Identification of a novel cell culture adaptation site on the capsid of foot-and-mouth disease virus.

Vaccination remains the most effective tool for control of foot-and-mouth disease both in endemic countries and as an emergency preparedness for new outbreaks. Foot-and-mouth disease vaccines are chemically inactivated virus preparations and the production of new vaccines is critically dependent upon cell culture adaptation of field viruses, which can prove problematic. A major driver of cell culture adaptation is receptor availability. Field isolates of foot-and-mouth disease virus (FMDV) use RGD-dependent integrins as receptors, whereas cell culture adaptation often selects for variants with altered receptor preferences. Previously, two independent sites on the capsid have been identified where mutations are associated with improved cell culture growth. One is a shallow depression formed by the three major structural proteins (VP1-VP3) where mutations create a heparan sulphate (HS)-binding site (the canonical HS-binding site). The other involves residues of VP1 and is located at the fivefold symmetry axis. For some viruses, changes at this site result in HS binding; for others, the receptors are unknown. Here, we report the identification of a novel site on VP2 where mutations resulted in an expanded cell tropism of a vaccine variant of A/IRN/87 (called A - ). Furthermore, we show that introducing the same mutations into a different type A field virus (A/TUR/2/2006) resulted in the same expanded cell culture tropism as the A/IRN/87 A - vaccine variant. These observations add to the evidence for multiple cell attachment mechanisms for FMDV and may be useful for vaccine manufacture when cell culture adaptation proves difficult.

Understanding foot-and-mouth disease virus transmission biology: identification of the indicators of infectiousness.

The control of foot-and-mouth disease virus (FMDV) outbreaks in non-endemic countries relies on the rapid detection and removal of infected animals. In this paper we use the observed relationship between the onset of clinical signs and direct contact transmission of FMDV to identify predictors for the onset of clinical signs and identify possible approaches to preclinical screening in the field. Threshold levels for various virological and immunological variables were determined using Receiver Operating Characteristic (ROC) curve analysis and then tested using generalized linear mixed models to determine their ability to predict the onset of clinical signs. In addition, concordance statistics between qualitative real time PCR test results and virus isolation results were evaluated. For the majority of animals (71%), the onset of clinical signs occurred 3-4 days post infection. The onset of clinical signs was associated with high levels of virus in the blood, oropharyngeal fluid and nasal fluid. Virus is first detectable in the oropharyngeal fluid, but detection of virus in the blood and nasal fluid may also be good candidates for preclinical indicators. Detection of virus in the air was also significantly associated with transmission. This study is the first to identify statistically significant indicators of infectiousness for FMDV at defined time periods during disease progression in a natural host species. Identifying factors associated with infectiousness will advance our understanding of transmission mechanisms and refine intra-herd and inter-herd disease transmission models.

Foot-and-mouth disease virus replicates only transiently in well-differentiated porcine nasal epithelial cells.

Three-dimensional (3D) porcine nasal mucosal and tracheal mucosal epithelial cell cultures were developed to analyze foot-and-mouth disease virus (FMDV) interactions with mucosal epithelial cells. The cells in these cultures differentiated and polarized until they closely resemble the epithelial layers seen in vivo. FMDV infected these cultures predominantly from the apical side, primarily by binding to integrin alphav beta6, in an Arg-Gly-Asp (RGD)-dependent manner. However, FMDV replicated only transiently without any visible cytopathic effect (CPE), and infectious progeny virus could be recovered only from the apical side. The infection induced the production of beta interferon (IFN-beta) and the IFN-inducible gene Mx1 mRNA, which coincided with the disappearance of viral RNA and progeny virus. The induction of IFN-beta mRNA correlated with the antiviral activity of the supernatants from both the apical and basolateral compartments. IFN-alpha mRNA was constitutively expressed in nasal mucosal epithelial cells in vitro and in vivo. In addition, FMDV infection induced interleukin 8 (IL-8) protein, granulocyte-macrophage colony-stimulating factor (GM-CSF), and RANTES mRNA in the infected epithelial cells, suggesting that it plays an important role in modulating the immune response.

Recovery of viral RNA and infectious foot-and-mouth disease virus from positive lateral-flow devices.

Foot-and-mouth disease Virus (FMDV) is an economically important, highly contagious picornavirus that affects both wild and domesticated cloven hooved animals. In developing countries, the effective laboratory diagnosis of foot-and-mouth disease (FMD) is often hindered by inadequate sample preservation due to difficulties in the transportation and storage of clinical material. These factors can compromise the ability to detect and characterise FMD virus in countries where the disease is endemic. Furthermore, the high cost of sending infectious virus material and the biosecurity risk it presents emphasises the need for a thermo-stable, non-infectious mode of transporting diagnostic samples. This paper investigates the potential of using FMDV lateral-flow devices (LFDs) for dry transportation of clinical samples for subsequent nucleic acid amplification, sequencing and recovery of infectious virus by electroporation. FMDV positive samples (epithelial suspensions and cell culture isolates) representing four FMDV serotypes were applied to antigen LFDs: after which it was possible to recover viral RNA that could be detected using real-time RT-PCR. Using this nucleic acid, it was also possible to recover VP1 sequences and also successfully utilise protocols for amplification of complete FMD virus genomes. It was not possible to recover infectious FMDV directly from the LFDs, however following electroporation into BHK-21 cells and subsequent cell passage, infectious virus could be recovered. Therefore, these results support the use of the antigen LFD for the dry, non-hazardous transportation of samples from FMD endemic countries to international reference laboratories.

Progress in the development of DNA vaccines against foot-and-mouth disease.

DNA vaccines are, in principle, the simplest yet most versatile methods of inducing protective humoral and cellular immune responses. Research involving this type of vaccine against veterinary diseases began in the early 1990s and has since seen the evaluation of more than 30 important viral pathogens, including the economically important foot-and-mouth disease. With the demonstration that DNA vaccines protect against foot-and-mouth disease in sheep and pigs, and the advantages these DNA vaccines have over the conventional formulations, this approach may provide a better solution to the control of this disease. In this review, we provide a comprehensive overview of DNA vaccination strategies for foot-and-mouth disease reported in the literature, in which we highlight the studies that have reported protection in the key target species.

IL-6 production following vaccination in pigs--an additional immune response parameter for assessing FMD vaccine efficacy?

Foot-and-mouth disease vaccine potency testing involving live virus challenge can be problematical in pigs. Alternative methods of assessing vaccine efficacy are therefore desirable. Here we investigate the link between IL-6 in blood at time of challenge and protection against challenge by carrying out statistical analyses utilising data from six separate potency tests performed in swine with the aim of assessing whether IL-6 could be exploited as an additional parameter for confirming vaccine efficacy in pigs. These analyses confirmed that systemic IL-6 levels increased when the administered vaccine dose increased and that the odds of protection against challenge increased as IL-6 levels increased. The link between increased protection and increased antibody was reaffirmed and a significant link between IL-6 levels and antibody levels was shown. We therefore conclude that quantifying the levels of IL-6 in serum could provide additional means of qualifying whether a vaccine will afford clinical protection or not in pigs, in the absence of an actual challenge, and thus offer the possibility of improved vaccine potency testing in pigs both in terms of animal welfare as well as cost.

Relationship between clinical signs and transmission of an infectious disease and the implications for control.

Control of many infectious diseases relies on the detection of clinical cases and the isolation, removal, or treatment of cases and their contacts. The success of such "reactive" strategies is influenced by the fraction of transmission occurring before signs appear. We performed experimental studies of foot-and-mouth disease transmission in cattle and estimated this fraction at less than half the value expected from detecting virus in body fluids, the standard proxy measure of infectiousness. This is because the infectious period is shorter (mean 1.7 days) than currently realized, and animals are not infectious until, on average, 0.5 days after clinical signs appear. These results imply that controversial preemptive control measures may be unnecessary; instead, efforts should be directed at early detection of infection and rapid intervention.

Longevity of protection in cattle following immunisation with emergency FMD A22 serotype vaccine from the UK strategic reserve.

To determine the longevity of protective immunity following a single administration of emergency vaccine, and establish whether the immune response could be enhanced by increasing the antigen payload even further, cattle were vaccinated with an A22 Iraq vaccine containing either 1x antigen payload (field dose) or 5x antigen payload. Six months post-immunisation all cattle received a homologous virus challenge. The magnitude of the virus neutralising antibody response elicited was consistent with the response to similarly formulated A serotype vaccines with a PD(50) greater than 32. All the vaccinated cattle, regardless of antigen payload, were protected from clinical disease following challenge although some cattle in both groups became sub-clinically infected. We conclude that immunisation with a single inoculation of vaccine from the UK emergency reserve can protect cattle from clinical disease for at least 6 months post-vaccination and that a boost may be unnecessary in an outbreak situation. Some animals may become sub-clinically infected but this is likely to be dependent on the severity of challenge. The study confirmed that a booster at 21 days post-vaccination was not necessary to maintain a cell-mediated response in cattle for 6 months. No increased benefits were recognised by increasing the antigen payload of this vaccine 5x.

Experimental evaluation of foot-and-mouth disease vaccines for emergency use in ruminants and pigs: a review.

Changes to foot-and-mouth disease (FMD) control policies since 2001 mean that emergency vaccination must be considered more readily as a control measure in the future. Since field application of vaccine for emergency use has only rarely been applied, the effectiveness of single dose administration, as a control measure in an outbreak situation, is poorly understood. In this review we consider all the available experimental data from studies utilizing either experimental or readily available, commercially produced vaccines, in order to assess their likely effectiveness as an additional means of controlling FMD transmission and spread in an emergency. Overall it is concluded that such vaccines offer an additional and valuable means of FMD control for both ruminants and pigs. They are able to reduce clinical disease, sub-clinical infection and excretion and onward transmission of virus. However, to be most effective, vaccination should be rapidly applied to give maximum opportunity for immunity to develop. We also identify areas for future research and emphasize the importance of vaccine efficacy studies in providing data for models that can help to predict the efficacy of differing FMD control strategies.

Dramatic improvement in FMD DNA vaccine efficacy and cross-serotype antibody induction in pigs following a protein boost.

To overcome the low and slow development of humoral antibody often observed with DNA vaccines we applied a prime-boost strategy. When FMD DNA vaccine P1-2A3C3D and pGM-CSF primed pigs were boosted with inactivated foot-and-mouth disease virus (FMDV) antigen and recombinant 3D (without adjuvant) an average 36-fold increase in the FMDV antibody response was observed compared to conventional vaccination, that included a log(10) virus neutralising titre increase. Most remarkably, a significant level of cross-serotype reactivity was observed against A, C and Asia1 in the virus neutralisation and ELISA tests. This prime-boost strategy fully protected pigs from a heterologous challenge.

Further evaluation of higher potency vaccines for early protection of cattle against FMDV direct contact challenge.

The effect of administering higher payload FMD vaccines 10 days prior to severe direct contact challenge on protection from clinical disease and sub-clinical infection was investigated in cattle using two antigen payloads (single strength and 10-fold). Regardless of antigen payload, vaccination was shown to significantly reduce the number of clinically infected animals, and significantly reduce virus excretion shortly after challenge, when compared with the unvaccinated group (P<0.05). Although FMDV transmission occurred from single strength vaccinated infected cattle to similarly vaccinated cattle held in indirect contact, no disease was induced in these animals. These studies further confirm that emergency vaccination does significantly reduce clinical disease and sub-clinical virus replication and excretion, particularly early post exposure, thereby reducing the possibility of transmission between animals and herds. To be most effective, however, the results also substantiate that time of vaccination prior to challenge significantly influences the number of animals becoming infected, so the decision to vaccinate should be made swiftly, to allow maximum opportunity for protective immunity to develop.

Enhancing immune responses against a plasmid DNA vaccine encoding a FMDV empty capsid from serotype O.

Various parameters have been examined for the improvement of immune responses induced by immunisation with foot-and-mouth disease virus (FMDV) DNA vaccine (pcDNA 3.1/P1-2A3C3D, P1) in swine. Our findings show that increasing the amount of P1 DNA plasmids and pGMCSF adjuvant plasmids induces stronger FMDV specific and neutralising antibody responses, as well as promoting cytokines IL-8 and IFNgamma secretion, in immunised pigs via multiple inoculation sites.

Secretory IgA as an indicator of oro-pharyngeal foot-and-mouth disease virus replication and as a tool for post vaccination surveillance.

A serotype-specific ELISA was developed to detect foot-and-mouth disease virus (FMDV) specific IgA antibody in the saliva of cattle, and the method was evaluated for its feasibility in detecting serotype O FMDV carrier animals, particularly amongst vaccinated cattle that had subsequently become sub-clinically infected. For this purpose, saliva samples were collected from naïve cattle (n = 173), FMDV challenged cattle (n = 10), FMDV vaccinated cattle (n = 40) and FMDV vaccinated-and-challenged cattle (n = 40). A subset of 29 cattle was sampled for 105-168 days after challenge. The FMDV infection status of each of the cattle was determined by virus isolation and RT-PCR tests on oesophago-pharyngeal fluids and the ability of the IgA test to detect viral infection and persistence was compared to an ELISA for the detection of serum antibodies against the 3ABC non-structural proteins of FMDV. Eleven out of twelve vaccinated cattle that were shown to be persistently infected with FMDV up to or beyond 28 days post challenge, were also detected by the IgA test on saliva. With some modification and further validation, this test could be useful in post-vaccination surveillance to help confirm the absence of sub-clinical infection in order to regain the FMD-free status of a region or country.

Foot-and-mouth disease vaccine potency testing: determination and statistical validation of a model using a serological approach.

European foot-and-mouth disease vaccine manufacturers are required to quantify the efficacy of their product in accordance with the European Pharmacopoeia (EP). The method used most often to establish the potency of foot-and-mouth disease vaccines requires viral challenge of vaccinated cattle. Alternative approaches, such as challenge-free serological assessments have many advantages over existing methods and could be used if robust statistical models could be developed that related antibody titres to protection from challenge. Logistic regression analysis of data from two independent research laboratories, representing six of the seven main serotypes of FMD, permitted the parameterisation of these models and indicated that a significant relationship existed between antibody titre and probability of protection. Furthermore, no significant differences were observed in the parameters of logistic models fitted to different strains within the serotypes A, O, and SAT-3, or when strains from serotypes A, O, and Asia-1, or SAT-1 and SAT-3, were combined. However, significant differences in the model parameters did exist between different laboratories. Using these models a bootstrap analysis suggested that for vaccines that induced consistently high titres, as few as six to eight individual animals could be used to establish with confidence the minimum protective doses that would protect 50% of vaccinated animals. We conclude that a serologically evaluated truncated test that eliminates the need to virus challenge cattle is a credible alternative for quantifying vaccine potency.