Assessing the value of PCR assays in oral fluid samples for detecting African swine fever, classical swine fever, and foot-and-mouth disease in U.S. swine.

INTRODUCTION: Oral fluid sampling and testing offers a convenient, unobtrusive mechanism for evaluating the health status of swine, especially grower and finisher swine. This assessment evaluates the potential testing of oral fluid samples with real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) to detect African swine fever, classical swine fever, or foot-and-mouth disease for surveillance during a disease outbreak and early detection in a disease-free setting. METHODS: We used a series of logical arguments, informed assumptions, and a range of parameter values from literature and industry practices to examine the cost and value of information provided by oral fluid sampling and rRT-PCR testing for the swine foreign animal disease surveillance objectives outlined above. RESULTS: Based on the evaluation, oral fluid testing demonstrated value for both settings evaluated. The greatest value was in an outbreak scenario, where using oral fluids would minimize disruption of animal and farm activities, reduce sample sizes by 23%-40%, and decrease resource requirements relative to current individual animal sampling plans. For an early detection system, sampling every 3 days met the designed prevalence detection threshold with 0.95 probability, but was quite costly. LIMITATIONS: Implementation of oral fluid testing for African swine fever, classical swine fever, or foot-and-mouth disease surveillance is not yet possible due to several limitations and information gaps. The gaps include validation of PCR diagnostic protocols and kits for African swine fever, classical swine fever, or foot-and-mouth disease on swine oral fluid samples; minimal information on test performance in a field setting; detection windows with low virulence strains of some foreign animal disease viruses; and the need for confirmatory testing protocol development.

Genetic Assessment of African Swine Fever Isolates Involved in Outbreaks in the Democratic Republic of Congo between 2005 and 2012 Reveals Co-Circulation of p72 Genotypes I, IX and XIV, Including 19 Variants.

African swine fever (ASF) is a devastating disease of domestic pigs. It is a socioeconomically important disease, initially described from Kenya, but subsequently reported in most Sub-Saharan countries. ASF spread to Europe, South America and the Caribbean through multiple introductions which were initially eradicated-except for Sardinia-followed by re­introduction into Europe in 2007. In this study of ASF within the Democratic Republic of the Congo, 62 domestic pig samples, collected between 2005-2012, were examined for viral DNA and sequencing at multiple loci: C-terminus of the B646L gene (p72 protein), central hypervariable region (CVR) of the B602L gene, and the E183L gene (p54 protein). Phylogenetic analyses identified three circulating genotypes: I (64.5% of samples), IX (32.3%), and XIV (3.2%). This is the first evidence of genotypes IX and XIV within this country. Examination of the CVR revealed high levels of intra-genotypic variation, with 19 identified variants.

Faustovirus, an asfarvirus-related new lineage of giant viruses infecting amoebae.

UNLABELLED: Giant viruses are protist-associated viruses belonging to the proposed order Megavirales; almost all have been isolated from Acanthamoeba spp. Their isolation in humans suggests that they are part of the human virome. Using a high-throughput strategy to isolate new giant viruses from their original protozoan hosts, we obtained eight isolates of a new giant viral lineage from Vermamoeba vermiformis, the most common free-living protist found in human environments. This new lineage was proposed to be the faustovirus lineage. The prototype member, faustovirus E12, forms icosahedral virions of ≈ 200 nm that are devoid of fibrils and that encapsidate a 466-kbp genome encoding 451 predicted proteins. Of these, 164 are found in the virion. Phylogenetic analysis of the core viral genes showed that faustovirus is distantly related to the mammalian pathogen African swine fever virus, but it encodes ≈ 3 times more mosaic gene complements. About two-thirds of these genes do not show significant similarity to genes encoding any known proteins. These findings show that expanding the panel of protists to discover new giant viruses is a fruitful strategy. IMPORTANCE: By using Vermamoeba, a protist living in humans and their environment, we isolated eight strains of a new giant virus that we named faustovirus. The genomes of these strains were sequenced, and their sequences showed that faustoviruses are related to but different from the vertebrate pathogen African swine fever virus (ASFV), which belongs to the family Asfarviridae. Moreover, the faustovirus gene repertoire is ≈ 3 times larger than that of ASFV and comprises approximately two-thirds ORFans (open reading frames [ORFs] with no detectable homology to other ORFs in a database).

Detection of African swine fever, classical swine fever, and foot-and-mouth disease viruses in swine oral fluids by multiplex reverse transcription real-time polymerase chain reaction.

African swine fever (ASF), classical swine fever (CSF), and foot-and-mouth disease (FMD) are highly contagious animal diseases of significant economic importance. Pigs infected with ASF and CSF viruses (ASFV and CSFV) develop clinical signs that may be indistinguishable from other diseases. Likewise, various causes of vesicular disease can mimic clinical signs caused by the FMD virus (FMDV). Early detection is critical to limiting the impact and spread of these disease outbreaks, and the ability to perform herd-level surveillance for all 3 diseases rapidly and cost effectively using a single diagnostic sample and test is highly desirable. This study assessed the feasibility of simultaneous ASFV, CSFV, and FMDV detection by multiplex reverse transcription real-time polymerase chain reaction (mRT-qPCR) in swine oral fluids collected through the use of chewing ropes. Animal groups were experimentally infected independently with each virus, observed for clinical signs, and oral fluids collected and tested throughout the course of infection. All animal groups chewed on the ropes readily before and after onset of clinical signs and before onset of lameness or serious clinical signs. ASFV was detected as early as 3 days postinoculation (dpi), 2-3 days before onset of clinical disease; CSFV was detected at 5 dpi, coincident with onset of clinical disease; and FMDV was detected as early as 1 dpi, 1 day before the onset of clinical disease. Equivalent results were observed in 4 independent studies and demonstrate the feasibility of oral fluids and mRT-qPCR for surveillance of ASF, CSF, and FMD in swine populations.

Development and validation of a multiplex, real-time RT PCR assay for the simultaneous detection of classical and African swine fever viruses.

A single-step, multiplex, real-time polymerase chain reaction (RT-PCR) was developed for the simultaneous and differential laboratory diagnosis of Classical swine fever virus (CSFV) and African swine fever virus (ASFV) alongside an exogenous internal control RNA (IC-RNA). Combining a single extraction methodology and primer and probe sets for detection of the three target nucleic acids CSFV, ASFV and IC-RNA, had no effect on the analytical sensitivity of the assay and the new triplex RT-PCR was comparable to standard PCR techniques for CSFV and ASFV diagnosis. After optimisation the assay had a detection limit of 5 CSFV genome copies and 22 ASFV genome copies. Analytical specificity of the triplex assay was validated using a panel of viruses representing 9 of the 11 CSFV subgenotypes, at least 8 of the 22 ASFV genotypes as well as non-CSFV pestiviruses. Positive and negative clinical samples from animals infected experimentally, due to field exposure or collected from the UK which is free from both swine diseases, were used to evaluate the diagnostic sensitivity and specificity for detection of both viruses. The diagnostic sensitivity was 100% for both viruses whilst diagnostic specificity estimates were 100% for CSFV detection and 97.3% for ASFV detection. The inclusion of a heterologous internal control allowed identification of false negative results, which occurred at a higher level than expected. The triplex assay described here offers a valuable new tool for the differential detection of the causative viruses of two clinically indistinguishable porcine diseases, whose geographical occurrence is increasingly overlapping.

Detection of African swine fever virus-like sequences in ponds in the Mississippi Delta through metagenomic sequencing.

Metagenomic characterization of water virome was performed in four Mississippi catfish ponds. Although differing considerably from African swine fever virus (ASFV), 48 of 446,100 sequences from 12 samples were similar enough to indicate that they represent new members in the family Asfarviridae. At present, ASFV is the only member of Asfarviridae, and this study presents the first indication of a similar virus in North America. At this point, there is no indication that the identified virus(es) pose a threat to human or animal health, and further study is needed to characterize their potential risks to both public health and agricultural development.

Molecular epidemiology of African swine fever in East Africa.

African swine fever (ASF) a lethal, viral hemorrhagic disease of domestic pigs, first reported from East Africa in 1921, is still widespread in this region. In order to assess field heterogeneity at the regional level, nucleotide sequences corresponding to the C-terminal end of the p72 gene were determined for 77 ASF viruses of diverse temporal and species origin occurring in eight East African countries. The number of sites completely conserved across all East African sequences characterized in this study was 84.2% and 86.8% on nucleotide and amino acid level, respectively. Phylogenetic analysis of a homologous 404 bp region revealed the presence of thirteen East African genotypes, of which eight appear to be country specific. An East African, pig-associated, homogeneous virus lineage linked to outbreaks in Mozambique, Zambia and Malawi over a 23 year period was demonstrated. In addition, genotype I (ESACWA) viruses were identified in East African sylvatic hosts for the first time which is significant as this genotype was previously thought to be restricted to the West African region where it occurs only in domestic pigs. The presence of discrete epidemiological cycles in East Africa and recovery of multiple genotypes affirms the epidemiological complexity of ASF in this region.

Co-circulation of two genetically distinct viruses in an outbreak of African swine fever in Mozambique: no evidence for individual co-infection.

In 1998, domestic pigs originating from villages within a 40 km radius of Ulongwe in the northern Tete Province of Mozambique were held in a quarantine facility for a 3-month period prior to their importation into South Africa. Eight of a total of 25 pigs died within the first 3 weeks of quarantine of what appeared clinically and on post mortem examination to be African swine fever (ASF). Organs were collected and preserved in formol-glycerosaline and the presence of ASF virus in these specimens was confirmed by three independent polymerase chain reaction (PCR) tests. Two gene regions were characterised, namely the C-terminus end of the major immunodominant protein VP72 and the central variable region (CVR) of the 9RL open reading frame (ORF). Results confirmed the presence of two genetically distinct viruses circulating simultaneously within a single outbreak focus. However, despite the pigs being housed within the same facility, no evidence of co-infection was observed within individual animals. Comparison of the two 1998 virus variants with viruses causing historical outbreaks of the disease in Mozambique revealed that these viruses belong to two distinct genotypes which are unrelated to viruses causing outbreaks between 1960 and 1994. In addition, the CVR and p72 gene regions of one of the 1998 Mozambique virus variants (variant-40) was shown to be identical to the virus recovered from an ASF outbreak in Madagascar in the same year, whilst the other (variant-92) was identical to a 1988 pig isolate from Zambia.