and-mouth disease virus of buffalo origin. Whereas intradermolingual inoculation of buffalo produced largely sub-clinical infection, inoculation in the dental pad produced vesicles in the mouth and on the feet. A buffalo infected via the dental pad transmitted infection to cattle and buffalo by direct contact with them for 24h. The contact-exposed buffalo developed (1) delayed-onset clinical signs, and (2) shedding of virus from the nose, commencing before the appearance of vesicles and continuing until the experiment was terminated 10 weeks after exposure. The covert nature of the disease in Indian buffalo, coupled with the prolonged shedding of virus, suggests that this species represents a host of epidemiological importance.

Evaluation of the Flinders Technology Associates Cards for Storage and Temperature Challenges in Field Conditions for Foot-and-Mouth Disease Virus Surveillance.

Foot-and-mouth disease virus (FMDV) samples transported to the laboratory from far and inaccessible areas for diagnosis and identification of FMDV pose a major problem in a tropical country like India, where wide fluctuation of temperature over a large geographical area is common. Inadequate storage methods lead to spoilage of FMDV samples collected from clinically positive animals in the field. Such samples are declared as non-typeable by the typing laboratories with the consequent loss of valuable epidemiological data. In this study, an attempt was made to evaluate the robustness of Flinders Technology Associates (FTA) cards for storage and transportation of FMDV samples in different climatic conditions which will be useful for FMDV surveillance. Simulation transport studies were conducted using FTA impregnated FMDV samples during post-monsoon (September-October 2010) and summer season (May-June 2012). FMDV genome or serotype could be identified from the FTA cards after the simulation transport studies with varying temperature (22-45°C) and relative humidity (20-100%). The stability of the viral RNA, the absence of infectivity and ease of processing the sample for molecular methods make the FTA cards an useful option for transport of FMDV genome for identification and type determination. The method can be used routinely for FMDV research as it is economical and the cards can be transported easily in envelopes by regular courier/postal systems. The absence of live virus in FTA card can be viewed as an advantage as it restricts the risk of transmission of live virus.

Antigenic and genetic comparison of foot-and-mouth disease virus serotype O Indian vaccine strain, O/IND/R2/75 against currently circulating viruses.

Foot-and-mouth disease (FMD) virus serotype O is the most common cause of FMD outbreaks in India and three of the six lineages that have been described are most frequently detected, namely Ind2001, PanAsia and PanAsia 2. We report the full capsid sequence of 21 serotype O viruses isolated from India between 2002 and 2012. All these viruses belong to the Middle East-South Asia (ME-SA) topotype. The serological cross-reactivity of a bovine post-vaccination serum pool raised against the current Indian vaccine strain, O/IND/R2/75,was tested by virus neutralisation test with the 23 Indian field isolates, revealing a good match between the vaccine and the field isolates. The cross reactivity of the O/IND/R2/75 vaccine with 19 field isolates from other countries (mainly from Asia and Africa) revealed a good match to 79% of the viruses indicating that the vaccine strain is broadly cross-reactive and could be used to control FMD in other countries. Comparison of the capsid sequences of the serologically non-matching isolates with the vaccine strain sequence identified substitutions in neutralising antigenic sites 1 and 2, which could explain the observed serological differences.

Development of anti-bovine IgA single chain variable fragment and its application in diagnosis of foot-and-mouth disease.

Recombinant antibody fragments like single chain variable fragments (scFvs) represent an attractive yet powerful alternative to immunoglobulins and hold great potential in the development of clinical diagnostic/therapeutic reagents. Structurally, scFvs are the smallest antibody fragments capable of retaining the antigen-binding capacity of whole antibodies and are composed of an immunoglobulin (Ig) variable light (VL) and variable heavy (VH) chain joined by a flexible polypeptide linker. In the present study, we constructed a scFv against bovine IgA from a hybridoma cell line IL-A71 that secretes a monoclonal antibody against bovine IgA using recombinant DNA technology. The scFv was expressed in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC). The binding activity and specificity of the scFv was established by its non-reactivity toward other classes of immunoglobulins as determined by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. Kinetic measurement of the scFv indicated that the recombinant antibody fragment had an affinity in picomolar range toward purified IgA. Furthermore, the scFv was used to develop a sensitive ELISA for the detection of foot and mouth disease virus (FMDV) carrier animals.

Transmission of foot-and-mouth disease virus from experimentally infected Indian buffalo (Bubalus bubalis) to in-contact naïve and vaccinated Indian buffalo and cattle.

This study investigated the transmission of foot-and-mouth disease virus (FMDV) from experimentally infected Indian buffalo to in-contact naïve and vaccinated cattle and buffalo. In each of six rooms, two donor buffalo that had been inoculated with FMDV were housed for five days with four recipient animals, comprising one vaccinated buffalo, one vaccinated calf, one unvaccinated buffalo and one unvaccinated calf. Vaccination was carried out with current Indian vaccine strain (O/IND/R2/75) and challenged on 28 days post-vaccination with an antigenically similar strain (O/HAS/34/05). All 12 donor buffalo and the six unvaccinated cattle and six unvaccinated calves developed clinical signs of foot-and-mouth disease (FMD). In contrast, all six vaccinated cattle (100%) and four out of six vaccinated buffalo (66.6%) were protected from disease but all became infected with FMDV. This confirms that buffalo have the potential to spread FMD by direct contact and that vaccination can block this spread. The numbers of animals in the study were too small to determine if the differences in clinical protection afforded by vaccination of cattle and buffalo are significant and warrant a different dose regime.

Development and evaluation of a real-time reverse transcription-loop-mediated isothermal amplification assay for rapid serotyping of foot-and-mouth disease virus.

A one-step, real-time reverse transcription-loop-mediated isothermal amplification assay (RT-LAMP) for rapid detection and serotyping of Indian foot-and-mouth disease virus (FMDV) is described. The RT-LAMP assay was found to be 10(3)-10(5) fold more sensitive in comparison with RT-PCR, with a detection limit ranging from 10(-3) to 10(-5) TCID(50) of virus samples of all three serotypes. The RT-LAMP assay and qRT-PCR could detect 100 percent of clinical samples of three serotypes, whereas the RT-PCR detected 69.7% of type O, 58.1% of type A and 60.0% of Asia 1 samples. The qRT-PCR has the same sensitivity as the RT-LAMP. The assay conditions with absence of cross reactivity within the three serotypes of FMDV and FMDV negative samples were established. The RT-LAMP assay could detect 100% of samples stored in FTA(®) cards. In comparison with the performance of the RT-PCR; the RT-LAMP appears to be more sensitive, rapid and specific, with the potential for use as a point-of-care (POC) test, especially in developing countries. The use of FTA(®) cards for the preservation of RNA samples coupled with the RT-LAMP assay for the identification of serotypes may help in achieving improved FMDV serotype identification both in the field and in the laboratory.

Genetic and antigenic characterization of Indian foot-and-mouth disease virus serotype O isolates collected during the period 2001 to 2012.

The phylogenetic analysis of VP1 sequences of the 39 type O foot and mouth virus (FMDV) isolates collected from different regions of India during the year of 2001-12 revealed that all isolates belonged to the Middle East - South Asia (ME-SA) topotype. Based on the amount of divergence among the isolates, the viruses were further classified into three distinct lineages namely Ind 2001, PanAsia and PanAsia-2 as well as a minor, unnamed group. Ind 2001 lineage viruses accounted for most of the current type O outbreaks. At the nucleotide level these isolates showed a divergence of 2% to 14% with an average sequence variation of ~9.9%. The serological spectrum of the current vaccine strain was studied by using bovine vaccinate serum (BVS) raised against O/IND/R2/75. All the current field isolates (n=24) were homologous ('r' value 0.4 to 1.0) to the vaccine strain. Examination of the amino acid sequences for selection pressure revealed the positive selection at amino acid sites 13 and 45.

Clinical protection, sub-clinical infection and persistence following vaccination with extinction payloads of O1 Manisa Foot-and-Mouth Disease monovalent vaccine and challenge in goats and comparison with sheep.

Small ruminants play an important role in the epidemiology of Foot-and-Mouth Disease (FMD). Small ruminants are vaccinated with one-half or one-third of cattle dose of oil-based or aqueous vaccines respectively. The extinction antigen payload in vaccine for protection in small ruminants is poorly studied. FMD seronegative Nellore sheep (n=30) and Osmanabadi goats (n=30) were vaccinated with different payloads of O(1) Manisa vaccine (0.45-5 µg). Vaccinated and sero-negative unvaccinated sheep (n=6) and goats (n=6) were challenged intradermally into the coronary band with O(1) Manisa virus. The sheep and goats were monitored for signs of FMD and samples were collected for measuring viraemia and virus associated with nasal swabs and probang samples. Clotted blood was collected for serology. Vaccines containing antigen payload up to 0.94 µg protected sheep and goats against challenge. Sheep and goats vaccinated with 0.45 µg antigen payload were poorly protected against challenge. An antigen payload of 0.94 µg was sufficient to offer complete protection and also absence of carrier status. Sheep and goats with no vaccination or with poor sero conversion to vaccination showed sub-clinical infection and became carriers. The results of the study suggest that vaccination offers protection from clinical disease even at a low payload of 0.94 µg and hence one-half of cattle dose of the oil-based vaccine formulations is sufficient to induce protective immune response in sheep and goats. Since no live virus could be isolated after 5 days post challenge from the nasal swab or probang samples even though viral RNA was detected, the risk of these animals transmitting disease was probably very low.

Development of foot-and-mouth disease virus (FMDV) serotype O virus-like-particles (VLPs) vaccine and evaluation of its potency.

Foot-and-mouth disease (FMD) is an economically significant viral disease that rampage dairy and other livestock industries in many countries. The disease is being controlled by the use of an inactivated vaccine. However, a recombinant marker vaccine, which avoids the use of live virus, may be an option for the unambiguous differentiation of infected animals from vaccinated animals. A recombinant baculovirus clone containing P1-2A-3C coding sequences of foot-and-mouth disease virus (FMDV) serotype O(1) Manisa was generated. The FMDV structural proteins along with the 3C protease were expressed in Sf9 cells and the generation of virus like particles (VLP) was studied. The recombinant protein was formulated as vaccine using an oil adjuvant, ISA 206 and potency of the vaccine was tested in cattle. The vaccine had a potency value (PD(50)) of 5.01 and most of the vaccinated animals exhibited neutralizing antibody titers after two immunizations.

Immune Response in Goats to Different Payloads of FMDV Monovalent Vaccine: Protection Against Virulent Challenge and Development of Carrier Status.

The relationship of Foot-and-Mouth Disease virus antigen payload and number of dose of vaccine conferring protection against virus challenge in goats was studied. Goats vaccinated with oil adjuvant Foot-and-Mouth Disease vaccines containing different antigen payloads with or without booster resisted virulent challenge at 21 days post-vaccination or 7 days after booster respectively. However, localized sub-clinical infection was observed in two vaccinated goats on 35 days post-challenge. RNA could be detected from 31.8% of vaccinated goats (10(2.69)-10(4.99) viral RNA copies per cotton swab of nasal secretions) on day 35 post-challenge. Since no live virus could be isolated after 5 days post-challenge, the risk of these animals transmitting the disease was probably very low. The finding showed that oil adjuvant Foot-and-Mouth Disease vaccines containing antigen payload of 1.88 µg may prevent or reduce the local virus replication at the oropharynx and shedding of virus from nasal secretions and thereby reduce the amount of virus released into the environment subsequent to exposure to live virus. This study also showed that goats with poor sero conversion to vaccination can be infected without overt clinical signs and became carriers like sheep.

Immune response in goats to two commercial foot-and-mouth disease vaccines and the assessment of maternal immunity in their kids.

In this investigation, the immune response of goats to two commercial foot-and-mouth disease vaccines (FMDV) was compared. Highest mean antibody titre was observed on days 60 and 21 in goats vaccinated with two doses of algel (group 1) and oil adjuvant (group 2) quadrivalent vaccines, respectively. There was no significant (P > 0.05) difference in mean antibody titre between the two vaccine groups. However, the antibody titres for type O fell below the protective titres by day 180 and 270 for groups 1 and 2, respectively. The mean maternal antibody titre was 0.610 +/- 0.0 immediately after birth. The highest mean maternal antibody titre was observed at 24 h after birth for all serotypes and then steadily declined. The maternal immunity of kids born to the vaccinated does was persistent up to 90 days after birth. There was no significant (P > 0.05) difference in mean maternal antibody titre between the two groups of goats for all four serotypes throughout the study period. The protective maternal antibody titre for serotype O was maintained only up to 1 week after birth, where for the other three serotypes A, C and Asia1 the protective maternal antibody titre was maintained up to 4 weeks of birth. Oil adjuvant vaccine may be used for control of FMDV in goats and the animals have to be revaccinated after 9 months, whereas the kids must be vaccinated at around 3-4 months after birth. Goats must be included in the FMDV control programmes and the same schedule for cattle can be followed.

Poly(lactide-co-glycolide) microspheres: a potent oral delivery system to elicit systemic immune response against inactivated rabies virus.

Rabies is an endemic, fatal zoonotic disease in the developing countries. Oral vaccination strategies are suitable for rabies control in developing countries. Studies were performed to investigate the suitability of poly(lactide-co-glycolide) (PLG) microspheres as an oral delivery system for beta-propiolactone inactivated concentrated rabies virus (CRV). Immune responses induced by encapsulated (PLG+CRV) and un-encapsulated inactivated rabies virus after oral and intraperitoneal route administrations were compared. The anti-rabies virus IgG antibody titer, virus neutralizing antibody (VNA) titers obtained by mouse neutralization test (MNT) and IgG2a and IgG1 titers of mice group immunized orally with PLG+CRV showed significantly (p<0.001) higher response than the group immunized orally with un-encapsulated CRV. There was no significant difference (p>0.05) between groups inoculated by intraperitoneal route. The stimulation index (SI) obtained by lymphoproliferation assay of PLG+CRV oral group also showed significantly (p<0.001) higher response than the group immunized orally with un-encapsulated CRV, suggesting that oral immunization activates Th1-mediated cellular immunity. Immunized mice of all experimental groups were challenged intracerebrally with a lethal dose of virulent rabies virus Challenge Virus Standard (CVS). The survival rates of mice immunized orally with PLG+CRV and CRV alone were 75% and 50%, respectively, whereas intraperitoneally immunized groups showed 100% protection. The overall results of humoral, cellular immune response and survival rates of mice immunized orally with PLG+CRV were significantly (p<0.001) higher than those of mice immunized orally with CRV alone. These data suggest that the PLG encapsulated inactivated rabies virus can be used for oral immunization against rabies.