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This study provides the first comprehensive report on the molecular characteristics of African swine fever virus (ASFV) variants in Serbia between 2019 and 2022. Since its first observation in July 2019, the disease has been found in wild boar and domestic swine. The study involved the analysis of 95 ASFV-positive samples collected from 12 infected administrative districts in Serbia. Partial four genomic regions were genetically characterized, including B646L, E183L, B602L, and the intergenic region (IGR) between the I73R-I329L genes. The results of the study suggest that multiple ASFV strains belonging to genotype II are circulating in Serbia, as evidenced by the analysis of the IGR between I73R-I329L genes that showed the most differences. Furthermore, the phylogenetic analysis of the B602L gene showed three different clades within the CVR I group of ASFV strains. Regarding the IGR, 98.4% were grouped into IGR II, with only one positive sample grouped into the IGR III group. These findings provide essential insights into the molecular characteristics of ASFV variants in Serbia and contribute to the knowledge of circulating strains of ASFV in Europe. However, further research is necessary to gain a better understanding of ASFV spread and evolution.

African swine fever (ASF) is a contagious viral disease of pigs and wild boar that poses a major threat to the global swine industry. The genotype II African swine fever virus (ASFV) entered the European Union (EU) in 2014 and since then fourteen countries have been affected, Italy and North Macedonia being the last in 2022. While whole genome sequencing remains the gold standard for the identification of new genetic markers, sequencing of multiple loci with significant variations could be used as a rapid and cost-effective alternative to track outbreaks and study disease evolution in endemic areas.

One way to mitigate risk of feed-based pathogens for swine diets is to quarantine feed ingredients before inclusion in complete diets. Data have been generated evaluating the stability of swine viruses in ingredients, but the stability of African swine fever virus (ASFV) in feed or in a feed manufacturing environment has not been well characterized. Therefore, this study aimed to determine the stability of ASFV DNA in swine feed and on mill surfaces over time. A pilot-scale feed mill was used to manufacture six sequential batches of feed consisting of a batch of ASFV-free feed, followed by a batch inoculated with ASFV (final concentration = 5.6 × 104 TCID50/g), and then four subsequent ASFV-free batches. After each batch, 10 feed samples were aseptically collected in a double “X” pattern. During feed manufacturing, 24 steel coupons were placed on the floor of the manufacturing area and allowed to collect dust during feed manufacturing. Once feed manufacturing was completed, feed samples and steel coupons were stored at room temperature. Three of each were randomly selected from storage on 3, 7, 14, 28, 60, 90, and 180 days after feed manufacturing and analyzed for ASFV DNA. For feed samples, there was evidence of a batch × day interaction (P ¼ 0:023) for the quantification of genomic copies/g of feed, indicating that the amount of ASFV DNA present was impacted by both the batch of feed and days held at room temperature. There were no differences of genomic copies/g in early batches, but quantity of detectable ASFV decreased with increasing storage time. In Batches 4–6, the greatest quantity of ASFV DNA was detected on the day of feed manufacturing. The lowest quantity was detected on Day 7 for Batch 4, Day 60 for Batch 5, and at 28 and 180 days for Batch 6. There was no evidence of ASFV degradation on environmental discs across holding times (P ¼ 0:433). In conclusion, the quarantining of feed may help reduce but not eliminate the presence of ASFV DNA in feed over t

African swine fever (ASF) is a highly lethal disease of domestic and wild swine caused by African swine fever virus (ASFV). The disease currently circulates in Africa, Europe, Asia and on the island of Hispaniola. The ongoing epizootics in Europe and Asia have produced millions of animal deaths and severe economic losses. No effective vaccine is available for ASF, making rapid and accurate detection of ASFV essential for disease mitigation strategies. Currently available diagnostics for ASFV possess significant limitations related to assay performance, deployability, and/or turn-around time; therefore there is an unmet need for pen-side diagnostic tests with sufficient sensitivity and specificity. A chromatographic lateral flow immunoassay (LFIA) was developed for the detection of ASFV antigen in EDTA-treated whole blood using monoclonal antibodies targeting the viral p30 protein. The assay requires only water to perform and provides results in 25 min, making it well-suited for field use. The LFIA was capable of detecting genotype I and genotype II strains of ASFV in EDTA blood from experimentally infected pigs at varying time-points after infection, though it was unable to detect a genotype X ASFV strain. Diagnostic sensitivity correlated with clinical disease severity, body temperature, and viral DNA levels, and was over 90% in animals showing moderate to severe ASF-related symptoms after challenge with virulent genotype II virus. The LFIA also showed a robust diagnostic specificity of over 98%, which is essential to field testing for a high consequence to foregin animal disease. The LFIA targeting the viral p30 protein can reliably detect ASFV in whole blood from animals showing moderate to severe clinical signs of infection with virulent genotype I and II isolates, making it a promising candidate for use as a field-deployable antigen detection assay. Additional evaluation using field samples and different virus strains is required to further assess the utility o

It is critical to have methods that can detect and mitigate the risk of African swine fever virus (ASFV) in potentially contaminated feed or ingredients bound for the United States. The purpose of this work was to evaluate feed batch sequencing as a mitigation technique for ASFV contamination in a feed mill, and to determine if a feed sampling method could identify ASFV following experimental inoculation. Batches of feed were manufactured in a BSL-3Ag room at Kansas State University's Biosafety Research Institute in Manhattan, Kansas. First, the pilot feed manufacturing system mixed, conveyed, and discharged an ASFV-free diet. Next, a diet was manufactured using the same equipment, but contained feed inoculated with ASFV for final concentration of 5.6 × 104 TCID50/g. Then, four subsequent ASFV-free batches of feed were manufactured. After discharging each batch into a collection container, 10 samples were collected in a double ‘X’ pattern. Samples were analysed using a qPCR assay for ASFV p72 gene then the cycle threshold (Ct) and Log10 genomic copy number (CN)/g of feed were determined. The qPCR Ct values (p < .0001) and the Log10 genomic CN/g (p < .0001) content of feed samples were impacted based on the batch of feed. Feed samples obtained after manufacturing the ASFV-contaminated diet contained the greatest amounts of ASFV p72 DNA across all criteria (p < .05). Quantity of ASFV p72 DNA decreased sequentially as additional batches of feed were manufactured, but was still detectable after batch sequence 4. This subsampling method was able to identify ASFV genetic material in feed samples using p72 qPCR. In summary, sequencing batches of feed decreases concentration of ASFV contamination in feed, but does not eliminate it. Bulk ingredients can be accurately evaluated for ASFV contamination by collecting 10 subsamples using the sampling method described herein. Future research is needed to evaluate if different mitigation techniques can reduce ASFV feed contaminatio

African swine fever (ASF) is a highly lethal infectious disease that affects domestic pigs and wild boar. Outbreaks of ASF have grown considerably in the last decade causing important economic consequences for the swine industry. Its control is hampered by the lack of an effective treatment or vaccine. In Europe, the wild boar is a key wild reservoir for ASF. The results of the oral vaccination trial of wild boar with Lv17/WB/Rie1 are hope for this problem. However, this vaccine candidate has certain safety concerns, since it is a naturally attenuated vaccine. Therefore, the current study aims to evaluate the safety of this vaccine candidate in terms of overdose (high dose) and repeated doses (revaccination) in wild boar. Low-dose orally vaccinated animals developed only a slight transient fever after vaccination and revaccination. This was also the case for most of the high-dose vaccinated wild boar, except for one of them which succumbed after revaccination. Although this fatality was related to hierarchical fights between animals, we consider that further studies are required for clarification. Considering these new results and the current epidemiological situation of ASF in wild boar, this vaccine prototype is a promising tool for the control of the disease in these wild populations, although further studies are needed.

African swine fever (ASF) is currently the major concern of the global swine industry, as a consequence of which a reconsideration of the containment and prevention measures taken to date is urgently required. A great interest in developing an effective and safe vaccine against ASF virus (ASFV) infection has, therefore, recently appeared. The objective of the present study is to test an inactivated ASFV preparation under a vaccination strategy that has not previously been tested in order to improve its protective effect. The following have been considered: (i) virus inactivation by using a low binary ethyleneimine (BEI) concentration at a low temperature, (ii) the use of new and strong adjuvants; (iii) the use of very high doses (6 × 109 haemadsorption in 50% of infected cultures (HAD50)), and (iv) simultaneous double inoculation by two different routes of administration: intradermal and intramuscular. Five groups of pigs were, therefore, inoculated with BEI- Pol16/DP/OUT21 in different adjuvant formulations, twice with a 4-week interval. Six weeks later, all groups were intramuscularly challenged with 10 HAD50 of the virulent Pol16/DP/OUT21 ASFV isolate. All the animals had clinical signs and pathological findings consistent with ASF. This lack of effectiveness supports the claim that an inactivated virus strategy may not be a viable vaccine option with which to fight ASF.

It is critical to understand the role feed manufacturing may have regarding potential African swine fever virus (ASFV) transmission, especially given the evidence that feed and/or ingredients may be potential vectors. The objective of the study was to evaluate the distribution of ASFV in a feed mill following manufacture of contaminated feed. To accomplish this, a pilot-scale feed mill consisting of a mixer, bucket elevator, and spouting was constructed in a BSL-3Ag facility. First, a batch of ASFV-free feed was manufactured, followed by a batch of feed that had an ASFV-contaminated ingredient added to feed, which was then mixed and discharged from the equipment. Subsequently, four additional ASFV-free batches of feed were manufactured using the same equipment. Environmental swabs from 18 locations within the BSL-3Ag room were collected after each batch of feed was discharged. The locations of the swabs were categorized into four zones: 1) feed contact surface, 2) non-feed contact surface < 1 meter away from feed, 3) non-feed contact surface > 1 meter from feed, and 4) transient surfaces. Environmental swabs were analyzed using a qPCR specific for the ASFV p72 gene and reported as genomic copy number (CN)/mL of environmental swab processing buffer. Genomic copies were transformed with a log10 function for statistical analysis. There was no evidence of a zone × batch interaction for log10 genomic CN/mL (P = 0.625) or cycle threshold (Ct) value (P = 0.608). Sampling zone impacted the log10 p72 genomic CN/mL (P < 0.0001) and Ct values (P < 0.0001), with a greater amount of viral genome detected on transient surfaces compared to other surfaces (P < 0.05). This study illustrates that once ASFV enters the feed mill environment it becomes widespread and movement of people can significantly contribute to the spread of ASFV in a feed mill environment.

This study aimed to compare the infection dynamics of three genotype II African swine fever viruses (ASFV) circulating in Europe. Eighteen domestic pigs divided into three groups were infected intramuscularly or by direct contact with two haemadsorbent ASFVs (HAD) from Poland (Pol16/DP/ OUT21) and Estonia (Est16/WB/Viru8), and with the Latvian non-HAD ASFV (Lv17/WB/Rie1). Parameters, such as symptoms, pathogenicity, and distribution of the virus in tissues, humoral immune response, and dissemination of the virus by blood, oropharyngeal and rectal routes, were investigated. The Polish ASFV caused a case of rapidly developing fatal acute disease, while the Estonian ASFV caused acute to sub-acute infections and two animals survived. In contrast, animals infected with the ASFV from Latvia developed a more subtle, mild, or even subclinical disease. Oral excretion was sporadic or even absent in the attenuated group, whereas in animals that developed an acute or sub-acute form of ASF, oral excretion began at the same time the ASFV was detected in the blood, or even 3 days earlier, and persisted up to 22 days. Regardless of virulence, blood was the main route of transmission of ASFV and infectious virus was isolated from persistently infected animals for at least 19 days in the attenuated group and up to 44 days in the group of moderate virulence. Rectal excretion was limited to the acute phase of infection. In terms of diagnostics, the ASFV genome was detected in contact pigs from oropharyngeal samples earlier than in blood, independently of virulence. Together with blood, both samples could allow to detect ASFV infection for a longer period. The results presented here provide quantitative data on the spread and excretion of ASFV strains of different virulence among domestic pigs that can help to better focus surveillance activities and, thus, increase the ability to detect ASF introductions earlier.

African swine fever (ASF) causes greater sanitary, social and economic impacts on swine herds than many other swine diseases. Although ASF was first described in 1921 and it has affected more than fifty countries in Africa, Europe and South America, several key issues about its pathogenesis, immune evasion and epidemiology remain uncertain. This article reviews the main characteristics of the causative virus, its molecular epidemiology, natural hosts, clinical features, epidemiology and control worldwide. It also identifies and prioritizes gaps in ASF from a horizontal point of view encompassing fields including molecular biology, epidemiology, prevention, diagnosis and vaccine development. The purpose of this review is to promote ASF research and enhance its control.

Since its arrival in the Caucasus and Russia in 2007, African swine fever virus (ASFV) has spread widely and has now affected the EU countries of Estonia, Latvia, Lithuania, Poland and, more recently, the Czech Republic and Romania. The ever-increasing evidence of seropositive wild boar in certain areas suggests that some animals may be surviving for some time or could even be recovering from the disease. This could be due to acquired immunity after the primary infection and/or the presence of related viruses of reduced virulence. To assess these hypotheses, two ASFV field strains from Estonia were studied in vivo in two groups of domestic pigs. After an incubation period of 4 ± 1.6 days, the pigs inoculated intramuscularly with Es15/WB-Tartu 14 ASFV (group 2) developed clinical signs associated with acute disease and succumbed 7 and 11 days post infection (dpi). Pigs inoculated with Es15/WB-Valga-14 ASFV (group 1) had longer incubation times (8 days) than those in group 2 and developed variable clinical signs and lesions compatible with subacute and chronic forms of ASF; they succumbed at 11 and 25 dpi. The in-contact pigs in both groups became infected 7-14 days after exposure and exhibited variable clinical manifestations and pathological findings ranging from acute to chronic disease. Two animals per group recovered completely after infection and were protected against a subsequent homologous virus challenge-exposure performed at 78 dpi. Under experimental conditions, no transmission occurred from the survivors to susceptible sentinel pigs housed together with the survivors 137 days after the primary infection.

African swine fever (ASF) is a complex infectious disease of swine that constitutes devastating impacts on animal health and the world economy. Here, we investigated the evolutionary epidemiology of ASF virus (ASFV) in Eurasia and Africa using the concatenated gene sequences of the viral protein 72 and the central variable region of isolates collected between 1960 and 2015. We used Bayesian phylodynamic models to reconstruct the evolutionary history of the virus, to identify virus population demographics and to quantify dispersal patterns between host species. Results suggest that ASFV exhibited a significantly high evolutionary rate and population growth through time since its divergence in the 18th century from East Africa, with no signs of decline till recent years. This increase corresponds to the growing pig trade activities between continents during the 19th century, and may be attributed to an evolutionary drift that resulted from either continuous circulation or maintenance of the virus within Africa and Eurasia. Furthermore, results implicate wild suids as the ancestral host species (root state posterior probability = 0.87) for ASFV in the early 1700s in Africa. Moreover, results indicate the transmission cycle between wild suids and pigs is an important cycle for ASFV spread and maintenance in pig populations, while ticks are an important natural reservoir that can facilitate ASFV spread and maintenance in wild swine populations. We illustrated the prospects of phylodynamic methods in improving risk-based surveillance, support of effective animal health policies, and epidemic preparedness in countries at high risk of ASFV incursion.

In 2014, African swine fever virus (ASFV) was introduced into the Baltic states and Poland. Since then, the disease has continued to spread within these regions, and recently, cases were reported in the Czech Republic and Romania. Currently, there is an increasing risk of ASFV introduction into Western Europe. Hence, there is an urgent need to assess current contingency plans. For this purpose, knowledge of modes-of-transmission and clinical outcome in pigs infected with new European ASFV strains is needed. In the present study, two experiments were conducted in pigs using an isolate of ASFV from Poland (designated here POL/2015/Podlaskie/Lindholm). In both studies, pigs were inoculated intranasally with the virus and contact pigs were exposed to the experimentally infected pigs, either directly (contact within and between pens) or by air. Pigs exposed to the virus by intranasal inoculation, by direct contact to infected animals and by aerosol developed acute disease characterized by viremia, fever and depression. Infectious virus was first detected in blood obtained from the inoculated pigs and then sequentially among the within-pen, between-pen and air-contact pigs. ASFV DNA and occasionally infectious virus was found in nasal-, oral-, and rectal swabs obtained from the pigs, and ASFV DNA was detected in air samples. No anti-ASFV antibodies were detected in sera. In conclusion, the study shows that the currently circulating strain of ASFV can be efficiently transmitted via direct contact and by aerosols. Also, the results provide quantitative transmission parameters and knowledge of infection stages in pigs infected with this ASFV.

African swine fever (ASF) is an important emerging transboundary animal disease (TAD), which currently has an impact on many countries in Africa, Eastern Europe, the Caucasus and the Russian Federation. The current situation in Europe shows the ability of the virus to rapidly spread, which stands to threaten the global swine industry. At present, there is no viable vaccine to minimize spread of the disease and stamping out is the main source of control. In February 2011, Ethiopia had reported its first suspected outbreaks of ASF. Genomic analyses of the collected ASF virus (ASFV) strains were undertaken using 23 tissue samples collected from domestic swine in Ethiopia from 2011 to 2014. The analysis of Ethiopian ASFVs partial p72 gene sequence showed the identification of a new genotype, genotype XXIII, that shares a common ancestor with genotypes IX and X, which comprise isolates circulating in Eastern African countries and the Republic of Congo. Analysis of the p54 gene also followed the p72 pattern and the deduced amino acid sequence of the central variable region (CVR) of the B602L gene showed novel tetramer repeats not previously characterized.

African swine fever (ASF) virus was introduced in Latvia in June 2014. Thirty-two outbreaks in domestic pigs and 217 cases in wild boar were notified in 2014. Twenty-eight outbreaks (87.5%) were primary outbreaks. The contagiosity within pig herds was low. Failure to use simple biosecurity measures to reduce the chance of virus introduction, for example by inadvertent feeding of locally produced virus contaminated fodder were the main causes for the outbreaks in backyard holdings. The infection in wild boar survived locally in two different areas with a low prevalence and a slow spread. The persistence of the infection in wild boar within an area was most probably linked to wild boar scavenging the carcasses of infected wild boar. © 2016 Elsevier Ltd

Classical swine fever (CSF) and African swine fever (ASF) are both highly contagious diseases of domestic pigs and wild boar and are clinically indistinguishable. For both diseases, antibody detection is an integral and crucial part of prevention and control measures. The purpose of our study was to develop and initially validate a duplex pen-side test for simultaneous detection and differentiation of specific antibodies against CSF virus (CSFV) and ASF virus (ASFV). The test was based on the major capsid protein VP72 of ASFV and the structural protein E2 of CSFV, both considered the most immunogenic proteins of these viruses. The performance of the pen-side test was evaluated using a panel of porcine samples consisting of experimental, reference, and field sera, with the latter collected from European farms free of both diseases. The new lateral flow assay was able to detect specific antibodies to ASFV or CSFV, showing good levels of sensitivity and specificity. These preliminary data indicate the potential of the newly developed pen-side test for rapid differential detection of antibodies found in the 2 diseases, which is of particular importance in the field and in front-line laboratories where equipment and skilled personnel are limited and control of ASF and CSF is crucial. © 2016, © 2016 The Author(s).

Background African swine fever (ASF) is a viral infectious disease of domestic and wild suids of all breeds and ages, causing a wide range of hemorrhagic syndromes and frequently characterized by high mortality. The disease is endemic in Sub-Saharan Africa and Sardinia. Since 2007, it has also been present in different countries of Eastern Europe, where control measures have not been effective so far. The continued spread poses a serious threat to the swine industry worldwide. In the absence of vaccine, early detection of infected animals is of paramount importance for control of the outbreak, to prevent the transmission of the virus to healthy animals and subsequent spreading of the disease. Current laboratory diagnosis is mainly based on virological methods (antigen and genome detection) and serodiagnosis. Results In the present work, a Lateral Flow Assay (LFA) for antigen detection has been developed and evaluated. The test is based on the use of a MAb against VP72 protein of ASFV, the major viral capsid protein and highly immunogenic. First experiments using VP72 viral and recombinant protein or inactivated culture virus showed promising results with a sensitivity similar to that of a commercially available Antigen-ELISA. Moreover, these strips were tested with blood from experimentally infected pigs and field animals and the results compared with those of PCR and Antigen-ELISA. For the experimentally infected samples, there was an excellent correlation between the LFA and the ELISA, while the PCR always showed to be more sensitive (38 % positive samples by PCR versus 27 % by LFA). The LFA was demonstrated to be positive for animals with circulating virus levels exceeding 104 HAU. With the field samples, once again, the PCR detected more positives than either the Antigen-ELISA or LFA, although here the number of positive samples scored by the LFA exceeded the values obtained with the Antigen-ELISA, showing 60 % positivity vs 48 % for the ELISA. For the two group

The performance of Whatman 3-MM filter papers for the collection, drying, shipment and long-term storage of blood at ambient temperature, and for the detection of African swine fever virus and antibodies was assessed. Conventional and real-time PCR, viral isolation and antibody detection by ELISA were performed on paired samples (blood/tissue versus dried-blood 3-MM filter papers) collected from experimentally infected pigs and from farm pigs in Madagascar and Côte d'Ivoire. 3-MM filter papers were used directly in the conventional and real-time PCR without previous extraction of nucleic acids. Tests that performed better with 3-MM filter papers were in descending order virus isolation, real-time UPL PCR and conventional PCR. The analytical sensitivity of real-time UPL PCR on filter papers was similar to conventional testing (virus isolation or conventional PCR) on organs or blood. In addition, blood-dried filter papers were tested in ELISA for antibody detection and the observed sensitivity was very close to conventional detection on serum samples and gave comparable results. Filter papers were stored up to 9 months at 20-25°C and for 2 months at 37°C without significant loss of sensitivity for virus genome detection. All tests on 3-MM filter papers had 100% specificity compared to the gold standards. Whatman 3-MM filter papers have the advantage of being cheap and of preserving virus viability for future virus isolation and characterization. In this study, Whatman 3-MM filter papers proved to be a suitable support for the collection, storage and use of blood in remote areas of tropical countries without the need for a cold chain and thus provide new possibilities for antibody testing and virus isolation. © 2014 Blackwell Verlag GmbH.

In the absence of effective vaccine(s), control of African swine fever caused by African swine fever virus (ASFV) must be based on early, efficient, cost-effective detection and strict control and elimination strategies. For this purpose, we developed an indirect ELISA capable of detecting ASFV antibodies in either serum or oral fluid specimens. The recombinant protein used in the ELISA was selected by comparing the early serum antibody response of ASFV-infected pigs (NHV-p68 isolate) to three major recombinant polypeptides (p30, p54, p72) using a multiplex fluorescent microbead-based immunoassay (FMIA). Nonhazardous (non-infectious) antibody-positive serum for use as plate positive controls and for the calculation of sample-to-positive (SP) ratios was produced by inoculating pigs with a replicon particle (RP) vaccine expressing the ASFV p30 gene. The optimized ELISA detected anti-p30 antibodies in serum and/or oral fluid samples from pigs inoculated with ASFV under experimental conditions beginning 8 to 12 days post inoculation. Tests on serum (n = 200) and oral fluid (n = 200) field samples from an ASFV-free population demonstrated that the assay was highly diagnostically specific. The convenience and diagnostic utility of oral fluid sampling combined with the flexibility to test either serum or oral fluid on the same platform suggests that this assay will be highly useful under the conditions for which OIE recommends ASFV antibody surveillance, i.e.;in ASFV-endemic areas and for the detection of infections with ASFV isolates of low virulence. © 2016 Giménez-Lirola et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Rift Valley fever (RVF) is a mosquito-borne viral zoonosis which affects humans and a wide range of domestic and wild ruminants. The large spread of RVF in Africa and its potential to emerge beyond its geographic range requires the development of surveillance strategies to promptly detect the disease outbreaks in order to implement efficient control measures, which could prevent the widespread of the virus to humans. The Animal Health Mediterranean Network (REMESA) linking some Northern African countries as Algeria, Egypt, Libya, Mauritania, Morocco, Tunisia with Southern European ones as France, Italy, Portugal and Spain aims at improving the animal health in the Western Mediterranean Region since 2009. In this context, a first assessment of the diagnostic capacities of the laboratories involved in the RVF surveillance was performed. The first proficiency testing (external quality assessment- EQA) for the detection of the viral genome and antibodies of RVF virus (RVFV) was carried out from October 2013 to February 2014. Ten laboratories participated from 6 different countries (4 from North Africa and 2 from Europe). Six laboratories participated in the ring trial for both viral RNA and antibodies detection methods, while four laboratories participated exclusively in the antibodies detection ring trial. For the EQA targeting the viral RNA detection methods 5 out of 6 laboratories reported 100% of correct results. One laboratory misidentified 2 positive samples as negative and 3 positive samples as doubtful indicating a need for corrective actions. For the EQA targeting IgG and IgM antibodies methods 9 out of the 10 laboratories reported 100% of correct results, whilst one laboratory reported all correct results except one false-positive. These two ring trials provide evidence that most of the participating laboratories are capable to detect RVF antibodies and viral RNA thus recognizing RVF infection in affected ruminants with the diagnostic methods currently availab

Twelve complete African swine fever virus (ASFV) genome sequences are currently publicly available and these include only one sequence from East Africa. We describe genome sequencing and annotation of a recent pig-derived p72 genotype IX, and a tick-derived genotype X isolate from Kenya using the Illumina platform and comparison with the Kenya 1950 isolate. The three genomes constitute a cluster that was phylogenetically distinct from other ASFV genomes, but 98–99 % conserved within the group. Vector-based compositional analysis of the complete genomes produced a similar topology. Of the 125 previously identified ‘core’ ASFV genes, two ORFs of unassigned function were absent from the genotype IX sequence which was 184 kb in size as compared to 191 kb for the genotype X. There were multiple differences among East African genomes in the 360 and 110 multicopy gene families. The gene corresponding to 360-19R has transposed to the 5′ variable region in both genotype X isolates. Additionally, there is a 110 ORF in the tick-derived genotype X isolate formed by fusion of 13L and 14L that is unique among ASFV genomes. In future, functional analysis based on the variations in the multicopy families may reveal whether they contribute to the observed differences in virulence between genotpye IX and X viruses. © 2015, Springer Science+Business Media New York.

African swine fever virus (ASFV) was first reported in eastern Europe/Eurasia in 2007. Continued spread of ASFV has placed central European countries at risk, and in 2014, ASFV was detected in Lithuania and Poland. Sequencing showed the isolates are identical to a 2013 ASFV from Belarus but differ from ASFV isolated in Georgia in 2007.

We describe a horizontal survey of African swine fever virus (ASFV) prevalence and risk factors associated with virus infection in domestic pigs in two contrasting production systems in Kenya. A free range/tethering, low input production system in Ndhiwa District of South-western Kenya is compared with a medium input stall fed production system in Kiambu District of Central Kenya. Analysis of variance (ANOVA) of data derived from cluster analysis showed that number of animals, number of breeding sows and number of weaner pigs were a significant factor in classifying farms in Nhiwa and Kiambu. Analysis of blood and serum samples using a PCR assay demonstrated an average animal level positivity to ASFV of 28% in two independent samplings in South-western Kenya and 0% PCR positivity in Central Kenya. No animals were sero-positive in either study site using the OIE indirect-ELISA and none of the animals sampled exhibited clinical symptoms of ASF. The farms that contained ASFV positive pigs in Ndhiwa District were located in divisions bordering the Ruma National Park from which bushpig (Potamochoerus larvatus) incursions into farms had been reported. ASFV prevalence (P<0.05) was significantly higher at distances between 6 and 16. km from the National Park than at distances closer or further away. One of the 8 bushpigs sampled from the park, from which tissues were obtained was PCR positive for ASFV. The data therefore indicated a potential role for the bushpig in virus transmission in South-western Kenya, but there was no evidence of a direct sylvatic virus transmission cycle in Central Kenya. ASF control strategies implemented in these areas will need to take these epidemiological findings into consideration. © 2012 Elsevier B.V.

The presence of antibodies against African swine fever (ASF), a complex fatal notifiable OIE disease of swine, is always indicative of previous infection, since there is no vaccine that is currently used in the field. The early appearance and subsequent long-term persistence of antibodies combined with cost-effectiveness make antibody detection techniques essential in control programmes. Recent reports appear to indicate that the serological tests recommended by the OIE for ASF monitoring are much less effective in East and Southern Africa where viral genetic and antigenic diversity is the greatest. We report herein an extensive analysis including more than 1000 field and experimental infection sera, in which the OIE recommended tests are compared with antigen-specific ELISAs and immuno-peroxidase staining of cells (IPT). The antibody detection results generated using new antigen-specific tests, developed in this study, which are based on production of antigen fractions generated by infection and virus purification from COS-1 cells, showed strong concordance with the OIE tests. We therefore conclude that the lack of success is not attributable to antigenic polymorphism and may be related to the specific characteristics of the local breeds African pigs. © 2012 Elsevier B.V.

African swine fever (ASF) is a complex, highly lethal, notifiable disease of swine. ASF is wide-spread in sub-Saharan Africa and East European countries and there is presently a great risk of spread to neighboring countries. Since there is no vaccine for ASF virus (ASFV), control is based on rapid and early detection of the disease via surveillance. This approach requires collecting blood samples from large number of animals. Laborious and expensive of itself, this process also presents an additional risk because ASFV is present at high concentrations in the blood. The objective of this study was to initiate studies into the potential use of oral fluid as an alternative to serum for ASF diagnosis, for latter studying its possible use in surveillance and control programs. To this end, oral fluid samples collected at different times post infection from eight pigs experimentally inoculated with an attenuated ASFV were assayed using modified protocols of the two validated serological techniques, the enzyme-immune-liked assay (ELISA) and immunoperoxidase technique (IPT). Antibodies against ASFV were detected in oral fluid samples of all animals from early post infection through the end of the experiment by ELISA and IPT. These results confirmed the presence of ASFV antibodies in swine oral fluids samples, the possibility of an oral fluid-based approach in ASF diagnosis and, potentially in ASF surveillance. © 2013 Elsevier B.V.

Summary African swine fever virus (ASFV) causes one of the most dreaded transboundary animal diseases (TADs) in Suidae. African swine fever (ASF) often causes high rates of morbidity and mortality, which can reach 100% in domestic swine. To date, serological diagnosis has the drawback of not being able to differentiate variants of this virus. Previous studies have identified the 22 genotypes based on sequence variation in the C-terminal region of the p72 gene, which has become the standard for categorizing ASFVs. This article describes a genotyping assay developed using a segment of PCR-amplified genomic DNA of approximately 450bp, which encompasses the C-terminal end of the p72 gene. Complementary paired DNA probes of 15 or 17bp in length, which are identical except for a single nucleotide polymorphism (SNP) in the central position, were designed to either individually or in combination differentiate between the 22 genotypes. The assay was developed using xMAP technology; probes were covalently linked to microspheres, hybridized to PCR product, labelled with a reporter and read in the Luminex 200 analyzer. Characterization of the sample was performed by comparing fluorescence of the paired SNP probes, that is, the probe with higher fluorescence in a complementary pair identified the SNP that a particular sample possessed. In the final assay, a total of 52 probes were employed, 24 SNP pairs and 4 for general detection. One or more samples from each of the 22 genotypes were tested. The assay was able to detect and distinguish all 22 genotypes. This novel assay provides a powerful novel tool for the simultaneous rapid diagnosis and genotypic differentiation of ASF. © 2012 Blackwell Verlag GmbH.

African swine fever is one of the most important viral diseases of pigs and which caused significant economic damage on the pig production worldwide. Nowadays, it is still present on the African continent, in Transcaucasus countries (TCC), on Island of Sardinia and in Russia. Outbreaks of the disease have been reported in Russia for the last four years, affected especially the Southern Federal District of the country. Since 2010, a new outbreak area has been observed in the Northwestern Federal District.In order to study the evolution of African swine fever virus (ASFV) isolates, strains were collected in the Russian Federation from 2007 to 2011 and investigated by means of partial sequencing and fragment length polymorphism. In detail, 7 variable regions, namely B646L, E183L, I196L, B602L, I73R/I329R, I78R/I215L and KP86R were investigated. Phylogenetic analyses revealed 100% nucleotide identity of B646L and E183L gene sequences of all examined isolates. All isolates formed one genetic cluster within genotype II. Moreover, no amplified fragment length polymorphism (AFLP) was observed for B602L, I196L, I73R/I329R, and I78R/I215L genes. The flanking primers used to amplify the KP86R gene failed to amplify a product in all the isolates. The obtained data strongly suggests that only one ASFV virus variant caused the outbreaks from 2007 to 2011 in the territory of the Russian Federation. © 2012 Elsevier B.V.

African swine fever (ASF) is an economically devastating disease for the pig industry, especially in Africa. Identifying what supports infection on pig farms in this region remains the key component in developing a risk-based approach to understanding the epidemiology of ASF and controlling the disease. Nigeria was used for this matched case-control study, because there is perpetual infection in some areas, while contiguous areas are intermittently infected. Risk factors and biosecurity practices in pig farms were evaluated in association with ASF infection. Subsets of farms located in high-density pig population areas and high-risk areas for ASF infection were randomly selected for analysis. Most plausible risk factor variables from the univariable analysis included in the multivariable analysis include owner of farm had regular contact with infected farms and other farmers, untested pigs were routinely purchased into the farm in the course of outbreaks, there was an infected neighbourhood, other livestock were kept alongside pigs, there was a presence of an abattoir/slaughter slab in pig communities, wild birds had free access to pig pens, tools and implements were routinely shared by pig farmers, there was free access to feed stores by rats, and feed was purchased from a commercial source. Only the presence of an abattoir in a pig farming community (OR=8.20; CI95%=2.73, 24.63; P<0.001) and the presence of an infected pig farm in the neighbourhood (OR=3.26; CI95%=1.20, 8.83; P=0.02) were significant. There was a marginally significant negative association (protective) between risk of ASF infection and sharing farm tools and equipment (OR=0.35; CI95%=0.12, 1.01; P=0.05).Of the 28 biosecurity measures evaluated, food and water control (OR=0.14; CI95%=0.04, 0.46; P<0.001), separation/isolation of sick pigs (OR=0.14; CI95%=0.04, 0.53; P=0.004) and washing and disinfection of farm equipment and tools (OR=0.27; CI95%=0.10, 0.78; P=0.02) were negatively associated

Three discrete regions of the African swine fever virus (ASFV) were analysed in the genomes of a wide range of isolates collected from wild and domestic pigs in Sardinia, over a 31-year period (1978-2009). The analysis was conducted by genotyping based on sequence data from three single copy ASF genes. The E183L gene encoding the structural protein p54 and part of the gene encoding the p72 protein were used to delineate genotypes, before intra-genotypic resolution of viral relationships by analysis of tetramer amino acid repeats within the hypervariable central variable region (CVR) of the B602L gene. The data revealed that these isolates did not show significant variation in their p72 and p54 sequence when compared between different isolates showing a remarkable genetic stability of these genome regions. In particular, the phylogeny revealed that all the Sardinian isolates belong to the same largest and most homogeneous p72 genotype I together with viruses from Europe, South America, the Caribbean and West Africa, and p54 genotype Ia which comprises viruses from Europe and America. The analysis of B602L gene revealed a minor difference in the number of tetramer repeats, placing the Sardinian isolates into two clusters, accordingly to their temporal distribution, namely sub-group III and sub-group X, this latter showing a deletion of 12 tetramer repeats located in the centre of the array. The genetic variation of this fragment suggests that one sub-group could be derived from the other supporting the hypothesis of a single introduction of ASFV in Sardinia. © 2011 Springer Science+Business Media, LLC.

A real-time polymerase chain reaction (PCR) assay for the rapid detection of African swine fever virus (ASFV), multiplexed for simultaneous detection of swine beta-actin as an endogenous control, has been developed and validated by four National Reference Laboratories of the European Union for African swine fever (ASF) including the European Union Reference Laboratory. Primers and a TaqMan ® probe specific for ASFV were selected from conserved regions of the p72 gene. The limit of detection of the new real-time PCR assay is 5.7-57 copies of the ASFV genome. High accuracy, reproducibility and robustness of the PCR assay (CV ranging from 0.7 to 5.4%) were demonstrated both within and between laboratories using different real-time PCR equipments. The specificity of virus detection was validated using a panel of 44 isolates collected over many years in various geographical locations in Europe, Africa and America, including recent isolates from the Caucasus region, Sardinia, East and West Africa. Compared to the OIE-prescribed conventional and real-time PCR assays, the sensitivity of the new assay with internal control was improved, as demonstrated by testing 281 field samples collected in recent outbreaks and surveillance areas in Europe and Africa (170 samples) together with samples obtained through experimental infections (111 samples). This is particularly evident in the early days following experimental infection and during the course of the disease in pigs sub-clinically infected with strains of low virulence (from 35 up to 70dpi). The specificity of the assay was also confirmed on 150 samples from uninfected pigs and wild boar from ASF-free areas. Measured on the total of 431 tested samples, the positive deviation of the new assay reaches 21% or 26% compared to PCR and real-time PCR methods recommended by OIE. This improved and rigorously validated real-time PCR assay with internal control will provide a rapid, sensitive and reliable molecular tool for ASFV detect

Samples from infected domestic pigs associated with an outbreak of African swine fever (ASF) in three districts of central Uganda in 2007 were confirmed as being infected with African swine fever virus (ASFV) using a P72 gene-based polymerase chain reaction amplification (PCR) assay combined with restriction analysis. None of the sera collected from pigs with clinical symptoms were positive using the OIE serological prescribed tests. However, seven haemadsorbing viruses were isolated in macrophage culture and genotyped by partial p72 and full length p54-gene sequencing. Four of these viruses were isolated directly from serum samples. All the viruses were classified within the domesticpig cycle-associated p72 and p54 genotype IX which also includes viruses responsible for ASF outbreaks in Kenya in 2006 and 2007 and Uganda in 2003. To define virus relationships at higher resolution, typing was performed by analysis of tetrameric amino acid repeat regions within the central variable region (CVR) of the B602L gene. Ugandan isolates sequences exhibited 100% identity to viruses isolated from outbreaks in Kenya in 2007. The identity was greater than the viruses obtained from an earlier outbreak in Kenya in 2006. This provides further evidence that genetically similar ASFV virus within p72 Genotype IX may be circulating between Kenya and Uganda. © 2011 Academic Journals.

African swine fever virus p72 genotype IX, associated with outbreaks in eastern Africa, is cocirculating in the Republic of the Congo with West African genotype I. Data suggest that viruses from eastern Africa are moving into western Africa, increasing the threat of outbreaks caused by novel viruses in this region.

One of the main criticisms to DNA vaccines is the poor immunogenicity that they confer on occasions, at least in large animals. Confirming this theory, immunization with plasmid DNA encoding two African swine fever virus genes in frame (pCMV-PQ), failed in inducing detectable immune responses in pigs, while it was successful in mice. Aiming to improve the immune responses induced in swine, a new plasmid was constructed, encoding the viral genes fused in frame with a single chain variable fragment of an antibody specific for a swine leukocyte antigen II (pCMV-APCH1PQ). Our results clearly demonstrate that targeting antigens to antigen professional cells exponentially enhanced the immune response induced in pigs, albeit that the DNA vaccine was not able to confer protection against lethal viral challenge. Indeed, a viremia exacerbation was observed in each of the pigs that received the pCMV-APCH1PQ plasmid, this correlating with the presence of non-neutralizing antibodies and antigen-specific SLA II-restricted T-cells. The implications of our discoveries for the development of future vaccines against African swine fever virus and other swine pathogens are discussed. © 2011 Elsevier Ltd.

The role of the ancestral sylvatic cycle of the African swine fever virus (ASFV) is not well understood in the endemic areas of eastern Africa. We therefore analysed the ASF infection status on samples collected from 51 free-ranging warthogs (Phacocherus africanus) and 1576 Ornithodorus porcinus ticks from 26 independent warthog burrows at a single ranch in Kenya. Abattoir samples from 83 domestic pigs without clinical symptoms, originating from specific locations with no recent reported ASF outbreaks were included in this study. All samples were derived from areas of central Kenya, where ASF outbreaks have been reported in the past. Infection with ASFV was confirmed in 22% of O. porcinus pools, 3.22% of adult warthog serum samples and 49% of domestic pig serum samples by using p72-based PCR. All of the warthog sera were positive for anti-ASFV antibodies, investigated by using ELISA, but none of the domestic pig sera were positive. Twenty O. porcinus-, 12 domestic pig- and three warthogderived viruses were genotyped at four polymorphic loci. The ASFV isolates from ticks and domestic pigs clustered within p72 genotype X. By contrast, ASF viruses genotyped directly from warthog sera, at same locality as the tick isolates, were within p72 genotype IX and genetically similar to viruses causing recent ASF outbreaks in Kenya and Uganda. This represents the first report of the co-existence of different ASFV genotypes in warthog burrow-associated ticks and adult wild warthogs. The data from this and earlier studies suggest transfer of viruses of at least two different p72 genotypes, from wild to domestic pigs in East Africa. © 2011 SGM.

In this study, a new in situ hybridisation (ISH) protocol has been developed to identify African swine fever virus (ASFV) genome in formalin-fixed, paraffin-embedded tissues. Different digoxigenin labelled ASFV-probes were tested, including single ASFV-specific oligonucleotides, an 18.5. kb restriction fragment from the viral genome and the entire ASFV genome. The latter showed the highest sensitivity in all tissues tested, independently of the virus used for challenge E75L or Ba71L. Although a similar ASFV genome distribution was observed, the number of ISH-positive cells was higher for Ba71L compared to E75L infected tissues. As expected, the monocyte-macrophage cell lineage was the main target cell for ASFV infection. Corresponding with the last stages of infection, ISH-positive signals were also found in other cell types, including endothelial cells, hepatocytes and neutrophils. Furthermore, two unexpected findings were also noticed the detection of a specific ISH-signal in lymphocytes and a tendency to find the signal in the nucleus of infected cells. In summary, the present findings demonstrate the utility of this new ISH protocol to study ASFV pathogenesis and its potential use as a diagnostic tool. © 2010 Elsevier B.V.

African swine fever (ASF) has had significant economic and social impact in Nigeria since 1997. However, there has been no effective national response to bring it under control. In this report, we confirm that ASF is still prevalent and widespread in Nigeria. Results from both serosurveillance and virological analyses indicated that ASF is present in most of the agro-ecological zones of the country. Nine per cent (9%) of serum samples and 48% of tissue samples were positive for ASF virus antibody and genome, respectively. Areas with high pig-related activities (marketing, consumption and farming) have higher prevalences compared with areas with less pig activities. Farm-gate buyers, marketing systems and transport of untested pigs within the country assist with the circulation of the virus. Only by putting in place a comprehensive routine surveillance and testing system, reorganizing the market and transportation systems for pigs, implementing on-farm bio-security protocols and considering the option of compensation will it be possible to achieve a significant reduction in ASF prevalence in Nigeria. ©2010 Blackwell Verlag GmbH.

Viral molecular epidemiology has traditionally analyzed variation in single genes. Whole genome phylogenetic analysis of 123 concatenated genes from 11 ASFV genomes, including E75, a newly sequenced virulent isolate from Spain, identified two clusters. One contained South African isolates from ticks and warthog, suggesting derivation from a sylvatic transmission cycle. The second contained isolates from West Africa and the Iberian Peninsula. Two isolates, from Kenya and Malawi, were outliers. Of the nine genomes within the clusters, seven were within p72 genotype 1. The 11 genomes sequenced comprised only 5 of the 22 p72 genotypes. Comparison of synonymous and non-synonymous mutations at the genome level identified 20 genes subject to selection pressure for diversification. A novel gene of the E75 virus evolved by the fusion of two genes within the 360 multicopy family. Comparative genomics reveals high diversity within a limited sample of the ASFV viral gene pool. © 2010 Elsevier Inc. All rights reserved.

The design of a 5' conjugated minor groove binder (MGB) probe real-time PCR assay is described for the rapid, sensitive and specific detection of African swine fever virus (ASFV) DNA. The assay is designed against the 9GL region and is capable of detecting 20 copies of a DNA standard. It does not detect any of the other common swine DNA viruses tested in this study. The assay can detect ASFV DNA in a range of clinical samples. Sensitivity was equivalent to the Office International des Epizooties (OIE) recommended TaqMan assay. In addition the assay was found to have a detection limit 10-fold more sensitive than the conventional PCR recommended by the OIE. Linear range was ten logs from 2×101 to 2×1010. The assay is rapid with an amplification time just over 2h. The development of this assay provides a useful tool for the specific diagnosis of ASF in statutory or emergency testing programs or for the detection of ASFV DNA in research applications. © 2010 Elsevier B.V.

Complete sequencing of p54-gene from 67 European, American, and West and East African Swine Fever virus (ASFV) isolates revealed that West African and European ASFV isolates classified within the predominant Genotype I according to partial sequencing of p72 were discriminated into four major sub-types on the basis of their p54 sequences. This highlighted the value of p54 gene sequencing as an additional, intermediate-resolution, molecular epidemiological tool for typing of ASFV viruses. We further evaluated p54-based genotyping, in combination with partial sequences of two other genes, for determining the genetic relationships and origin of viruses responsible for disease outbreaks in Kenya. Animals from Western and central Kenya were confirmed as being infected with ASFV using a p72 gene-based PCR assay, following outbreaks of severe hemorrhagic disease in domestic pigs in 2006 and 2007. Eleven hemadsorbing viruses were isolated in macrophage culture and genotyped using a combination of full-length p54-gene sequencing, partial p72-gene sequencing, and analysis of tetrameric amino acid repeat regions within the variable region of the B602L gene (CVR). The data revealed that these isolates were identical in their p72 and p54 sequence to viruses responsible for ASF outbreaks in Uganda in 2003. There was a minor difference in the number of tetrameric repeats within the B602L sequence of the Kenyan isolates that caused the second Kenyan outbreak in 2007. A practical implication of the genetic similarity of the Kenyan and Ugandan viral isolates is that ASF control requires a regional approach. © 2008 Springer Science+Business Media, LLC.

African swine fever (ASF) is an infectious and economically important disease of domestic pigs. The absence of vaccine renders the diagnostic test the only tool that can be used for the control of new outbreaks of the disease. At present, the enzyme-linked immunosorbent assay (ELISA) test is the most useful method for large-scale ASF serological studies, although false positives have been detected, mainly on poorly preserved sera. In order to improve the current diagnostic test available for ASF, we have studied the antigenic properties of the ASF virus polyprotein pp62 and its suitability for use in a novel ELISA. Two well-known antigenic proteins of ASF virus, p32 and p54, were also included in this study. These proteins were expressed in the baculovirus expression system and used as antigens in ASF serological tests. Our results indicate that the use of these recombinant proteins as antigens in the ELISAs improves the sensitivity and specificity obtained with the conventional diagnosis test used to detect antibodies against ASF virus. Furthermore, the use of polyprotein pp62 in an ELISA for testing poorly preserved sera allows performance of the diagnosis of ASF without the need to confirm the results by the immunoblot test. These features make pp62 one of the most interesting viral proteins to be used for serological ASF diagnosis. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

We describe the validation of an enzyme-linked immunosorbent assay (ELISA) and confirmatory immunoblotting assays based on a recombinant p30 protein (p30r) produced in insect larvae using a baculovirus vector. Such validation included the following (i) the scaling up and standardization of p30r production and the associated immunoassays, (ii) a broad immunological analysis using a large number of samples (a total of 672) from Spain and different African locations, and (iii) the detection of the ASF virus (ASFV)-antibody responses at different times after experimental infection. Yields of p30r reached up to 15% of the total protein recovered from the infected larvae at 3 days postinfection. Serological analysis of samples collected in Spain revealed that the p30r-based ELISA presented similar sensitivity to and higher specificity than the conventional Office International des Epizooties-approved ASFV ELISA. Moreover, the p30r ELISA was more sensitive than the conventional ELISA test in detecting ASFV-specific antibodies in experimentally infected animals at early times postinfection. Both the recombinant and conventional ELISAs presented variable rates of sensitivity and specificity with African samples, apparently related to their geographical origin. Comparative analyses performed on the sequences, predicted structures, and antigenicities of p30 proteins from different Spanish and African isolates suggested that variability among isolates might correlate with changes in antigenicity, thus affecting detection by the p30r ELISA. Our estimations indicate that more than 40,000 ELISA determinations and 2,000 confirmatory immunoblotting tests can be performed with the p30r protein obtained from a single infected larva, making this a feasible and inexpensive strategy for production of serological tests with application in developing countries. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Variable regions of the African swine fever virus genome, which contain arrays of tandem repeats, were compared in the genomes of isolates obtained over a 40-year period. Comparison of the size of products generated by polymerase chain reaction (PCR) from four different genome regions, within the B602L and KP86R genes and intergenic regions J286L and BtSj, placed 43 closely related isolated from Europe, the Caribbean, West and Central Africa into 17 different virus sub-groups. Sequence analysis of the most variable fragment, within the B602L gene, from 81 different isolates distinguished 31 sub-groups of virus isolates which varied in sequence and number of a tandem repeat encoding 4 amino acids. Thus, each of these analysis methods enabled isolates, which were previously grouped together by sequencing of a more conserved genome region, to be separated into multiple sub-groups. This provided additional information about strains of viruses circulating in different countries. The methods could be used in future to study the epidemiology and evolution of virus isolates and to trace the sources of disease outbreaks. © 2006 Springer-Verlag.