Characterization of the Protective Cellular Immune Response in Pigs Immunized Intradermally with the Live Attenuated African Swine Fever Virus (ASFV) Lv17/WB/Rie1.

Candidate vaccines against African swine fever virus (ASFV) based on naturally attenuated or genetically modified viruses have the potential to generate protective immune responses, although there is no consensus on what defines a protective immune response against ASFV. Studies, especially in sensitive host species and focused on unravelling protective mechanisms, will contribute to the development of safer and more effective vaccines. The present study provides a detailed analysis of phenotypic and functional data on cellular responses induced by intradermal immunization and subsequent boosting of domestic pigs with the naturally attenuated field strain Lv17/WB/Rie1, as well as the mechanisms underlying protection against intramuscular challenge with the virulent genotype II Armenia/07 strain. The transient increase in IL-8 and IL-10 in serum observed after immunization might be correlated with survival. Protection was also associated with a robust ASFV-specific polyfunctional memory T-cell response, where CD4CD8 and CD8 T cells were identified as the main cellular sources of virus-specific IFNγ and TNFα. In parallel with the cytokine response, these T-cell subsets also showed specific cytotoxic activity as evidenced by the increased expression of the CD107a degranulation marker. Along with virus-specific multifunctional CD4CD8 and CD8 T-cell responses, the increased levels of antigen experienced in cytotoxic CD4 T cells observed after the challenge in immunized pigs might also contribute to controlling virulent infection by killing mechanisms targeting infected antigen-presenting cells. Future studies should elucidate whether the memory T-cell responses evidenced in the present study persist and provide long-term protection against further ASFV infections.

Clinical, Virological and Immunological Responses after Experimental Infection with African Horse Sickness Virus Serotype 9 in Immunologically Naïve and Vaccinated Horses.

This study described the clinical, virological, and serological responses of immunologically naïve and vaccinated horses to African horse sickness virus (AHSV) serotype 9. Naïve horses developed a clinical picture resembling the cardiac form of African horse sickness. This was characterized by inappetence, reduced activity, and hyperthermia leading to lethargy and immobility-recumbency by days 9-10 post-infection, an end-point criteria for euthanasia. After challenge, unvaccinated horses were viremic from days 3 or 4 post-infection till euthanasia, as detected by serogroup-specific (GS) real time RT-PCR (rRT-PCR) and virus isolation. Virus isolation, antigen ELISA, and GS-rRT-PCR also demonstrated high sensitivity in the post-mortem detection of the pathogen. After infection, serogroup-specific VP7 antibodies were undetectable by blocking ELISA (b-ELISA) in 2 out of 3 unvaccinated horses during the course of the disease (9-10 dpi). Vaccinated horses did not show significant side effects post-vaccination and were largely asymptomatic after the AHSV-9 challenge. VP7-specific antibodies could not be detected by the b-ELISA until day 21 and day 30 post-inoculation, respectively. Virus neutralizing antibody titres were low or even undetectable for specific serotypes in the vaccinated horses. Virus isolation and GS-rRT-PCR detected the presence of AHSV vaccine strains genomes and infectious vaccine virus after vaccination and challenge. This study established an experimental infection model of AHSV-9 in horses and characterized the main clinical, virological, and immunological parameters in both immunologically naïve and vaccinated horses using standardized bio-assays.

Dynamics of African swine fever virus (ASFV) infection in domestic pigs infected with virulent, moderate virulent and attenuated genotype II ASFV European isolates.

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.

High Doses of Inactivated African Swine Fever Virus Are Safe, but Do Not Confer Protection against a Virulent Challenge.

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.

First Oral Vaccination of Eurasian Wild Boar Against African Swine Fever Virus Genotype II.

African swine fever (ASF), the most significant threat to the pig industry worldwide, has spread to more than 55 countries on three continents, and it affects more than 77% of the world swine population. In the European Union, wild boar (Sus scrofa) is the most severely affected host. The main reasons for the unprecedented and constant spread of ASF in Europe are the trade activities, the continuous movement of infected-wild boar populations among regions and the lack of vaccine to prevent ASF infection. In this study, we demonstrate that oral immunization of wild boar with a non-hemadsorbing, attenuated ASF virus of genotype II isolated in Latvia in 2017 (Lv17/WB/Rie1) conferred 92% protection against challenge with a virulent ASF virus isolate (Arm07). This is, to our knowledge, the first report of a promising vaccine against ASF virus in wild boar by oral administration. Further studies should assess the safety of repeated administration and overdose, characterize long-term shedding and verify the genetic stability of the vaccine virus to confirm if Lv17/WB/Rie1 could be used for free-ranging wild boar in ASF control programs.

Attenuated and non-haemadsorbing (non-HAD) genotype II African swine fever virus (ASFV) isolated in Europe, Latvia 2017.

A non-haemadsorbing (non-HAD) ASF virus (ASFV) genotype II, namely Lv17/WB/Rie1, was isolated from a hunted wild boar in Latvia in 2017. Domestic pigs experimentally infected with the non-HAD ASFV developed a nonspecific or subclinical form of the disease. Two months later, these animals were fully protected when exposed to other domestic pigs infected with a related virulent HAD genotype II ASFV.

Phylodynamics and evolutionary epidemiology of African swine fever p72-CVR genes in Eurasia and Africa.

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.

Approaches and Perspectives for Development of African Swine Fever Virus Vaccines.

African swine fever (ASF) is a complex disease of swine, caused by a large DNA virus belonging to the family Asfarviridae. The disease shows variable clinical signs, with high case fatality rates, up to 100%, in the acute forms. ASF is currently present in Africa and Europe where it circulates in different scenarios causing a high socio-economic impact. In most affected regions, control has not been effective in part due to lack of a vaccine. The availability of an effective and safe ASFV vaccines would support and enforce control-eradication strategies. Therefore, work leading to the rational development of protective ASF vaccines is a high priority. Several factors have hindered vaccine development, including the complexity of the ASF virus particle and the large number of proteins encoded by its genome. Many of these virus proteins inhibit the host's immune system thus facilitating virus replication and persistence. We review previous work aimed at understanding ASFV-host interactions, including mechanisms of protective immunity, and approaches for vaccine development. These include live attenuated vaccines, and "subunit" vaccines, based on DNA, proteins, or virus vectors. In the shorter to medium term, live attenuated vaccines are the most promising and best positioned candidates. Gaps and future research directions are evaluated.

Usefulness of an intra-gastric balloon before bariatric surgery.

INTRODUCTION: There are only few reports regarding the use of intragastric-balloons (IGB®) to achieve weight loss and subsequently decrease surgical complications. In this study, we try to assess whether presurgery weight loss using IGB decreases the postsurgical mortality after bariatric surgery. METHODS: This is a prospective case-control study. We matched 1:1 by gender, age (± 10 y-o) and type of surgery (sleeve resection [LSG] or gastric bypass [LGBP]), matching cases (A) and controls (H, from a historic cohort). Morbidly obese patients with an indication for bariatric surgery were included in the study. Cases (A) were recruited from an ongoing clinical trial, and the controls (H) came from a historic cohort prior to the start of the clinic trial. The presurgical weight loss in group A was reached by IGB combined with diet, versus only diet in group H. RESULTS: We included 58 patients, 65.5% women, 69% LGBP/31% LSG. The mean age of group A was 42 and 43.4 years old for group H. ASA III of 24.1% group A vs 58.6% group H, p = 0.012. The mean total weight loss (TWL) before surgery was greater in group A (16.2 kg, SD 9.75) than in group H (1.2 kg, SD 6.4), p < 0.0001. The % of EWL before surgery was 23.5 (SD 11.6) in group A vs 2.4 (SD 8) in group H, p < 0.001. Hospital stay was seven days for group A, and eight days for group H, p = 0.285. The rate of unsuccessful IGB treatment to accomplish the scheduled weight loss was 34.5%. The balloon morbidity was 17.2% (6.9% severe). All in all, morbidity (due to bariatric surgery and IGB) was 41% in both groups. Postsurgical morbidity moderate-severe was 20.3% in group A (6.9% severe) and 27.3% in group H (17.2% severe) without statistical significance. One patient died in group H (mortality rate, 3.44%). CONCLUSION: Preoperative IGB treatment in morbid obesity has not been found to be effective at decreasing postsurgical morbidity LSG and LGBP, despite the fact that it acheives a greater weight loss than diet and exercise.

Comparative analysis of the complete genome sequences of Kenyan African swine fever virus isolates within p72 genotypes IX and X.

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.

Genetic variation among African swine fever genotype II viruses, eastern and central Europe.

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.

Potential use of oral fluid samples for serological diagnosis of African swine fever.

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.

Development and inter-laboratory validation study of an improved new real-time PCR assay with internal control for detection and laboratory diagnosis of African swine fever virus.

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 detection in pigs in newly infected areas, control in endemic areas and surveillance in ASF-free areas.

African swine fever virus p72 genotype IX in domestic pigs, Congo, 2009.

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.

Genetic characterisation of African swine fever viruses from recent and historical outbreaks in Sardinia (1978-2009).

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.

African swine fever viruses with two different genotypes, both of which occur in domestic pigs, are associated with ticks and adult warthogs, respectively, at a single geographical site.

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 warthog-derived 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.

Experimental infection of European red deer (Cervus elaphus) with bluetongue virus serotypes 1 and 8.

Bluetongue (BT) is a climate change-related emerging infectious disease in Europe. Outbreaks of serotypes 1, 2, 4, 6, 8, 9, 11, and 16 are challenging Central and Western Europe since 1998. Measures to control or eradicate bluetongue virus (BTV) from Europe have been implemented, including movement restrictions and vaccination of domestic BTV-susceptible ruminants. However, these measures are difficult to apply in wild free-ranging hosts of the virus, like red deer (Cervus elaphus), which could play a role in the still unclear epidemiology of BT in Europe. We show for the first time that BTV RNA can be detected in European red deer blood for long periods, comparable to those of domestic ruminants, after experimental infection with BTV-1 and BTV-8. BTV RNA was detected in experimentally infected red deer blood up to the end of the study (98-112 dpi). BTV-specific antibodies were found in serum both by enzyme-linked immunosorbent assay (ELISA) and virus neutralization (VNT) from 8 to 12 dpi to the end of the study, peaking at 17-28 dpi. Our results indicate that red deer can be infected with BTV and maintain BTV RNA for long periods, remaining essentially asymptomatic. Thus, unvaccinated red deer populations have the potential to be a BT reservoir in Europe, and could threaten the success of the European BTV control strategy. Therefore, wild and farmed red deer should be taken into account for BTV surveillance, and movement restrictions and vaccination schemes applied to domestic animals should be adapted to include farmed or translocated red deer.

Phylogenomic analysis of 11 complete African swine fever virus genome sequences.

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.

Enhanced discrimination of African swine fever virus isolates through nucleotide sequencing of the p54, p72, and pB602L (CVR) genes.

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.

Novel gel-based and real-time PCR assays for the improved detection of African horse sickness virus.

In order to improve, ensure and accelerate the diagnosis of African horse sickness, a highly devastating, transboundary animal disease listed by the World Animal Health Organisation, (OIE) three novel diagnostic PCR assays were developed and tested in this study. The reverse transcription-PCR (RT-PCR) tests were the following: (a) a conventional, gel-based RT-PCR, (b) a real-time PCR with SYBR-Green-named rRT-PCR SYBR-Green-, and (c) a real-time PCR rRT-PCR with TaqMan probe (termed rRT-PCR TaqMan). The same pair of primers-directed against African Horse Sickness Virus (AHSV) segment 5, encoding the non-structural protein NS1, is used in the three tests listed above. The three PCR assays detected similarly the nine AHSV serotypes from cultivated viral suspensions of different origins. The RT-PCR assays provided high sensitivity ranging from 0.1 to 1.2TCID(50)/ml. The specificity was also high, considering that related viruses, such as Bluetongue virus, and other equine viruses, such as West Nile Virus, remained negative for RT-PCR amplification. The detection of AHSV virus can be completed within 2-3h. These results indicate that the novel PCR methods described in this paper provide robust and versatile tools that allow rapid and highly specific, simultaneous detection of all AHSV serotypes.