Exposure to 17α-Ethinylestradiol Results in Differential Susceptibility of Largemouth Bass (Micropterus salmoides) to Bacterial Infection.

Disease outbreaks, skin lesions, mortality events, and reproductive abnormalities have been observed in wild populations of centrarchids. The presence of estrogenic endocrine disrupting compounds (EEDCs) has been implicated as a potential causal factor for these effects. The effects of prior EEDC exposure on immune response were examined in juvenile largemouth bass (Micropterus salmoides) exposed to a potent synthetic estrogen (17α-ethinylestradiol, EE2) at a low (EE2Low, 0.87 ng/L) or high (EE2High, 9.08 ng/L) dose for 4 weeks, followed by transfer to clean water and injection with an LD40 dose of the Gram-negative bacteria Edwardsiella piscicida. Unexpectedly, this prior exposure to EE2High significantly increased survivorship at 10 d post-infection compared to solvent control or EE2Low-exposed, infected fish. Both prior exposure and infection with E. piscicida led to significantly reduced hepatic glycogen levels, indicating a stress response resulting in depletion of energy stores. Additionally, pathway analysis for liver and spleen indicated differentially expressed genes associated with immunometabolic processes in the mock-injected EE2High treatment that could underlie the observed protective effect and metabolic shift in EE2High-infected fish. Our results demonstrate that exposure to a model EEDC alters metabolism and immune function in a fish species that is ecologically and economically important in North America.

The Probiotic Bacterium Phaeobacter inhibens Downregulates Virulence Factor Transcription in the Shellfish Pathogen Vibrio coralliilyticus by N-Acyl Homoserine Lactone Production.

Phaeobacter inhibens S4Sm acts as a probiotic bacterium against the oyster pathogen Vibrio coralliilyticus Here, we report that P. inhibens S4Sm secretes three molecules that downregulate the transcription of major virulence factors, metalloprotease genes, in V. coralliilyticus cultures. The effects of the S4Sm culture supernatant on the transcription of three genes involved in protease activity, namely, vcpA, vcpB, and vcpR (encoding metalloproteases A and B and their transcriptional regulator, respectively), were examined by reverse transcriptase quantitative PCR (qRT-PCR). The expression of vcpB and vcpR were reduced to 36% and 6.6%, respectively, compared to that in an untreated control. We constructed a V. coralliilyticus green fluorescent protein (GFP) reporter strain to detect the activity of inhibitory compounds. Using a bioassay-guided approach, the molecules responsible for V. coralliilyticus protease inhibition activity were isolated from S4Sm supernatant and identified as three N-acyl homoserine lactones (AHLs). The three AHLs are N-(3-hydroxydecanoyl)-l-homoserine lactone, N-(dodecanoyl-2,5-diene)-l-homoserine lactone, and N-(3-hydroxytetradecanoyl-7-ene)-l-homoserine lactone, and their half maximal inhibitory concentrations (IC50s) against V. coralliilyticus protease activity were 0.26 µM, 3.7 µM, and 2.9 µM, respectively. Our qRT-PCR data demonstrated that exposures to the individual AHLs reduced the transcription of vcpR and vcpB Combinations of the three AHLs (any two or all three AHLs) on V. coralliilyticus produced additive effects on protease inhibition activity. These AHL compounds may contribute to the host protective effects of S4Sm by disrupting the quorum sensing pathway that activates protease transcription of V. coralliilyticusIMPORTANCE Probiotics represent a promising alternative strategy to control infection and disease caused by marine pathogens of aquaculturally important species. Generally, the beneficial effects of probiotics include improved water quality, control of pathogenic bacteria and their virulence, stimulation of the immune system, and improved animal growth. Previously, we isolated a probiotic bacterium, Phaeobacter inhibens S4Sm, which protects oyster larvae from Vibrio coralliilyticus RE22Sm infection. We also demonstrated that both antibiotic secretion and biofilm formation play important roles in S4Sm probiotic activity. Here, we report that P. inhibens S4Sm, an alphaproteobacterium and member of the Roseobacter clade, also secretes secondary metabolites that hijack the quorum sensing ability of V. coralliilyticus RE22Sm, suppressing virulence gene expression. This finding demonstrates that probiotic bacteria can exert their host protection by using a multipronged array of behaviors that limit the ability of pathogens to become established and cause infection.

Isocitrate dehydrogenase mutation in Vibrio anguillarum results in virulence attenuation and immunoprotection in rainbow trout (Oncorhynchus mykiss).

BACKGROUND: Vibrio anguillarum is an extracellular bacterial pathogen that is a causative agent of vibriosis in finfish and crustaceans with mortality rates ranging from 30% to 100%. Mutations in central metabolism (glycolysis and the TCA cycle) of intracellular pathogens often result in attenuated virulence due to depletion of required metabolic intermediates; however, it was not known whether mutations in central metabolism would affect virulence in an extracellular pathogen such as V. anguillarum. RESULTS: Seven central metabolism mutants were created and characterized with regard to growth in minimal and complex media, expression of virulence genes, and virulence in juvenile rainbow trout (Oncorhynchus mykiss). Only the isocitrate dehydrogenase (icd) mutant was attenuated in virulence against rainbow trout challenged by either intraperitoneal injection or immersion. Further, the icd mutant was shown to be immunoprotective against wild type V. anguillarum infection. There was no significant decrease in the expression of the three hemolysin genes detected by qRT-PCR. Additionally, only the icd mutant exhibited a significantly decreased growth yield in complex media. Growth yield was directly related to the abundance of glutamate. A strain with a restored wild type icd gene was created and shown to restore growth to a wild type cell density in complex media and pathogenicity in rainbow trout. CONCLUSIONS: The data strongly suggest that a decreased growth yield, resulting from the inability to synthesize α-ketoglutarate, caused the attenuation despite normal levels of expression of virulence genes. Therefore, the ability of an extracellular pathogen to cause disease is dependent upon the availability of host-supplied nutrients for growth. Additionally, a live vaccine strain could be created from an icd deletion strain.

Reclassification of the larval pathogen for marine bivalves Vibrio tubiashii subsp. europaeus as Vibrio europaeus sp. nov.

The Orientalis clade has a relevant significance for bivalve aquaculture since it includes the pathogens Vibrio bivalvicida, Vibrio tubiashii subsp. tubiashii and Vibrio tubiashii subsp. europaeus. However, the previous taxonomic description of the subspecies of V. tubiashii shows some incongruities that should be emended. In the genomic age, the comparison between genome assemblies is the key to clarify the taxonomic position of both subspecies. With this purpose, we have tested the ability of multilocus sequence analysis based on eight housekeeping gene sequences (gapA, gyrB, ftsZ, mreB, pyrH, recA, rpoA and topA), different in silico genome-to-genome comparisons, chemotaxonomic features and phenotypic traits to reclassify the subspecies V. tubiashii subsp. europaeus within the Orientalis clade. This polyphasic approach clearly demonstrated that this subspecies is phylogenetically and phenotypically distinct from V. tubiashii and should be elevated to the rank of species as Vibrio europaeus sp. nov. This reclassification allows us to update the Orientalis clade (V. bivalvicida,V. brasiliensis, V. crosai, V. hepatarius, V. orientalis, V. sinaloensis, V. tubiashii and V. europaeus sp. nov.) and reconstruct a better phylogeny of the genus Vibrio. An emended description of V. tubiashii is provided. Finally, the proposed novel species is represented by emergent bivalve pathogens [type strain PP-638T (=CECT 8136T=DSM 27349T), PP2-843 and 07/118 T2] responsible for high mortalities in Spanish and French hatcheries.

Following the infection process of vibriosis in Manila clam (Ruditapes philippinarum) larvae through GFP-tagged pathogenic Vibrio species.

Vibriosis represents the main bottleneck for the larval production process in shellfish aquaculture. While the signs of this disease in bivalve larvae are well known, the infection process by pathogenic Vibrio spp. during episodes of vibriosis has not been elucidated. To investigate the infection process in bivalves, the pathogens of larvae as V. tubiashii subsp. europaensis, V. neptunius and V. bivalvicida were tagged with green fluorescent protein (GFP). Larvae of Manila clam (Ruditapes philippinarum) were inoculated with the GFP-labeled pathogens in different infection assays and monitored by microscopy. Manila clam larvae infected by distinct GFP-tagged Vibrio spp. in different challenges showed the same progression in the infection process, defining three infection stages. GFP-tagged Vibrio spp. were filtered by the larvae through the vellum and entered in the digestive system through the esophagus and stomach and colonized the digestive gland and particularly the intestine, where they proliferated during the first 2h of contact (Stage I), suggesting a chemotactic response. Then, GFP-tagged Vibrio spp. expanded rapidly to the surrounding organs in the body cavity from the dorsal to ventral region (Stage II; 6-8h), colonizing the larvae completely at the peak of infection (Stage III) (14-24h). Results demonstrated for the first time that the vibriosis is asymptomatic in Manila clam larvae during the early infection stages. Thus, the early colonization and the rapid proliferation of Vibrio pathogens within the body cavity supported the sudden and fatal effect of the vibriosis, since the larvae exhibited the first signs of disease when the infection process is advanced. As a first step in the elucidation of the potential mechanisms of bacterial pathogenesis in bivalve larvae the enzymatic activities of the extracellular products released from the wild type V. neptunius, V. tubiashii subsp. europaensis and V. bivalvicida were determined and their cytotoxicity was demonstrated in fish and homeothermic cell lines for the first time. That activity was lost after heat treatment.

Analysis of the Unique Historical Isolate of African Swine Fever Virus Isolate Spencer from Outbreaks in 1951.

African swine fever (ASF) is a deadly hemorrhagic disease of domestic and wild swine that was first described in the early 20th century after the introduction of European pigs to Kenya. The etiological agent, the African swine fever virus (ASFV), is a large DNA virus within the Asfarviridae family that is broadly categorized epidemiologically into genotypes based on the nucleotide sequence of B646L, the gene encoding the major capsid protein p72. ASF outbreaks in Africa have been linked historically to 25 genotypes by p72 nucleotide analysis and, recently, to 6 genotypes by amino acid comparison, whereas global outbreaks of ASF outside of Africa have only been linked to 2 genotypes: genotype I, which led to an outbreak in Europe during the 1960s that later spread to South America, and genotype II, responsible for the current pandemic that began in Georgia in 2007 and has since spread to Europe, Asia, and Hispaniola. Here, we present an analysis of the genome of ASFV Spencer, an isolate that was collected in 1951 near Johannesburg, South Africa. While nucleotide analysis of Spencer indicates the p72 coding sequence is unique, differentiating from the closest reference by five nucleotides, the predicted amino acid sequence indicates that it is 100% homologous to contemporary genotype 1. Full genome analysis reveals it is more similar to Mkuzi1979 and encodes genes that share similarity with either genotype 1 or genotype 2 outbreak strains.

African swine fever virus P72 genotyping tool.

Historically, genotyping of African swine fever virus was based on partial sequencing of B646L (p72). Until recently, the number of differences that defined genotypes was ambiguous. This tool allows a sequence to be uploaded and will report its closest matches along with its likely p72 genotype.

African Swine Fever Virus Protein-Protein Interaction Prediction.

The African swine fever virus (ASFV) is an often deadly disease in swine and poses a threat to swine livestock and swine producers. With its complex genome containing more than 150 coding regions, developing effective vaccines for this virus remains a challenge due to a lack of basic knowledge about viral protein function and protein-protein interactions between viral proteins and between viral and host proteins. In this work, we identified ASFV-ASFV protein-protein interactions (PPIs) using artificial intelligence-powered protein structure prediction tools. We benchmarked our PPI identification workflow on the Vaccinia virus, a widely studied nucleocytoplasmic large DNA virus, and found that it could identify gold-standard PPIs that have been validated in vitro in a genome-wide computational screening. We applied this workflow to more than 18,000 pairwise combinations of ASFV proteins and were able to identify seventeen novel PPIs, many of which have corroborating experimental or bioinformatic evidence for their protein-protein interactions, further validating their relevance. Two protein-protein interactions, I267L and I8L, I267L__I8L, and B175L and DP79L, B175L__DP79L, are novel PPIs involving viral proteins known to modulate host immune response.

Full-Length ASFV B646L Gene Sequencing by Nanopore Offers a Simple and Rapid Approach for Identifying ASFV Genotypes.

African swine fever (ASF) is an acute, highly hemorrhagic viral disease in domestic pigs and wild boars. The disease is caused by African swine fever virus, a double stranded DNA virus of the Asfarviridae family. ASF can be classified into 25 different genotypes, based on a 478 bp fragment corresponding to the C-terminal sequence of the B646L gene, which is highly conserved among strains and encodes the major capsid protein p72. The C-terminal end of p72 has been used as a PCR target for quick diagnosis of ASF, and its characterization remains the first approach for epidemiological tracking and identification of the origin of ASF in outbreak investigations. Recently, a new classification of ASF, based on the complete sequence of p72, reduced the 25 genotypes into only six genotypes; therefore, it is necessary to have the capability to sequence the full-length B646L gene (p72) in a rapid manner for quick genotype characterization. Here, we evaluate the use of an amplicon approach targeting the whole B646L gene, coupled with nanopore sequencing in a multiplex format using Flongle flow cells, as an easy, low cost, and rapid method for the characterization and genotyping of ASF in real-time.

Recombinant Vaccine Strain ASFV-G-Δ9GL/ΔUK Produced in the IPKM Cell Line Is Genetically Stable and Efficacious in Inducing Protection in Pigs Challenged with the Virulent African Swine Fever Virus Field Isolate Georgia 2010.

We have previously reported that the recombinant African Swine Fever (ASF) vaccine candidate ASFV-G-Δ9GL/ΔUK efficiently induces protection in domestic pigs challenged with the virulent strain Georgia 2010 (ASFV-G). As reported, ASFV-G-Δ9GL/ΔUK induces protection, while intramuscularly (IM), administered at doses of 104 HAD50 or higher, prevents ASF clinical disease in animals infected with the homologous ASFV g strain. Like other recombinant vaccine candidates obtained from ASFV field isolates, ASFV-G-Δ9GL/ΔUK stocks need to be produced in primary cultures of swine macrophages, which constitutes an important limitation in the production of large virus stocks at the industrial level. Here, we describe the development of ASFV-G-Δ9GL/ΔUK stocks using IPKM (Immortalized Porcine Kidney Macrophage) cells, which are derived from swine macrophages. We show that ten successive passages of ASFV-G-Δ9GL/ΔUK in IPKM cells induced small changes in the virus genome. The produced virus, ASFV-G-Δ9GL/ΔUKp10, presented a similar level of replication in swine macrophages cultures to that of the original ASFV-G-Δ9GL/ΔUK (ASFV-G-Δ9GL/ΔUKp0). The protective efficacy of ASFV-G-Δ9GL/ΔUKp10 was evaluated in pigs that were IM-inoculated with either 104 or 106 HAD50 of ASFV-G-Δ9GL/ΔUKp10. While animals inoculated with 104 HAD50 present a partial protection against the experimental infection with the virulent parental virus ASFV-G, those inoculated with 106 HAD50 were completely protected. Therefore, as was just recently reported for another ASF vaccine candidate, ASFV-G-ΔI177L, IPKM cells are an effective alternative to produce stocks for vaccine strains which only grow in swine macrophages.

Near-complete genome sequences of multiple genotype 1 African swine fever virus isolates from 2016 to 2018 in Cameroon.

African swine fever virus has been endemic in Cameroon since 1982. Here, we announce the sequences of Cameroon/2016/C1, Cameroon/2016/C5, Cameroon/2017/C-A2, Cameroon/2018/C02, and Cameroon/2018/CF3, five genotype 1 African swine fever virus genomes collected from domestic pigs between 2016 and 2018.

Deletion of the EP402R Gene from the Genome of African Swine Fever Vaccine Strain ASFV-G-∆I177L Provides the Potential Capability of Differentiating between Infected and Vaccinated Animals.

The African swine fever virus (ASFV) mutant ASFV-G-∆I177L is a safe and efficacious vaccine which induces protection against the challenge of its parental virus, the Georgia 2010 isolate. Although a genetic DIVA (differentiation between infected and vaccinated animals) assay has been developed for this vaccine, still there is not a serological DIVA test for differentiating between animals vaccinated with ASFV-G-∆I177L and those infected with wild-type viruses. In this report, we describe the development of the ASFV-G-∆I177L mutant having deleted the EP402R gene, which encodes for the viral protein responsible for mediating the hemadsorption of swine erythrocytes. The resulting virus, ASFV-G-∆I177L/∆EP402R, does not have a decreased ability to replicates in swine macrophages when compared with the parental ASFV-G-∆I177L. Domestic pigs intramuscularly (IM) inoculated with either 102 or 106 HAD50 of ASFV-G-∆I177L/∆EP402R remained clinically normal, when compared with a group of mock-vaccinated animals, indicating the absence of residual virulence. Interestingly, an infectious virus could not be detected in the blood samples of the ASFV-G-∆I177L/∆EP402R-inoculated animals in either group at any of the time points tested. Furthermore, while all of the mock-inoculated animals presented a quick and lethal clinical form of ASF after the intramuscular inoculation challenge with 102 HAD50 of highly virulent parental field isolate Georgia 2010 (ASFV-G), all of the ASFV-G-∆I177L/∆EP402R-inoculated animals were protected, remaining clinically normal until the end of the observational period. Most of the ASFV-G-∆I177L/∆EP402R-inoculated pigs developed strong virus-specific antibody responses against viral antigens, reaching maximum levels at 28 days post inoculation. Importantly, all of the sera collected at that time point in the ASFV-G-∆I177L/∆EP402R-inoculated pigs did not react in a direct ELISA coated with the recombinant EP402R protein. Conversely, the EP402R protein was readily recognized by the pool of sera from the animals immunized with recombinant live attenuated vaccine candidates ASFV-G-∆I177L, ASFV-G-∆MGF, or ASFV-G-∆9GL/∆UK. Therefore, ASFV-G-∆I177L/∆EP402R is a novel, safe and efficacious candidate with potential to be used as an antigenically DIVA vaccine.

A Retrospective Analysis Reveals That the 2021 Outbreaks of African Swine Fever Virus in Ghana Were Caused by Two Distinct Genotypes.

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF), a highly infectious and lethal disease of domesticated swine. Outbreaks of ASF have been mostly restricted to the continent of Africa. The outbreaks that have occurred outside of Africa were controlled by extensive depopulation of the domesticated pig population. However, in 2007, an outbreak occurred in the country of Georgia, where ASFV infected wild pigs and quickly spread across eastern Europe. Since the reintroduction of ASF into Europe, variants of the current pandemic strain, ASFV Georgia 2007/01 (ASFV-G), which is classified as Genotype 2 based on p72 sequencing, have been reported in countries within western Europe, Asia, and the island of Hispaniola. Additionally, isolates collected in 2020 confirmed the presence of variants of ASFV-G in Nigeria. Recently, we reported similar variants of ASFV-G collected from domestic pigs suspected of dying of ASF in Ghana in 2022. Here, we retroactively report, based on full-length sequencing, that similar variants were present in Ghana in 2021. The SNP analysis revealed derivatives of ASFV with distinct genetic markers. Furthermore, we identified three full-length ASFV genomes as Genotype 1, indicating that there were two genotypes circulating in proximity during the 2021 ASF outbreaks in Ghana.

A Standardized Pipeline for Assembly and Annotation of African Swine Fever Virus Genome.

Obtaining a complete good-quality sequence and annotation for the long double-stranded DNA genome of the African swine fever virus (ASFV) from next-generation sequencing (NGS) technology has proven difficult, despite the increasing availability of reference genome sequences and the increasing affordability of NGS. A gap analysis conducted by the global African swine fever research alliance (GARA) partners identified that a standardized, automatic pipeline for NGS analysis was urgently needed, particularly for new outbreak strains. Whilst there are several diagnostic and research labs worldwide that collect isolates of the ASFV from outbreaks, many do not have the capability to analyze, annotate, and format NGS data from outbreaks for submission to NCBI, and some publicly available ASFV genomes have missing or incorrect annotations. We developed an automated, standardized pipeline for the analysis of NGS reads that directly provides users with assemblies and annotations formatted for their submission to NCBI. This pipeline is freely available on GitHub and has been tested through the GARA partners by examining two previously sequenced ASFV genomes; this study also aimed to assess the accuracy and limitations of two strategies present within the pipeline: reference-based (Illumina reads) and de novo assembly (Illumina and Nanopore reads) strategies.

H-NS is a negative regulator of the two hemolysin/cytotoxin gene clusters in Vibrio anguillarum.

Hemolysins produced by Vibrio anguillarum have been implicated in the development of hemorrhagic septicemia during vibriosis, a fatal fish disease. Previously, two hemolysin gene clusters responsible for the hemolysis and cytotoxicity of V. anguillarum were identified: the vah1-plp gene cluster and the rtxACHBDE gene cluster. In this study, we identified the hns gene, which encodes the H-NS protein and acts as a negative regulator of both gene clusters. The V. anguillarum H-NS protein shares strong homology with other bacterial H-NS proteins. An hns mutant exhibited increased hemolytic activity and cytotoxicity compared to the wild-type strain. Complementation of the hns mutation restored hemolytic activity and cytotoxicity levels to nearly wild-type levels. Furthermore, expression of rtxA, rtxH, rtxB, vah1, and plp increased in the hns mutant and decreased in the hns-complemented mutant strain compared to expression in the wild-type strain. Additionally, experiments using DNase I showed that purified recombinant H-NS protected multiple sites in the promoter regions of both gene clusters. The hns mutant also exhibited significantly attenuated virulence against rainbow trout. Complementation of the hns mutation restored virulence to wild-type levels, suggesting that H-NS regulates many genes that affect fitness and virulence. Previously, we showed that HlyU is a positive regulator of expression for both gene clusters. In this study, we demonstrate that upregulation by hlyU is hns dependent, suggesting that H-NS acts to repress or silence both gene clusters and HlyU acts to relieve that repression or silencing.

Full genome sequence for the African swine fever virus outbreak in the Dominican Republic in 1980.

African swine fever is a lethal disease of domestic pigs, geographically expanding as a pandemic, that is affecting countries across Eurasia and severely damaging their swine production industry. After more than 40 years of being absent in the Western hemisphere, in 2020 ASF reappeared in the Dominican Republic and Haiti. The recent outbreak strain in the Dominican Republic has been identified as a genotype II ASFV a derivative of the ASF strain circulating in Asia and Europe. However, to date no full-length genome sequence from either the 1978-1980 Here we report the complete genome sequence of an African swine fever virus (ASFV) (DR-1980) that was previously isolated from blood collected in 1980 from the Dominican Republic at the end of the last outbreak, before culling of all swine on the island of Hispaniola and stored in the Plum Island Animal Disease Center ASFV repository. A contig representing the full-length genome (183,687 base pairs) was de novo assembled into a single contig using both Nanopore and Illumina sequences. DR-1980 was determined to belong to genotype I and, as determined by full genome comparison, a close relative to the sequenced Sardinia viruses that were causing outbreaks at this time.

Draft Genome Sequence of Vibrio parahaemolyticus PSU5579, Isolated during an Outbreak of Acute Hepatopancreatic Necrosis Disease in Thailand.

Here, we announce the draft genome sequence of Vibrio parahaemolyticus strain PSU5579, isolated from a shrimp hatchery in southern Thailand during an outbreak of acute hepatopancreatic necrosis disease (AHPND). The genome contains 44 contigs with a sequence length of 5,229,426 bp, 4,861 coding sequences, and a G+C content of 45.3%.

Evaluation of the Function of ASFV Gene E66L in the Process of Virus Replication and Virulence in Swine.

African swine fever virus (ASFV) is the etiological agent of an economically important disease of swine currently affecting large areas of Africa, Eurasia and the Caribbean. ASFV has a complex structure harboring a large dsDNA genome which encodes for more than 160 proteins. One of the proteins, E66L, has recently been involved in arresting gene transcription in the infected host cell. Here, we investigate the role of E66L in the processes of virus replication in swine macrophages and disease production in domestic swine. A recombinant ASFV was developed (ASFV-G-∆E66L), from the virulent parental Georgia 2010 isolate (ASFV-G), harboring the deletion of the E66L gene as a tool to assess the role of the gene. ASFV-G-∆E66L showed that the E66L gene is non-essential for ASFV replication in primary swine macrophages when compared with the parental highly virulent field isolate ASFV-G. Additionally, domestic pigs infected with ASFV-G-∆E66L developed a clinical disease undistinguishable from that produced by ASFV-G. Therefore, E66L is not involved in virus replication or virulence in domestic pigs.

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.

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.