Analysis of acidified feed components containing African swine fever virus.

Mitigation of African swine fever (ASF) virus in contaminated feed materials would assist control activities. Various finely-ground pig feed ingredients (5 cereals, 4 plant proteins, 2 animal proteins, 1 oil, 1 compound) were sprayed and mixed thoroughly with a buffered formic acid formulation (0, 1 or 2% vol/vol) to produce a consistent and durable level of formate (1% or 2%) with consistent acidification of cereal ingredients to less than pH 4. No such acidification was noted in other ingredients. Selected representative feed ingredients were further mixed with infectious ASF virus (106 TCID50) or media alone and incubated for 0, 6, 12, 24, 48, 72 or 168 h. The residual ASF virus at each timepoint was quantified using qPCR and a cell culture based TCID50 assay to determine survivability. Maize, rice bran and compound feed (with or without formate) all reduced infectious ASF virus to levels below the detection threshold of the cell culture assay (101.3 TCID50/mL). A consistent reduction in ASF virus DNA levels was observed by qPCR assay when maize containing ASF virus was mixed with 1% or 2% buffered formic acid. This reduction in viral DNA corresponded to the acidifying pH effect measured. No such reduction in ASF virus DNA levels was noted in non-cereal ingredients containing ASF virus, in which the pH had not been lowered below pH 4 following treatment. Interestingly, residual ASF virus levels in spiked meat/bone meal were greater than control levels, suggesting a buffering effect of that feed ingredient.

Complete Genome Sequence of African Swine Fever Virus Isolated from a Domestic Pig in Timor-Leste, 2019.

Here, we report the complete genome sequence of the African swine fever virus (ASFV) isolate ASFV/Timor-Leste/2019/1, isolated from a domestic pig during the first outbreak of ASF in Timor-Leste in 2019. Using target enrichment short-read Illumina data combined with long-read Oxford Nanopore data, we assembled a full-length genome sequence of 192,237 bp.

Bluetongue virus serotype 12 enters Australia - a further incursion of novel western lineage genome segments.

Bluetongue virus (BTV) is an arbovirus (genus: Orbivirus) that occurs worldwide. It infects domestic and wild ruminant species and can cause disease in livestock, producing high economic impact. Recently, it gained extra prominence throughout Europe, with disease occurring in regions traditionally free of BTV. BTV enters Australia from Southeast Asia via wind-borne infected Culicoides spp. The first Australian isolation was 1975 (BTV-20) and further serotypes were isolated between 1979-86 (BTV-1, -3, -9, -15, -16, -21, -23). Despite increased, more sensitive, monitoring, no more were detected in over two decades, implying a stable BTV episystem of eastern ancestry. Isolations of BTV-2, -7 and -5 then occurred between 2007-15, with the latter two possessing genome segments with high sequence identity to western isolates. We report on the first isolation and genomic characterization of BTV-12, which revealed that three more novel western topotype gene segments have entered northern Australia.

Identification and genomic characterization of the first isolate of bluetongue virus serotype 5 detected in Australia.

Bluetongue virus (BTV), transmitted by midges (Culicoides sp), is distributed worldwide and causes disease in ruminants. In particular, BT can be a debilitating disease in sheep causing serious trade and socio-economic consequences at both local and global levels. Across Australia, a sentinel cattle herd surveillance program monitors the BTV activity. Prior to 2014, BTV-1, -2, -3, -7, -9, -15, -16, -20, -21 and -23 had been isolated in Australia, but no bluetongue disease has occurred in a commercial Australian flock. We routinely use a combination of serology, virus isolation, RT-PCR and next generation and conventional nucleotide sequencing technologies to detect and phylogenetically characterize incursions of novel BTV strains into Australia. Screening of Northern Territory virus isolates in 2015 revealed BTV-5, a serotype new to Australia. We derived the complete genome of this isolate and determined its phylogenetic relationship with exotic BTV-5 isolates. Gene segments 2, 6, 7 and 10 exhibited a close relationship with the South African prototype isolate RSArrrr/5. This was the first Australian isolation of a Western topotype of segment 10. Serological surveillance data highlighted the antigenic cross-reactivity between BTV-5 and BTV-9. Phylogenetic investigation of segments 2 and 6 of these serotypes confirmed their unconventional relationships within the BTV serogroup. Our results further highlighted a need for a revision of the current serologically based system for BTV strain differentiation and importantly, implied a potential for genome segments of pathogenic Western BTV strains to rapidly enter Southeast Asia. This emphasized a need for continued high-level surveillance of vectors and viruses at strategic locations in the north of Australia The expansion of routine characterization and classification of BTV to a whole genome approach is recommended, to better monitor the presence and level of establishment of novel Western topotype segments within the Australian episystem.

Evaluation of an African swine fever (ASF) vaccine strategy incorporating priming with an alphavirus-expressed antigen followed by boosting with attenuated ASF virus.

In this study, an alphavirus vector platform was used to deliver replicon particles (RPs) expressing African swine fever virus (ASFV) antigens to swine. Alphavirus RPs expressing ASFV p30 (RP-30), p54 (RP-54) or pHA-72 (RP-sHA-p72) antigens were constructed and tested for expression in Vero cells and for immunogenicity in pigs. RP-30 showed the highest expression in Vero cells and was the most immunogenic in pigs, followed by RP-54 and RP-sHA-p72. Pigs primed with two doses of the RP-30 construct were then boosted with a naturally attenuated ASFV isolate, OURT88/3. Mapping of p30 identified an immunodominant region within the amino acid residues 111-130. However, the principal effect of the prime-boost was enhanced recognition of an epitope covered by the peptide sequence 61-110. The results suggest that a strategy incorporating priming with a vector-expressed antigen followed by boosting with an attenuated live virus may broaden the recognition of ASFV epitopes.

Assessment of a Rabies Virus Rapid Diagnostic Test for the Detection of Australian Bat Lyssavirus.

Australian bat lyssavirus (ABLV) is closely related to the classical rabies virus and has been associated with three human fatalities and two equine fatalities in Australia. ABLV infection in humans causes encephalomyelitis, resulting in fatal disease, but has no effective therapy. The virus is maintained in enzootic circulation within fruit bats (Pteropid spp.) and at least one insectivorous bat variety (Saccolaimus flaviventris). Most frequently, laboratory testing is conducted on pteropodid bat brains, either following a potential human exposure through bites, scratches and other direct contacts with bats, or as opportunistic assessment of sick or dead bats. The level of medical intervention and post-exposure prophylaxis is largely determined on laboratory testing for antigen/virus as the demonstrable infection status of the in-contact bat. This study evaluates the comparative diagnostic performance of a lateral flow test, Anigen Rabies Ag detection rapid test (RDT), in pteropodid variant of ABLV-infected bat brain tissues. The RDT demonstrated 100% agreement with the reference standard fluorescent antibody test on 43 clinical samples suggesting a potential application in rapid diagnosis of pteropodid variant of ABLV infection. A weighted Kappa value of 0.95 confirmed a high level of agreement between both tests.

Development and validation of an immunoperoxidase antigen detection test for improved diagnosis of rabies in Indonesia.

Rabies continues to pose a significant threat to human and animal health in regions of Indonesia. Indonesia has an extensive network of veterinary diagnostic laboratories and the 8 National laboratories are equipped to undertake diagnostic testing for rabies using the commercially-procured direct fluorescent antibody test (FAT), which is considered the reference (gold standard) test. However, many of the Indonesian Provincial diagnostic laboratories do not have a fluorescence microscope required to undertake the FAT. Instead, certain Provincial laboratories continue to screen samples using a chemical stain-based test (Seller's stain test, SST). This test has low diagnostic sensitivity, with negative SST-tested samples being forwarded to the nearest National laboratory resulting in significant delays for completion of testing and considerable additional costs. This study sought to develop a cost-effective and diagnostically-accurate immunoperoxidase antigen detection (RIAD) test for rabies that can be readily and quickly performed by the resource-constrained Provincial laboratories. This would reduce the burden on the National laboratories and allow more rapid diagnoses and implementation of post-exposure prophylaxis. The RIAD test was evaluated using brain smears fixed with acetone or formalin and its performance was validated by comparison with established rabies diagnostic tests used in Indonesia, including the SST and FAT. A proficiency testing panel was distributed between Provincial laboratories to assess the reproducibility of the test. The performance of the RIAD test was improved by using acetone fixation of brain smears rather than formalin fixation such that it was of equivalent accuracy to that of the World Organisation for Animal Health (OIE)-recommended FAT, with both tests returning median diagnostic sensitivity and specificity values of 0.989 and 0.993, respectively. The RIAD test and FAT had higher diagnostic sensitivity than the SST (median = 0.562). Proficiency testing using a panel of 6 coded samples distributed to 16 laboratories showed that the RIAD test had good reproducibility with an overall agreement of 97%. This study describes the successful development, characterisation and use of a novel RIAD test and its fitness for purpose as a screening test for use in provincial Indonesian veterinary laboratories.

Gene expression analysis of whole blood RNA from pigs infected with low and high pathogenic African swine fever viruses.

African swine fever virus (ASFV) is a macrophage-tropic virus responsible for ASF, a transboundary disease that threatens swine production world-wide. Since there are no vaccines available to control ASF after an outbreak, obtaining an understanding of the virus-host interaction is important for developing new intervention strategies. In this study, a whole transcriptomic RNA-Seq method was used to characterize differentially expressed genes in pigs infected with a low pathogenic ASFV isolate, OUR T88/3 (OURT), or the highly pathogenic Georgia 2007/1 (GRG). After infection, pigs infected with OURT showed no or few clinical signs; whereas, GRG produced clinical signs consistent with acute ASF. RNA-Seq detected the expression of ASFV genes from the whole blood of the GRG, but not the OURT pigs, consistent with the pathotypes of these strains and the replication of GRG in circulating monocytes. Even though GRG and OURT possess different pathogenic properties, there was significant overlap in the most upregulated host genes. A small number of differentially expressed microRNAs were also detected in GRG and OURT pigs. These data confirm previous studies describing the response of macrophages and lymphocytes to ASFV infection, as well as reveal unique gene pathways upregulated in response to infection with GRG.

Analysis of Arbovirus Isolates from Australia Identifies Novel Bunyaviruses Including a Mapputta Group Virus from Western Australia That Links Gan Gan and Maprik Viruses.

The Mapputta group comprises antigenically related viruses indigenous to Australia and Papua New Guinea that are included in the family Bunyaviridae but not currently assigned to a specific genus. We determined and analyzed the genome sequences of five Australian viruses isolated from mosquitoes collected during routine arbovirus surveillance in Western Australia (K10441, SW27571, K13190, and K42904) and New South Wales (12005). Based on matching sequences of all three genome segments to prototype MRM3630 of Trubanaman virus (TRUV), NB6057 of Gan Gan virus (GGV), and MK7532 of Maprik virus (MPKV), isolates K13190 and SW27571 were identified as TRUV, 12005 as GGV, and K42904 as a Mapputta group virus from Western Australia linking GGV and MPKV. The results confirmed serum neutralization data that had linked SW27571 to TRUV. The fifth virus, K10441 from Willare, was most closely related to Batai orthobunyavirus, presumably representing an Australian variant of the virus. Phylogenetic analysis also confirmed the close relationship of our TRUV and GGV isolates to two other recently described Australian viruses, Murrumbidgee virus and Salt Ash virus, respectively. Our findings indicate that TRUV has a wide circulation throughout the Australian continent, demonstrating for the first time its presence in Western Australia. Similarly, the presence of a virus related to GGV, which had been linked to human disease and previously known only from the Australian southeast, was demonstrated in Western Australia. Finally, a Batai virus isolate was identified in Western Australia. The expanding availability of genomic sequence for novel Australian bunyavirus variants supports the identification of suitably conserved or diverse primer-binding target regions to establish group-wide as well as virus-specific nucleic acid tests in support of specific diagnostic and surveillance efforts throughout Australasia.

Recent emergence and spread of an Arctic-related phylogenetic lineage of rabies virus in Nepal.

Rabies is a zoonotic disease that is endemic in many parts of the developing world, especially in Africa and Asia. However its epidemiology remains largely unappreciated in much of these regions, such as in Nepal, where limited information is available about the spatiotemporal dynamics of the main etiological agent, the rabies virus (RABV). In this study, we describe for the first time the phylogenetic diversity and evolution of RABV circulating in Nepal, as well as their geographical relationships within the broader region. A total of 24 new isolates obtained from Nepal and collected from 2003 to 2011 were full-length sequenced for both the nucleoprotein and the glycoprotein genes, and analysed using neighbour-joining and maximum-likelihood phylogenetic methods with representative viruses from all over the world, including new related RABV strains from neighbouring or more distant countries (Afghanistan, Greenland, Iran, Russia and USA). Despite Nepal's limited land surface and its particular geographical position within the Indian subcontinent, our study revealed the presence of a surprising wide genetic diversity of RABV, with the co-existence of three different phylogenetic groups: an Indian subcontinent clade and two different Arctic-like sub-clades within the Arctic-related clade. This observation suggests at least two independent episodes of rabies introduction from neighbouring countries. In addition, specific phylogenetic and temporal evolution analysis of viruses within the Arctic-related clade has identified a new recently emerged RABV lineage we named as the Arctic-like 3 (AL-3) sub-clade that is already widely spread in Nepal.