Genetic and antigenic characterization of serotype O FMD viruses from East Africa for the selection of suitable vaccine strain.

Foot-and-mouth disease (FMD) is endemic in Eastern Africa with circulation of multiple serotypes of the virus in the region. Most of the outbreaks are caused by serotype O followed by serotype A. The lack of concerted FMD control programmes in Africa has provided little incentive for vaccine producers to select vaccines that are tailored to circulating regional isolates creating further negative feedback to deter the introduction of vaccine-based control schemes. In this study a total of 80 serotype O FMD viruses (FMDV) isolated from 1993 to 2012 from East and North Africa were characterized by virus neutralisation tests using bovine antisera to three existing (O/KEN/77/78, O/Manisa and O/PanAsia-2) and three putative (O/EA/2002, O/EA/2009 and O/EA/2010) vaccine strains and by capsid sequencing. Genetically, these viruses were grouped as either of East African origin with subdivision into four topotypes (EA-1, 2, 3 and 4) or of Middle-East South Asian (ME-SA) topotype. The ME-SA topotype viruses were mainly detected in Egypt and Libya reflecting the trade links with the Middle East countries. There was good serological cross-reactivity between the vaccine strains and most of the field isolates analysed, indicating that vaccine selection should not be a major constraint for control of serotype O FMD by vaccination, and that both local and internationally available commercial vaccines could be used. The O/KEN/77/78 vaccine, commonly used in the region, exhibited comparatively lower percent in vitro match against the predominant topotypes (EA-2 and EA-3) circulating in the region whereas O/PanAsia-2 and O/Manisa vaccines revealed broader protection against East African serotype O viruses, even though they genetically belong to the ME-SA topotype.

Selection of vaccine strains for serotype O foot-and-mouth disease viruses (2007-2012) circulating in Southeast Asia, East Asia and Far East.

Foot-and-mouth disease (FMD) is endemic in Southeast Asia (SEA) and East Asia with circulation of multiple serotypes and multiple genotypes within each serotype of the virus. Although countries like Japan and South Korea in the Far East were free of FMD, in 2010 FMD serotype O (O/Mya-98) outbreaks were recorded and since then South Korea has experienced several FMD outbreaks despite regular vaccination. In this study a total of 85 serotype O FMD viruses (FMDV) isolated from 2007 to 2012 from SEA, East Asia and Far East were characterized by virus neutralisation tests using antisera to four existing (O/HKN/6/83, O/IND/R2/75, O/SKR/2010 and O/PanAsia-2) and one putative (O/MYA/2009) vaccine strains, and by full capsid sequencing. Serological studies revealed broad cross-reactivity with the vaccine strains; O/PanAsia-2 exhibited a good match with 95.3%, O/HKN/6/83 with 91.8%, O/IND/R2/75 with 80%, and the putative strain O/MYA/2009 with 89.4% isolates employed in this study. Similarly O/PanAsia-2 and O/IND/R2/75 vaccines showed a good match with all eight viruses belonging to O-Ind-2001d sublineage whereas the vaccines of O/Mya-98 lineage, O/MYA/2009 and O/SKR/2010 exhibited the lowest match indicating their unsuitability to protect infections from O-Ind-2001d viruses. A Bayesian analysis of the capsid sequence data indicated these circulating viruses (n = 85) to be of either SEA or Middle East-South Asian (ME-SA) topotype. The ME-SA topotype viruses were mainly detected in Lao PDR, Vietnam, Myanmar and Thailand reflecting the trade links with the Indian subcontinent, and also within the SEA countries. Implications of these results in the context of FMD control in SEA and East Asian countries are discussed.

Experimental Infection of Ornithodoros erraticus sensu stricto with Two Portuguese African Swine Fever Virus Strains. Study of Factors Involved in the Dynamics of Infection in Ticks.

African swine fever (ASF) is a frequently devastating hemorrhagic disease of domestic pigs and wild boar and Ornithodoros erraticus sensu stricto argasid ticks are the only biological vectors of African swine fever virus (ASFV) known to occur in Europe. Recently this disease emerged in Eastern Europe and Russian Federation, showing a huge potential for a rapid spread between countries. There is some risk of re-emergence of ASF in the countries where these ticks exist, that can contribute for the persistence of infection and compromise control measures. In this study we aimed to identify factors that determine the probability of infection and its dynamics in the tick vector Ornithodoros erraticus sensu stricto, with two Portuguese strains of ASFV. Our results suggest that these ticks have a high likelihood of excreting the two haemadsorbing ASF viruses of different host origins and that, in field surveys, the analysis of adults and 5th nymphal stage can provide the best chance of detecting virus infection. The results also indicate that infection of pigs with highly virulent ASF viruses will promote higher rates of infection and a higher likelihood for virus excretion by ticks. Nevertheless, there is also a risk, although lower, that ticks can become infected on pigs that have overcome the acute phase of infection, which was simulated in our study by membrane feeding ticks with low titres of virus. We believe these results can be valuable in designing and interpreting the results of ASF control programmes, and future work can also be undertaken as our dataset is released under open access, to perform studies in risk assessment for ASFV persistence in a region where O. erraticus sensu stricto ticks are present.

The persistence of African swine fever virus in field-infected Ornithodoros erraticus during the ASF endemic period in Portugal.

African swine fever (ASF) is an important disease of pigs and outbreaks of ASF have occurred in Europe on multiple occasions. To explore the period for which the European soft tick species Ornithodoros erraticus (Acari: Argasidae) is able to act as a reservoir of African swine fever virus (ASFV) after infected hosts are removed, we collected specimens from farms in the provinces of Alentejo and Algarve in Portugal during the endemic period and tested them subsequently using cell culture and experimental infection. We show that ticks from previously infected farms may contain infectious virus for at least five years and three months after the removal of infectious hosts. Furthermore, in two cases infectious virus was successfully isolated from ticks on restocked farms that had not yet suffered a re-emergence of disease. Experimental transmission to pigs was demonstrated in batches tested up to 380 days after an outbreak. These results clarify the epidemiological role of O. erraticus ticks in the persistence of ASFV in the field, provide additional evidence to support its role in the re-emergence of a sporadic outbreak of ASF in Portugal in 1999 and suggest that the current quarantine legislation and restocking advice when these ticks are present on the pig farm premises is appropriate.

Protection of European domestic pigs from virulent African isolates of African swine fever virus by experimental immunisation.

African swine fever (ASF) is an acute haemorrhagic disease of domestic pigs for which there is currently no vaccine. We showed that experimental immunisation of pigs with the non-virulent OURT88/3 genotype I isolate from Portugal followed by the closely related virulent OURT88/1 genotype I isolate could confer protection against challenge with virulent isolates from Africa including the genotype I Benin 97/1 isolate and genotype X Uganda 1965 isolate. This immunisation strategy protected most pigs challenged with either Benin or Uganda from both disease and viraemia. Cross-protection was correlated with the ability of different ASFV isolates to stimulate immune lymphocytes from the OURT88/3 and OURT88/1 immunised pigs.

The CD2v protein enhances African swine fever virus replication in the tick vector, Ornithodoros erraticus.

The NH/P68 non-haemadsorbing (non-HAD) African swine fever virus (ASFV) isolate contains frameshift mutations in the EP402R and adjacent EP153R genes. These encode, respectively, the protein (CD2v) that is required for the haemadsorption (HAD) of swine erythrocytes to ASFV-infected cells and a C-type lectin protein. Two recombinant HAD viruses were constructed in this parental strain. In one of these the intact EP153R gene sequence was restored. Although restoration of the HAD phenotype did not increase virus virulence in pigs, a significant increase was observed in the number of pigs which developed viraemia. These HAD recombinant viruses replicated to titres approximately 1000-fold higher than the parental non-HAD isolate when membrane fed to Ornithodoros erraticus ticks. Inoculation of the non-HAD isolate across the gut wall increased viral replication to levels comparable to that of the HAD recombinant viruses. These results demonstrate a novel role for the CD2v protein in virus replication in ticks.

African swine fever virus isolate, Georgia, 2007.

African swine fever (ASF) is widespread in Africa but is rarely introduced to other continents. In June 2007, ASF was confirmed in the Caucasus region of Georgia, and it has since spread to neighboring countries. DNA fragments amplified from the genome of the isolates from domestic pigs in Georgia in 2007 were sequenced and compared with other ASF virus (ASFV) isolates to establish the genotype of the virus. Sequences were obtained from 4 genome regions, including part of the gene B646L that encodes the p72 capsid protein, the complete E183L and CP204L genes, which encode the p54 and p30 proteins and the variable region of the B602L gene. Analysis of these sequences indicated that the Georgia 2007 isolate is closely related to isolates belonging to genotype II, which is circulating in Mozambique, Madagascar, and Zambia. One possibility for the spread of disease to Georgia is that pigs were fed ASFV-contaminated pork brought in on ships and, subsequently, the disease was disseminated throughout the region.

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.

Molecular epidemiology of the foot-and-mouth disease virus outbreak in the United Kingdom in 2001.

The objective of this study was to quantify the extent to which the genetic diversity of foot-and-mouth disease virus (FMDV) arising over the course of infection of an individual animal becomes fixed, is transmitted to other animals, and thereby accumulates over the course of an outbreak. Complete consensus sequences of 23 genomes (each of 8,200 nucleotides) of FMDV were recovered directly from epithelium tissue acquired from 21 farms infected over a nearly 7-month period during the 2001 FMDV outbreak in the United Kingdom. An analysis of these consensus sequences revealed very few apparently ambiguous sites but clear evidence of 197 nucleotide substitutions at 191 different sites. We estimated the rate of nucleotide substitution to be 2.26 x 10(-5) per site per day (95% confidence interval [CI], 1.75 x 10(-5) to 2.80 x 10(-5)) and nucleotide substitutions to accrue in the consensus sequence at an average rate of 1.5 substitutions per farm infection. This is a sufficiently high rate showing that detailed histories of the transmission pathways can be reliably reconstructed. Coalescent methods indicated that the date at which FMDV first infected livestock in the United Kingdom was 7 February 2001 (95% CI, 20 January to 19 February 2001), which was identical to estimates obtained on the basis of purely clinical evidence. Nucleotide changes appeared to have occurred evenly across the genome, and within the open reading frame, the ratio of nonsynonymous-to-synonymous change was 0.09. The ability to recover particular transmission pathways of acutely acting RNA pathogens from genetic data will help resolve uncertainties about how virus is spread and could help in the control of future epidemics.

Detection of foot-and-mouth disease virus: comparative diagnostic sensitivity of two independent real-time reverse transcription-polymerase chain reaction assays.

Rapid and accurate diagnosis is central to the effective control of foot-and-mouth disease (FMD). It is now recognized that reverse-transcription polymerase chain reaction (RT-PCR) assays can play an important role in the routine detection of FMD virus (FMDV) in clinical samples. The aim of this study was to compare the ability of 2 independent real-time RT-PCR (rRT-PCR) assays targeting the 5' untranslated region (5'UTR) and RNA polymerase (3D) to detect FMDV in clinical samples. There was concordance between the results generated by the 2 assays for 88.1% (347 of 394) of RNA samples extracted from suspensions of epithelial tissue obtained from suspect FMD cases. The comparison between the 2 tests highlighted 19 FMDV isolates (13 for the 5'UTR and 6 for the 3D assay), which failed to produce a signal in 1 assay but gave a positive signal in the other. The sequence of the genomic targets of selected isolates highlighted nucleotide substitutions in the primer or probe regions, thereby providing an explanation for negative results generated in the rRT-PCR assays. These data illustrate the importance of the continuous monitoring of circulating FMDV field strains to ensure the design of the rRT-PCR assay remains fit for purpose and suggest that the use of multiple diagnostic targets could further enhance the sensitivity of molecular methods for the detection of FMDV.

Sensitivity of primary cells immortalised by oncogene transfection for the detection and isolation of foot-and-mouth disease and swine vesicular disease viruses.

Primary cells derived from calf thyroid (CTY), calf kidney (CK) and piglet kidney (PK) were immortalised by oncogene transfection and their susceptibility to infection by foot-and-mouth disease (FMD) virus and swine vesicular disease (SVD) virus examined. Eighty-five immortalised cell lines (47 CTY, 20 CK and 18 PK) proved stable upon repeated cell culture passage and many supported the growth of FMD virus and several of the PK cell lines supported SVD virus. However, none of the immortalised lines exhibited either the degree of sensitivity or the specificity for all virus serotypes and strains as shown by primary CTY and IB-RS-2 cell cultures which are routinely employed for vesicular virus diagnosis.