Epidemiological investigation of ASF outbreaks in Kerala (India): detection, source tracing and economic implications.

This study investigates suspected African swine fever (ASF) outbreaks in two villages of Kannur district in Kerala, India, with the aim of identifying the causative agent and its genotype, the source of infection, and estimating the economic losses due to the outbreaks. Clinically, the disease was acute with high mortality, while gross pathology was characterized by widespread haemorrhages in various organs, especially the spleen, which was dark, enlarged and had friable cut surfaces with diffuse haemorrhages. Notably, histopathological examination revealed multifocal, diffuse haemorrhages in the splenic parenchyma and lymphoid depletion accompanied by lymphoid cell necrosis. The clinico-pathological observations were suggestive of ASF, which was confirmed by PCR. The source of outbreak was identified as swill and it was a likely point source infection as revealed by epidemic curve analysis. The phylogenetic analysis of p72 gene identified the ASFV in the current outbreak as genotype-II and IGR II variant consistent with ASFVs detected in India thus far. However, the sequence analysis of the Central Variable Region (CVR) of the B602L gene showed that the ASFVs circulating in Kerala (South India) formed a separate clade along with those found in Mizoram (North East India), while ASFVs circulating in Arunachal Pradesh and Assam states of India grouped in to different clade. This study represents the first investigation of ASF outbreak in South India, establishing the genetic relatedness of the ASFV circulating in this region with that in other parts of the country. The study also underscores the utility of the CVR of the B602L gene in genetically characterizing highly similar Genotype II ASFVs to understand the spread of ASF within the country.

Assessment of the Impact of a Toll-like Receptor 2 Agonist Synthetic Lipopeptide on Macrophage Susceptibility and Responses to African Swine Fever Virus Infection.

Toll-like receptor 2 (TLR2) ligands are attracting attention as prophylactic and immunopotentiator agents against pathogens, including viruses. We previously reported that a synthetic diacylated lipopeptide (Mag-Pam2Cys_P48) polarized porcine macrophages towards a proinflammatory antimicrobial phenotype. Here, we investigated its role in modulating monocyte-derived macrophage (moMΦ) responses against African swine fever virus (ASFV), the etiological agent of one of the greatest threats to the global pig industry. Two ASFV isolates were compared: the attenuated NH/P68 and the virulent 26544/OG10. No effect on virus infection nor the modulation of surface markers' expression (MHC I, MHC II DR, CD14, CD16, and CD163) were observed when Mag-Pam2Cys_P48 treated moMΦ were infected using a multiplicity of infection (MOI) of 1. Mag-Pam2Cys_P48 treated moMΦ released higher levels of IL-1α, IL-1ß, IL-1Ra, and IL-18 in response to infection with NH/P68 ASFV compared to 26544/OG10-infected and mock-infected controls. Surprisingly, when infected using a MOI of 0.01, the virulent ASFV 26544/OG10 isolate replicated even slightly more efficiently in Mag-Pam2Cys_P48 treated moMΦ. These effects also extended to the treatment of moMΦ with two other lipopeptides: Mag-Pam2Cys_P80 and Mag-Pam2Cys_Mag1000. Our data suggested limited applicability of TLR2 agonists as prophylactic or immunopotentiator agents against virulent ASFV but highlighted the ability of the virulent 26544/OG10 to impair macrophage defenses.

Research progress on the proteins involved in African swine fever virus infection and replication.

African swine fever (ASF) is an acute, hemorrhagic and highly contagious infectious disease caused by African swine fever virus (ASFV), which infects domestic pigs or wild boars. It is characterized by short course of disease, high fever and hemorrhagic lesions, with mortality of up to 100% from acute infection. Up to now, the lack of commercial vaccines and effective drugs has seriously threatened the healthy economic development of the global pig industry. ASFV is a double-stranded DNA virus and genome varies between about 170-194 kb, which encodes 150-200 viral proteins, including 68 structural proteins and more than 100 non-structural proteins. In recent years, although the research on structure and function of ASFV-encoded proteins has been deepened, the structure and infection process of ASFV are still not clear. This review summarizes the main process of ASFV infection, replication and functions of related viral proteins to provide scientific basis and theoretical basis for ASFV research and vaccine development.

Simplifying sampling for African swine fever surveillance: Assessment of antibody and pathogen detection from blood swabs.

African swine fever (ASF) is a notifiable disease with serious socio-economic consequences that has been present in wild boar in the Baltic States and Poland since 2014. An introduction of ASF is usually accompanied by increased mortality, making fallen wild boar and hunted animals with signs of disease the main target for early warning and passive surveillance. It is difficult, however, to encourage hunters and foresters to report and take samples from these cases. A pragmatic and easy sampling approach with quick-drying swabs could facilitate this. In this study, we further evaluated the use of dry blood swabs for the detection of ASFV antibody and genome with samples from animal trials and diagnostic submissions (blood, bone and organs) from Estonia. Compared to serum samples, dried blood swabs yielded 93.1% (95% confidence interval: [83.3, 98.1]) sensitivity and 100% [95.9, 100.0] specificity in a commercial ASFV antibody ELISA. Similarly, the swabs gave a sensitivity of 98.9% [93.4, 100.0] and a specificity of 98.1% [90.1, 100.0] for genome detection by a standard ASFV p72 qPCR when compared to EDTA blood. The same swabs were tested in a VP72-antibody lateral flow device, with a sensitivity of 94.7% [85.4, 98.9] and specificity of 96.1% [89.0, 99.2] compared to the serum ELISA. When GenoTube samples tested in ELISA and LFD were compared, the sensitivity was 96.3% [87.3, 99.5] and the specificity was 93.8% [86.0, 97.9]. This study demonstrates reliable detection of ASFV antibody and genome from swabs. A field test of the swabs with decomposed wild boar carcasses in an endemic area in Estonia also gave promising results. Thus, this technique is a practical approach for surveillance of ASF in both free and endemic areas.

In situ hybridization with labeled probes: assessment of african Swine Fever virus in formalin-fixed paraffin-embedded tissues.

In situ hybridization (ISH) has become a very valuable molecular diagnostic tool to detect specific DNA or RNA sequences in biological samples through the use of complementary DNA- or RNA-labeled probes. Here, we describe an optimized in situ hybridization protocol to detect African swine fever virus (ASFV) DNA in formalin-fixed, paraffin-embedded tissues using digoxigenin-labeled probes.

First molecular assessment of the African swine fever virus status of Ornithodoros ticks from Swaziland.

African swine fever (ASF) is an economically significant haemorrhagic disease of domestic pigs. It is caused by the African swine fever virus (ASFV), a deoxyribonucleic acid (DNA)arbovirus. Argasid ticks of the genus Ornithodoros, which are widely distributed throughout southern Africa, play a primary role in virus maintenance and spread within the endemic sylvatic cycle. The ASF status of Swaziland is unknown, but this land-locked country is surrounded by ASF-positive countries, has a burgeoning pig industry and sylvatic cycle hosts present within its borders. In this first assessment of ASF status, warthog burrows in seven nature reserves and game management areas in Swaziland were investigated for tick and virus presence. Tick infestation rates of between 33.3% - 88.8% were recovered for the four Ornithodoros-infested reserves. A total of 562 ticks were screened for virus genome presence using a duplex Polymerase Chain Reaction (PCR) that targets the C-terminal end of the p72 gene of the ASFV and confirms DNA integrity through amplification of the 16S rRNA tick host gene. All samples were negative for virus genome presence and positive for the tick genome target. Nucleotide sequencing of the latter confirmed that Ornithodoros ticks from Swaziland are identical to those from the Kruger National Park in South Africa across the gene region characterised. Whilst this first evaluation of ASF presence in Swaziland indicates that the virus does not appear to be present in the key virus vector, the presence of sylvatic cycle hosts, together with the country's proximity to ASF-affected countries calls for expanded investigations and regular monitoring of the ASF status of Swaziland.

A host species-informative internal control for molecular assessment of African swine fever virus infection rates in the African sylvatic cycle Ornithodoros vector.

African swine fever virus (ASFV) infection in adult Ornithodoros porcinus (Murry 1877, sensuWalton 1979) ticks collected from warthog burrows in southern and East Africa was assessed using a duplex genomic amplification approach that is informative with respect to the invertebrate host species and infecting sylvatic cycle virus. DNA extracted from individual ticks was used as template for the simultaneous amplification of a C-terminal 478-bp ASFV p72 gene region and a approximately 313-bp fragment of the tick mitochondrial 16S rRNA gene, under optimized reaction conditions. Within-warthog burrow infection rates ranged from 0% to 43% using this approach, and phylogenetic analysis of 16S gene sequences revealed the presence of three geographically discrete O. porcinus lineages, but no support for subspecies recognition. False negatives are precluded by the inclusion of host species-informative primers that ensure the DNA integrity of cytoplasmically located genome extracts. In addition, infection rate estimates are further improved as false positives arising from carry-over contamination when performing a two-step nested polymerase chain reaction are negated by the one-step approach. Phylogenetic comparison of full-length virus gene sequences with the partial C-terminal p72 gene target confirmed the epidemiological utility of the latter in a sylvatic setting. The method is therefore of particular value in studies assessing the prevalence and diversity of ASFV in relation to the African sylvatic tick vector and holds potential for investigating the role of alternative tick species in virus maintenance and transmission.