Characterization of an African Swine Fever Virus Field Isolate from Vietnam with Deletions in the Left Variable Multigene Family Region.

In this paper, we report the characterization of a genetically modified live-attenuated African swine fever virus (ASFV) field strain isolated from Vietnam. The isolate, ASFV-GUS-Vietnam, belongs to p72 genotype II, has six multi-gene family (MGF) genes deleted, and an Escherichia coli GusA gene (GUS) inserted. When six 6-8-week-old pigs were inoculated with ASFV-GUS-Vietnam oro-nasally (2 × 105 TCID50/pig), they developed viremia, mild fever, lethargy, and inappetence, and shed the virus in their oral and nasal secretions and feces. One of the pigs developed severe clinical signs and was euthanized 12 days post-infection, while the remaining five pigs recovered. When ASFV-GUS-Vietnam was inoculated intramuscularly (2 × 103 TCID50/pig) into four 6-8 weeks old pigs, they also developed viremia, mild fever, lethargy, inappetence, and shed the virus in their oral and nasal secretions and feces. Two contact pigs housed together with the four intramuscularly inoculated pigs, started to develop fever, viremia, loss of appetite, and lethargy 12 days post-contact, confirming horizontal transmission of ASFV-GUS-Vietnam. One of the contact pigs died of ASF on day 23 post-contact, while the other one recovered. The pigs that survived the exposure to ASFV-GUS-Vietnam via the mucosal or parenteral route were fully protected against the highly virulent ASFV Georgia 2007/1 challenge. This study showed that ASFV-GUS-Vietnam field isolate is able to induce complete protection in the majority of the pigs against highly virulent homologous ASFV challenge, but has the potential for horizontal transmission, and can be fatal in some animals. This study highlights the need for proper monitoring and surveillance when ASFV live-attenuated virus-based vaccines are used in the field for ASF control in endemic countries.

Detection of Recombinant African Swine Fever Virus Strains of p72 Genotypes I and II in Domestic Pigs, Vietnam, 2023.

African swine fever virus (ASFV) genotype II is endemic to Vietnam. We detected recombinant ASFV genotypes I and II (rASFV I/II) strains in domestic pigs from 6 northern provinces in Vietnam. The introduction of rASFV I/II strains could complicate ongoing ASFV control measures in the region.

Oral Fluids for the Early Detection of Classical Swine Fever in Commercial Level Pig Pens.

The early detection of classical swine fever (CSF) remains a key challenge, especially when outbreaks are caused by moderate and low-virulent CSF virus (CSFV) strains. Oral fluid is a reliable and cost-effective sample type that is regularly surveilled for endemic diseases in commercial pig herds in North America. Here, we explored the possibility of utilizing oral fluids for the early detection of CSFV incursions in commercial-size pig pens using two independent experiments. In the first experiment, a seeder pig infected with the moderately-virulent CSFV Pinillos strain was used, and in the second experiment, a seeder pig infected with the highly-virulent CSFV Koslov strain was used. Pen-based oral fluid samples were collected daily and individual samples (whole blood, swabs) every other day. All samples were tested by a CSFV-specific real-time RT-PCR assay. CSFV genomic material was detected in oral fluids on the seventh and fourth day post-introduction of the seeder pig into the pen, in the first and second experiments, respectively. In both experiments, oral fluids tested positive before the contact pigs developed viremia, and with no apparent sick pigs in the pen. These results indicate that pen-based oral fluids are a reliable and convenient sample type for the early detection of CSF, and therefore, can be used to supplement the ongoing CSF surveillance activities in North America.

Molecular and Pathological Characterization of Classical Swine Fever Virus Genotype 2 Strains Responsible for the 2013-2018 Outbreak in Colombia.

Classical swine fever (CSF) is a highly contagious transboundary viral disease of domestic and wild pigs. Despite mass vaccination and continuous eradication programs, CSF remains endemic in Asia, some countries in Europe, the Caribbean and South America. Since June 2013, Northern Colombia has reported 137 CSF outbreaks, mostly in backyard production systems with low vaccination coverage. The purpose of this study was to characterize the virus responsible for the outbreak. Phylogenetic analysis based on the full-length E2 sequence shows that the virus is closely related to CSF virus (CSFV) genotype 2.6 strains circulating in Southeast Asia. The pathotyping experiment suggests that the virus responsible is a moderately virulent strain. The 190 nucleotide stretch of the E2 hypervariable region of these isolates also shows high similarity to the CSFV isolates from Colombia in 2005 and 2006, suggesting a common origin for the CSF outbreaks caused by genotype 2.6 strains. The emergence of genotype 2.6 in Colombia suggests a potential transboundary spread of CSFV from Asia to the Americas, complicating the ongoing CSF eradication efforts in the Americas, and emphasizes the need for continuous surveillance in the region.

A Re-Evaluation of African Swine Fever Genotypes Based on p72 Sequences Reveals the Existence of Only Six Distinct p72 Groups.

The African swine fever virus (ASFV) is currently causing a world-wide pandemic of a highly lethal disease in domestic swine and wild boar. Currently, recombinant ASF live-attenuated vaccines based on a genotype II virus strain are commercially available in Vietnam. With 25 reported ASFV genotypes in the literature, it is important to understand the molecular basis and usefulness of ASFV genotyping, as well as the true significance of genotypes in the epidemiology, transmission, evolution, control, and prevention of ASFV. Historically, genotyping of ASFV was used for the epidemiological tracking of the disease and was based on the analysis of small fragments that represent less than 1% of the viral genome. The predominant method for genotyping ASFV relies on the sequencing of a fragment within the gene encoding the structural p72 protein. Genotype assignment has been accomplished through automated phylogenetic trees or by comparing the target sequence to the most closely related genotyped p72 gene. To evaluate its appropriateness for the classification of genotypes by p72, we reanalyzed all available genomic data for ASFV. We conclude that the majority of p72-based genotypes, when initially created, were neither identified under any specific methodological criteria nor correctly compared with the already existing ASFV genotypes. Based on our analysis of the p72 protein sequences, we propose that the current twenty-five genotypes, created exclusively based on the p72 sequence, should be reduced to only six genotypes. To help differentiate between the new and old genotype classification systems, we propose that Arabic numerals (1, 2, 8, 9, 15, and 23) be used instead of the previously used Roman numerals. Furthermore, we discuss the usefulness of genotyping ASFV isolates based only on the p72 gene sequence.

Generation and characterization of a monoclonal antibody against an African swine fever virus protein encoded by the A137R gene.

The ongoing African swine fever (ASF) pandemic continues to have a major impact on global pork production and trade. Since ASF cannot be distinguished from other swine hemorrhagic fevers clinically, ASF-specific laboratory diagnosis is critical. Thus ASF virus (ASFV)-specific monoclonal antibodies (mAbs) are critical for the development of laboratory diagnostics. In this study, we report one ASFV-specific mAb, F88ASF-55, that was generated and characterized. This mAb recognizes the ASFV A137R-encoded protein (pA137R). Epitope mapping results revealed a highly conserved linear epitope recognized by this mAb, corresponding to amino acids 111-125 of pA137R. We explored the potential use of this mAb in diagnostic applications. Using F88ASF-55 as the detection antibody, six ASFV strains were detected in an enzyme-linked immunosorbent assay (ELISA) with low background. In immunohistochemistry (IHC) assays, this mAb specifically recognized ASFV antigens in the submandibular lymph nodes of animals experimentally infected with different ASFV strains. Although not all ASFV genotypes were tested in this study, based on the conserved ASFV epitope targeted by F88ASF-55, it has the potential to detect multiple ASFV genotypes. In conclusion, this newly generated ASFV pA137R-specific mAb has potential value in ASF diagnostic tool development. It can be used in ELISA, IHC, and possibly-immunochromatographic strip assays for ASFV detection. It also suggests that pA137R may be a good target for diagnostic assays to detect ASFV infection.

The 2022 Outbreaks of African Swine Fever Virus Demonstrate the First Report of Genotype II in Ghana.

African swine fever (ASF) is a lethal disease of domestic pigs that has been causing outbreaks for over a century in Africa ever since its first discovery in 1921. Since 1957, there have been sporadic outbreaks outside of Africa; however, no outbreak has been as devastating and as far-reaching as the current pandemic that originated from a 2007 outbreak in the Republic of Georgia. Derivatives with a high degree of similarity to the progenitor strain, ASFV-Georgia/2007, have been sequenced from various countries in Europe and Asia. However, the current strains circulating in Africa are largely unknown, and 24 different genotypes have been implicated in different outbreaks. In this study, ASF isolates were collected from samples from swine suspected of dying from ASF on farms in Ghana in early 2022. While previous studies determined that the circulating strains in Ghana were p72 Genotype I, we demonstrate here that the strains circulating in 2022 were derivatives of the p72 Genotype II pandemic strain. Therefore, this study demonstrates for the first time the emergence of Genotype II ASFV in Ghana.

Abundance and diversity of Culicoides Latreille (Diptera: Ceratopogonidae) in southern Ontario, Canada.

BACKGROUND: Culicoides Latreille (Diptera: Ceratopogonidae) is a genus of hematophagous midges feeding on various vertebrate hosts and serving as a vector for numerous pathogens important to livestock and wildlife health. North American pathogens include bluetongue (BT) and epizootic hemorrhagic disease (EHD) viruses. Little is known about Culicoides spp. distribution and abundance and species composition in Ontario, Canada, despite bordering numerous U.S. states with documented Culicoides spp. and BT and EHD virus activity. We sought to characterize Culicoides spp. distribution and abundance and to investigate whether select meteorological and ecological risk factors influenced the abundance of Culicoides biguttatus, C. stellifer, and the subgenus Avaritia trapped throughout southern Ontario. METHODS: From June to October of 2017 to 2018, CDC-type LED light suction traps were placed on twelve livestock-associated sites across southern Ontario. Culicoides spp. collected were morphologically identified to the species level when possible. Associations were examined using negative binomial regression among C. biguttatus, C. stellifer, and subgenus Avaritia abundance, and select factors: ambient temperature, rainfall, primary livestock species, latitude, and habitat type. RESULTS: In total, 33,905 Culicoides spp. midges were collected, encompassing 14 species from seven subgenera and one species group. Culicoides sonorensis was collected from three sites during both years. Within Ontario, the northern trapping locations had a pattern of seasonal peak abundance in August (2017) and July (2018), and the southern locations had abundance peaks in June for both years. Culicoides biguttatus, C. stellifer, and subgenus Avaritia were significantly more abundant if ovine was the primary livestock species at trapping sites (compared to bovine). Culicoides stellifer and subgenus Avaritia were significantly more abundant at mid- to high-temperature ranges on trap days (i.e., 17.3-20.2 and 20.3-31.0 °C compared to 9.5-17.2 °C). Additionally, subgenus Avaritia were significantly more abundant if rainfall 4 weeks prior was between 2.7 and 20.1 mm compared to 0.0 mm and if rainfall 8 weeks prior was between 0.1 and 2.1 mm compared to 0.0 mm. CONCLUSIONS: Results from our study describe Culicoides spp. distribution in southern Ontario, the potential for spread and maintenance of EHD and BT viruses, and concurrent health risks to livestock and wildlife in southern Ontario in reference to certain meteorological and ecological risk factors. We identified that Culicoides spp. are diverse in this province, and appear to be distinctly distributed spatially and temporally. The livestock species present, temperature, and rainfall appear to have an impact on the abundance of C. biguttatus, C. stellifer, and subgenus Avaritia trapped. These findings could help inform targeted surveillance, control measures, and the development of management guides for Culicoides spp. and EHD and BT viruses in southern Ontario, Canada.

Characterization of a Novel African Swine Fever Virus p72 Genotype II from Nigeria.

African swine fever (ASF) is a high-consequence transboundary hemorrhagic fever of swine. It continues to spread across the globe causing socio-economic issues and threatening food security and biodiversity. In 2020, Nigeria reported a major ASF outbreak, killing close to half a million pigs. Based on the partial sequences of the genes B646L (p72) and E183L (p54), the virus responsible for the outbreak was identified as an African swine fever virus (ASFV) p72 genotype II. Here, we report further characterization of ASFV RV502, one of the isolates obtained during the outbreak. The whole genome sequence of this virus revealed a deletion of 6535 bp between the nucleotide positions 11,760-18,295 of the genome, and an apparent reverse complement duplication of the 5' end of the genome at the 3' end. Phylogenetically, ASFV RV502 clustered together with ASFV MAL/19/Karonga and ASFV Tanzania/Rukwa/2017/1 suggesting that the virus responsible for the 2020 outbreak in Nigeria has a South-eastern African origin.

Pathological Characteristics of Domestic Pigs Orally Infected with the Virus Strain Causing the First Reported African Swine Fever Outbreaks in Vietnam.

African swine fever (ASF) is currently Vietnam's most economically significant swine disease. The first ASF outbreak in Vietnam was reported in February 2019. In this study, VNUA/HY/ASF1 strain isolated from the first ASF outbreak was used to infect 10 eight-week-old pigs orally with 103 HAD50 per animal. The pigs were observed daily for clinical signs, and whole blood samples were collected from each animal for viremia detection. Dead pigs were subjected to full post-mortem analyses. All 10 pigs displayed acute or subacute clinical signs and succumbed to the infection between 10 to 27 (19.8 ± 4.66) days post-inoculation (dpi). The onset of clinical signs started around 4-14 dpi. Viremia was observed in pigs from 6-16 dpi (11.2 ± 3.55). Enlarged, hyperemic, and hemorrhagic lymph nodes, enlarged spleen, pneumonia, and hydropericardium were observed at post-mortem examinations.

Emergence of a novel intergenic region (IGR) IV variant of african swine fever virus genotype II in domestic pigs in Vietnam.

African swine fever virus (ASFV) causes African swine fever (ASF), a deadly disease affecting both domestic pigs and wild boars. ASF has become endemic in Vietnam since its first appearance in early 2019. Our previous molecular surveillance studies revealed that all the ASFV strains circulating in Vietnam belong to p72 genotype II, p54 genotype II, CD2v serogroup 8, and CVR of B602L gene variant type I. However, the genetic analysis based on the tandem repeat sequences located between I73R and I329L genes revealed three different intergenic region (IGR) variants; I, II, and III. In this study, using ASFV field isolates collected from September 24th to December 27th, 2021, we report, for the first time, novel IGR IV variants circulating in the Vietnamese pig population.

Cell-Free Dot Blot: an Ultra-Low-Cost and Practical Immunoassay Platform for Detection of Anti-SARS-CoV-2 Antibodies in Human and Animal Sera.

Since its emergence in late 2019, the coronavirus disease 2019 (COVID-19) pandemic has caused severe disruption to key aspects of human life globally and highlighted the need for timely, adaptive, and accessible pandemic response strategies. Here, we introduce the cell-free dot blot (CFDB) method, a practical and ultra-low-cost immune diagnostic platform capable of rapid response and mass immunity screening for the current and future pandemics. Similar in mechanism to the widely used enzyme-linked immunosorbent assays (ELISAs), our method is novel and advantageous in that (i) it uses linear DNA to produce the target viral antigen fused to a SpyTag peptide in a cell-free expression system without the need for traditional cloning and antigen purification, (ii) it uses SpyCatcher2-Apex2, an Escherichia coli-produced peroxidase conjugate as a universal secondary detection reagent, obviating the need for commercial or sophisticated enzyme conjugates, and (iii) sera are spotted directly on a nitrocellulose membrane, enabling a simple "dipping" mechanism for downstream incubation and washing steps, as opposed to individual processing of wells in a multiwell plate. To demonstrate the utility of our method, we performed CFDB to detect anti-severe acute respiratory syndrome coronavirus 2 nucleocapsid protein antibodies in precharacterized human sera (23 negative and 36 positive for COVID-19) and hamster sera (16 negative and 36 positive for COVID-19), including independent testing at a collaborating laboratory, and we show assay performance comparable to that of conventional ELISAs. At a similar capacity to 96-well plate ELISA kits, one CFDB assay costs only ~$3 USD. We believe that CFDB can become a valuable pandemic response tool for adaptive and accessible sero-surveillance in human and animal populations. IMPORTANCE The recent COVID-19 pandemic has highlighted the need for diagnostic platforms that are rapidly adaptable, affordable, and accessible globally, especially for low-resource settings. To address this need, we describe the development and functional validation of a novel immunoassay technique termed the cell-free dot blot (CFDB) method. Based on the principles of cell-free synthetic biology and alternative dot blotting procedures, our CFDB immunoassay is designed to provide for timely, practical, and low-cost responses to existing and emerging public health threats, such as the COVID-19 pandemic, at a similar throughput and comparable performance as conventional ELISAs. Notably, the molecular detection reagents used in CFDB can be produced rapidly in-house, using established protocols and basic laboratory infrastructure, minimizing reliance on strained commercial reagents. In addition, the materials and imaging instruments required for CFDB are the same as those used for common Western blotting experiments, further expanding the reach of CFDB in decentralized facilities.

Reclassification of ASFV into 7 Biotypes Using Unsupervised Machine Learning.

In 2007, an outbreak of African swine fever (ASF), a deadly disease of domestic swine and wild boar caused by the African swine fever virus (ASFV), occurred in Georgia and has since spread globally. Historically, ASFV was classified into 25 different genotypes. However, a newly proposed system recategorized all ASFV isolates into 6 genotypes exclusively using the predicted protein sequences of p72. However, ASFV has a large genome that encodes between 150-200 genes, and classifications using a single gene are insufficient and misleading, as strains encoding an identical p72 often have significant mutations in other areas of the genome. We present here a new classification of ASFV based on comparisons performed considering the entire encoded proteome. A curated database consisting of the protein sequences predicted to be encoded by 220 reannotated ASFV genomes was analyzed for similarity between homologous protein sequences. Weights were applied to the protein identity matrices and averaged to generate a genome-genome identity matrix that was then analyzed by an unsupervised machine learning algorithm, DBSCAN, to separate the genomes into distinct clusters. We conclude that all available ASFV genomes can be classified into 7 distinct biotypes.

Estimation of basic reproduction number (R0) of African swine fever (ASF) in mid-size commercial pig farms in Vietnam.

African swine fever (ASF) is a devastating disease affecting the global swine industry. Recently, it has spread to many countries in Africa, Europe, Asia, and the Caribbean, leaving severe damage to local, regional, national, and global economies. Due to its highly complex molecular characteristics and pathogenesis, the development of a successful vaccine has been an unmet challenge. Therefore, ASF control relies solely on biosecurity, rapid detection, and elimination. Epidemiological information obtained from natural ASF outbreaks is critical for designing and implementing ASF control measures. Basic reproduction number (R0), an epidemiological metric used to describe the contagiousness or transmissibility of infectious agents, is an important epidemiological tool. In this study, we have calculated R0 for the in-farm spread of ASF among fattening pigs and sows in two midsize commercial pig farms, HY1 and HY2, that practice the spot removal approach in controlling ASF outbreaks in Vietnam. The R0 values for the sows and fattening pigs were 1.78 (1.35-2.35) and 4.76 (4.18-5.38) for HY1 and 1.55 (1.08-2.18) and 3.8 (3.33-4.28) for HY2. This is the first study to evaluate the transmission potential of ASF in midsize commercial pig farms in Vietnam. Based on the R0 values, we predict that the spot removal approach could be used to successfully control ASF outbreaks in midsize commercial sow barns but not in fattening pens.

Mitochondrial genome sequencing, mapping, and assembly benchmarking for Culicoides species (Diptera: Ceratopogonidae).

BACKGROUND: Mitochondrial genomes are the most sequenced genomes after bacterial and fungal genomic DNA. However, little information on mitogenomes is available for multiple metazoan taxa, such as Culicoides, a globally distributed, megadiverse genus containing 1,347 species. AIM:  Generating novel mitogenomic information from single Culicoides sonorensis and C. biguttatus specimens, comparing available mitogenome mapping and de novo assembly tools, and identifying the best performing strategy and tools for Culicoides species. RESULTS: We present two novel and fully annotated mitochondrial haplotypes for two Culicoides species, C. sonorensis and C. biguttatus. We also annotated or re-annotated the only available reference mitogenome for C. sonorensis and C. arakawae. All species present a high similarity in mitogenome organization. The general gene arrangement for all Culicoides species was identical to the ancestral insect mitochondrial genome. Only short spacers were found in C. sonorensis (up to 30 bp), contrary to C. biguttatus (up to 114 bp). The mitochondrial genes ATP8, NAD2, NAD6, and LSU rRNA exhibited the highest nucleotide diversity and pairwise interspecific p genetic distance, suggesting that these genes might be suitable and complementary molecular barcodes for Culicoides identification in addition to the commonly utilized COI gene. We observed performance differences between the compared mitogenome generation strategies. The mapping strategy outperformed the de novo assembly strategy, but mapping results were partially biased in the absence of species-specific reference mitogenome. Among the utilized tools, BWA performed best for C. sonorensis while SPAdes, MEGAHIT, and MitoZ were among the best for C. biguttatus. The best-performing mitogenome annotator was MITOS2. Additionally, we were able to recover exogenous mitochondrial DNA from Bos taurus (biting midges host) from a C. biguttatus blood meal sample. CONCLUSIONS: Two novel annotated mitogenome haplotypes for C. sonorensis and C. biguttatus using High-Throughput Sequencing are presented. Current results are useful as the baseline for mitogenome reconstruction of the remaining Culicoides species from single specimens to HTS and genome annotation. Mapping to a species-specific reference mitogenome generated better results for Culicoides mitochondrial genome reconstruction than de novo assembly, while de novo assembly resulted better in the absence of a closely related reference mitogenome. These results have direct implications for molecular-based identification of these vectors of human and zoonotic diseases, setting the basis for using the whole mitochondrial genome as a marker in Culicoides identification.

Molecular and histopathological characterization of lumpy skin disease in cattle in northern Vietnam during the 2020-2021 outbreaks.

Lumpy skin disease (LSD) is a serious emerging infectious disease in cattle caused by a virus of the family Poxviridae. According to the Department of Animal Health, LSD first occurred in Vietnam at the end of October 2020 in Cao Bang and Lang Son provinces. So far, the disease has infected over 63,000 animals, resulting in 9170 deaths occurring in 32 different provinces in northern and central Vietnam. In this study, skin samples from lumpy skin disease virus (LSDV)-infected cattle from the northern provinces of Vietnam displaying clinical symptoms including fever (> 40 °C), runny nose, drooling, and skin lesions were used for genetic characterization and histopathology. Genetic analysis of the partial P32 (LSDV074), partial F (LSDV117), complete RPO30 (LSDV035), and complete G-protein-coupled-chemokine-like receptor (GPCR) (LSDV011) genes showed that all Vietnamese LSDV strains belonged to the genus Capripoxvirus and were closely related to LSDV strains isolated in China. Microscopic examination of the skin lesions showed thickening of the epidermal layer of the skin and hair follicles, hyperplasia of sebaceous glands, intracytoplasmic inclusion bodies, and hemorrhages in the mesoderm.

Evaluation of a Lateral Flow Assay for Rapid Detection of African Swine Fever Virus in Multiple Sample Types.

Antibody-based lateral flow assay (LFA) is a quick and inexpensive tool used to detect pathogens in field samples, especially in hard-to-reach remote areas that may have limited access to central laboratories during an outbreak or surveillance. In this study, we investigated the ability of a commercially available LFA, PenCheck®, to detect African swine fever virus (ASFV) in clinical samples derived from pigs infected with highly virulent ASFV strains. The assay was specific and positively identified the majority of pigs showing high fever during the early stages (between 3 and 5 days) of infection. PenCheck® LFA also detected ASFV in serum and tissue samples collected from pigs that succumbed to experimental ASFV infection and whole blood, plasma, and tissue samples from the field. The limit of detection of the assay was ASFV titer 107.80 TCID50/mL, corresponding to ASFV real-time PCR values below 23 Ct. Although the sensitivity of the assay is less than that of the laboratory-based real-time PCR assays, the results obtained with the PenCheck® LFA in this study suggest that it can be used as a herd-level, field-deployable, and easy-to-use diagnostic tool to identify ASF-affected farms when access to portable molecular assays or central laboratories is not possible.

Multiple variants of African swine fever virus circulating in Vietnam.

African swine fever (ASF) is a contagious and deadly viral disease affecting swine of all ages. ASF was first reported in Vietnam in February 2019, and it is now considered endemic in Vietnam. In this study, 122 ASF-positive samples collected from domestic pigs in 28 different provinces of northern, central, and southern Vietnam during outbreaks in 2019-2021 were genetically characterized. The findings confirmed that all ASF virus (ASFV) strains circulating in Vietnam belonged to p72 genotype II, p54 genotype II, CD2v serogroup 8, and CVR gene variant type I. However, further analysis based on the tandem repeat sequences located between I73R and I329L genes revealed that there were three different variants of ASFV, IGR I, II, and III, circulating in the domestic pig population in Vietnam. The IGR II variants were the most prevalent (117/122 strains) and were detected in pigs in all of the provinces tested, followed by IGR III (4/122 strains) and IGR I (1/122 strains). This study confirms for the first time the presence of IGR III variants in Vietnam.

Superficial Inguinal Lymph Nodes for Screening Dead Pigs for African Swine Fever.

African swine fever (ASF) has spread across the globe and has reached closer to North America since being reported in the Dominican Republic and Haiti. As a result, surveillance measures have been heightened and the utility of alternative samples for herd-level monitoring and dead pig sampling have been investigated. Passive surveillance based on the investigation of dead pigs, both domestic and wild, plays a pivotal role in the early detection of an ASF incursion. The World Organization for Animal Health (OIE)-recommended samples for dead pigs are spleen, lymph nodes, bone marrow, lung, tonsil and kidney. However, obtaining these samples requires opening up the carcasses, which is time-consuming, requires skilled labour and often leads to contamination of the premises. As a result, we investigated the suitability of superficial inguinal lymph nodes (SILNs) for surveillance of dead animals. SILNs can be collected in minutes with no to minimum environmental contamination. Here, we demonstrate that the ASF virus (ASFV) genome copy numbers in SILNs highly correlate with those in the spleen and, by sampling SILN, we can detect all pigs that succumb to highly virulent and moderately virulent ASFV strains (100% sensitivity). ASFV was isolated from all positive SILN samples. Thus, sampling SILNs could be useful for routine surveillance of dead pigs on commercial and backyard farms, holding pens and dead on arrival at slaughter houses, as well as during massive die-offs of pigs due to unknown causes.

Development of a Novel Loop Mediated Isothermal Amplification Assay (LAMP) for the Rapid Detection of Epizootic Haemorrhagic Disease Virus.

Epizootic haemorragic disease (EHD) is an important disease of white-tailed deer and can cause a bluetongue-like illness in cattle. A definitive diagnosis of EHD relies on molecular assays such as real-time RT-qPCR or conventional PCR. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a cost-effective, specific, and sensitive technique that provides an alternative to RT-qPCR. We designed two sets of specific primers targeting segment-9 of the EHD virus genome to enable the detection of western and eastern topotypes, and evaluated their performance in singleplex and multiplex formats using cell culture isolates (n = 43), field specimens (n = 20), and a proficiency panel (n = 10). The limit of detection of the eastern and western RT-LAMP assays was estimated as ~24.36 CT and as ~29.37 CT in relation to real-time RT-qPCR, respectively, indicating a greater sensitivity of the western topotype singleplex RT-LAMP. The sensitivity of the western topotype RT-LAMP assay, relative to the RT-qPCR assay, was 72.2%, indicating that it could be theoretically used to detect viraemic cervines and bovines. For the first time, an RT-LAMP assay was developed for the rapid detection of the EHD virus that could be used as either a field test or high throughput screening tool in established laboratories to control the spread of EHD.