Molecular Characterization of African Swine Fever Viruses from Outbreaks in Peri-Urban Kampala, Uganda

African swine fever (ASF) is an infectious transboundary disease of domestic pigs and wild swine and is currently the most serious constraint to piggery in Uganda. The causative agent of ASF is a large double-stranded linear DNA virus with a complex structure. There are twenty-four ASFV genotypes described to date; however, in Uganda, only genotypes IX and X have been previously described. Inadequate ASF outbreak investigation has contributed to the delayed establishment of effective interventions to aid the control of ASF. Continuous virus characterization enhances the understanding of ASF epidemiology in terms of viral genome variations, extent, severity, and the potential source of the viruses responsible for outbreaks. We collected samples from pigs that had died of a hemorrhagic disease indicative of ASF. DNA was extracted from all samples and screened with the OIE recommended diagnostic PCR for ASF. Partial B646L (p72), full-length E183L (p54) genes, and CVR region of the P72 gene were amplified, purified, and sequenced. Web-based BLAST and MEGA X software were used for sequence analysis. ASF was confirmed in 10 of the 15 suspected pig samples. Phylogenetic analysis confirmed circulation of genotype IX by both full-length E183 (p54) and partial B646L (p72) gene sequencing. Intragenotypic resolution of the CVR region revealed major deletions in the virus genome, in some isolates of this study. The marked reduction in the number of tetrameric tandem repeats in some isolates of this study could potentially play a role in influencing the virulence of this particular genotype IX in Uganda.

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Identification of Promiscuous African Swine Fever Virus T-Cell Determinants Using a Multiple Technical Approach

Vaccines ◽

10.3390/vaccines9010029 ◽

2021 ◽

Vol 9 (1) ◽

pp. 29

Author(s):

Laia Bosch-Camós ◽

Elisabet López ◽

María Jesús Navas ◽

Sonia Pina-Pedrero ◽

Francesc Accensi ◽

...

Keyword(s):

T Cell ◽

Mononuclear Cells ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Cd8 T Cell ◽

Protective Role ◽

Full Length ◽

T Cell Epitopes ◽

Peripheral Blood Mononuclear

The development of subunit vaccines against African swine fever (ASF) is mainly hindered by the lack of knowledge regarding the specific ASF virus (ASFV) antigens involved in protection. As a good example, the identity of ASFV-specific CD8+ T-cell determinants remains largely unknown, despite their protective role being established a long time ago. Aiming to identify them, we implemented the IFNγ ELISpot as readout assay, using as effector cells peripheral blood mononuclear cells (PBMCs) from pigs surviving experimental challenge with Georgia2007/1. As stimuli for the ELISpot, ASFV-specific peptides or full-length proteins identified by three complementary strategies were used. In silico prediction of specific CD8+ T-cell epitopes allowed identifying a 19-mer peptide from MGF100-1L, as frequently recognized by surviving pigs. Complementarily, the repertoire of SLA I-bound peptides identified in ASFV-infected porcine alveolar macrophages (PAMs), allowed the characterization of five additional SLA I-restricted ASFV-specific epitopes. Finally, in vitro stimulation studies using fibroblasts transfected with plasmids encoding full-length ASFV proteins, led to the identification of MGF505-7R, A238L and MGF100-1L as promiscuously recognized antigens. Interestingly, each one of these proteins contain individual peptides recognized by surviving pigs. Identification of the same ASFV determinants by means of such different approaches reinforce the results presented here.

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Computational Analysis of African Swine Fever Virus Protein Space for the Design of an Epitope-Based Vaccine Ensemble

Pathogens ◽

10.3390/pathogens9121078 ◽

2020 ◽

Vol 9 (12) ◽

pp. 1078 ◽

Cited By ~ 1

Author(s):

Albert Ros-Lucas ◽

Florencia Correa-Fiz ◽

Laia Bosch-Camós ◽

Fernando Rodriguez ◽

Julio Alonso-Padilla

Keyword(s):

T Cell ◽

Vaccine Development ◽

De Novo ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Economic Losses ◽

Virus Protein ◽

Hemorrhagic Disease ◽

Starting Point

African swine fever virus is the etiological agent of African swine fever, a transmissible severe hemorrhagic disease that affects pigs, causing massive economic losses. There is neither a treatment nor a vaccine available, and the only method to control its spread is by extensive culling of pigs. So far, classical vaccine development approaches have not yielded sufficiently good results in terms of concomitant safety and efficacy. Nowadays, thanks to advances in genomic and proteomic techniques, a reverse vaccinology strategy can be explored to design alternative vaccine formulations. In this study, ASFV protein sequences were analyzed using an in-house pipeline based on publicly available immunoinformatic tools to identify epitopes of interest for a prospective vaccine ensemble. These included experimentally validated sequences from the Immune Epitope Database, as well as de novo predicted sequences. Experimentally validated and predicted epitopes were prioritized following a series of criteria that included evolutionary conservation, presence in the virulent and currently circulating variant Georgia 2007/1, and lack of identity to either the pig proteome or putative proteins from pig gut microbiota. Following this strategy, 29 B-cell, 14 CD4+ T-cell and 6 CD8+ T-cell epitopes were selected, which represent a starting point to investigating the protective capacity of ASFV epitope-based vaccines.

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African Swine Fever Laboratory Diagnosis—Lessons Learned from Recent Animal Trials

Pathogens ◽

10.3390/pathogens10020177 ◽

2021 ◽

Vol 10 (2) ◽

pp. 177

Author(s):

Jutta Pikalo ◽

Paul Deutschmann ◽

Melina Fischer ◽

Hanna Roszyk ◽

Martin Beer ◽

...

Keyword(s):

African Swine Fever Virus ◽

Clinical Signs ◽

African Swine Fever ◽

Laboratory Diagnosis ◽

Lessons Learned ◽

Hemorrhagic Disease ◽

Passive Surveillance ◽

Animal Trials ◽

Fit For Purpose ◽

The Impact

African swine fever virus (ASFV) causes a hemorrhagic disease in pigs with high socio-economic consequences. To lower the impact of disease incursions, early detection is crucial. In the context of experimental animal trials, we evaluated diagnostic workflows for a high sample throughput in active surveillance, alternative sample matrices for passive surveillance, and lateral flow devices (LFD) for rapid testing. We could demonstrate that EDTA blood is significantly better suited for early ASFV detection than serum. Tissues recommended by the respective diagnostic manuals were in general comparable in their performance, with spleen samples giving best results. Superficial lymph nodes, ear punches, and different blood swabs were also evaluated as potential alternatives. In summary, all matrices yielded positive results at the peak of clinical signs and could be fit for purpose in passive surveillance. However, weaknesses were discovered for some matrices when it comes to the early phase of infection or recovery. The antigen LFD showed variable results with best performance in the clinical phase. The antibody LFD was quite comparable with ELISA systems. Concluding, alternative approaches are feasible but have to be embedded in control strategies selecting test methods and sample materials following a “fit-for-purpose” approach.

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Recombinant cDNA Probe from Bluetongue Virus Genome Segment 10 for Identification of Bluetongue Virus

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063878900100308 ◽

1989 ◽

Vol 1 (3) ◽

pp. 237-241 ◽

Cited By ~ 9

Author(s):

Carlos Alberto de Mattos ◽

Cecilia Cristina de Mattos ◽

Bennie Irve Osburn

Keyword(s):

Bluetongue Virus ◽

Cdna Probe ◽

Prototype Strain ◽

Virus Genome ◽

Genome Segment ◽

Hemorrhagic Disease ◽

Cross Hybridization ◽

Double Stranded Rna ◽

Field Isolates ◽

Serotype 1

Genome segment 10 of bluetongue virus (BTV) serotype 11 UC8 strain was cloned and subsequently hybridized to viral double-stranded RNA extracted from 90 field isolates of BTV serotypes 10, 11, 13, and 17; the prototype strains of BTV 2, 10, 11, 13, and 17; the prototype strain epizootic hemorrhagic disease virus (EHDV) serotype 1; and 4 field isolates of EHDV serotype 2. The 90 field isolates were obtained from different counties in California, Louisiana, and Idaho during the years 1979, 1980, and 1981. The cloned genetic probe hybridized with all the BTV samples tested, showing different degrees of cross-hybridization at the stringency conditions used in this study. This indicated that BTV genome segment 10 has conserved nucleotide sequences among the BTV serotypes 2, 10, 11, 13, and 17. No cross-hybridization signals were detected between the cloned genome segment 10 of BTV 11 UC8 strain and the prototype strain of EHDV serotype 1 and the field isolates of serotype 2. This probe recognized a wide variety of BTV isolates.

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Genome Size Estimation and Full-Length Transcriptome of Sphingonotus tsinlingensis: Genetic Background of a Drought-Adapted Grasshopper

Frontiers in Genetics ◽

10.3389/fgene.2021.678625 ◽

2021 ◽

Vol 12 ◽

Author(s):

Lu Zhao ◽

Hang Wang ◽

Ping Li ◽

Kuo Sun ◽

De-Long Guan ◽

...

Keyword(s):

Genome Size ◽

Genetic Background ◽

Tandem Repeats ◽

Full Length ◽

Arid Environments ◽

Size Estimation ◽

Sequencing Data ◽

Protein Coding ◽

Grasshopper Species ◽

Hsp Genes

Sphingonotus Fieber, 1852 (Orthoptera: Acrididae), is a grasshopper genus comprising approximately 170 species, all of which prefer dry environments such as deserts, steppes, and stony benchlands. In this study, we aimed to examine the adaptation of grasshopper species to arid environments. The genome size of Sphingonotus tsinlingensis was estimated using flow cytometry, and the first high-quality full-length transcriptome of this species was produced. The genome size of S. tsinlingensis is approximately 12.8 Gb. Based on 146.98 Gb of PacBio sequencing data, 221.47 Mb full-length transcripts were assembled. Among these, 88,693 non-redundant isoforms were identified with an N50 value of 2,726 bp, which was markedly longer than previous grasshopper transcriptome assemblies. In total, 48,502 protein-coding sequences were identified, and 37,569 were annotated using public gene function databases. Moreover, 36,488 simple tandem repeats, 12,765 long non-coding RNAs, and 414 transcription factors were identified. According to gene functions, 61 cytochrome P450 (CYP450) and 66 heat shock protein (HSP) genes, which may be associated with drought adaptation of S. tsinlingensis, were identified. We compared the transcriptomes of S. tsinlingensis and two other grasshopper species which were less tolerant to drought, namely Mongolotettix japonicus and Gomphocerus licenti. We observed the expression of CYP450 and HSP genes in S. tsinlingensis were higher. We produced the first full-length transcriptome of a Sphingonotus species that has an ultra-large genome. The assembly characteristics were better than those of all known grasshopper transcriptomes. This full-length transcriptome may thus be used to understand the genetic background and evolution of grasshoppers.

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Two Novel Multigene Families, 530 and 300, in the Terminal Variable Regions of African Swine Fever Virus Genome

Virology ◽

10.1006/viro.1994.1426 ◽

1994 ◽

Vol 202 (2) ◽

pp. 997-1002 ◽

Cited By ~ 27

Author(s):

T. Yozawa ◽

G.F. Kutish ◽

C.L. Afonso ◽

Z. Lu ◽

D.L. Rock

Keyword(s):

African Swine Fever Virus ◽

African Swine Fever ◽

Virus Genome ◽

Fever Virus ◽

Multigene Families ◽

Variable Regions

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Characterization of the African swine fever virus protein p49: a new late structural polypeptide

Microbiology ◽

10.1099/0022-1317-81-1-59 ◽

2000 ◽

Vol 81 (1) ◽

pp. 59-65 ◽

Cited By ~ 8

Author(s):

Inmaculada Galindo ◽

Eladio Viñuela ◽

Angel L. Carrascosa

Keyword(s):

Cell Attachment ◽

African Swine Fever Virus ◽

Rabbit Antiserum ◽

African Swine Fever ◽

Virus Genome ◽

Fever Virus ◽

Virus Protein ◽

Significant Similarity ◽

Reading Frame ◽

Cell Extracts

The open reading frame B438L, located within the EcoRI B fragment of the African swine fever virus genome, is predicted to encode a protein of 438 amino acids with a molecular mass of 49·3 kDa. It presents a cell attachment RGD (Arg–Gly–Asp) motif but no other significant similarity to protein sequences in databases. Northern blot and primer extension analysis showed that B438L is transcribed only at late times during virus infection. The B438L gene product has been expressed in Escherichia coli, purified and used as an antigen for antibody production. The rabbit antiserum specific for pB438L recognized a protein of about 49 kDa in virus-infected cell extracts. This protein was synthesized late in infection by all the virus strains tested, was located in cytoplasmic virus factories and appeared as a structural component of purified virus particles.

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Bos grunniens papillomavirus type 1: a novel deltapapillomavirus associated with fibropapilloma in yak

Journal of General Virology ◽

10.1099/vir.0.046086-0 ◽

2013 ◽

Vol 94 (1) ◽

pp. 159-165 ◽

Cited By ~ 9

Author(s):

Wei Zhu ◽

Jian-Bao Dong ◽

Jun Zhang ◽

Kazuyuki Uchida ◽

Ken-ichi Watanabe ◽

...

Keyword(s):

Virus Genome ◽

Diagnostic Methods ◽

Diagnostic Pcr ◽

Bos Grunniens ◽

Nucleotide Sequence Alignment ◽

The Family ◽

Pcr Products ◽

Full Length Genome ◽

First Time

Papillomaviruses (PVs) have been widely identified among vertebrates, but have not yet been reported to infect yaks. We report, for the first time, a novel deltapapillomavirus that was associated with fibropapilloma in yak herds on the Qinghai–Tibetan Plateau. Six skin papilloma samples were collected and examined using histopathology, immunohistochemistry and PCR assays. The samples were identified as fibropapilloma and were found to contain PV antigens. Sequencing of diagnostic PCR products and the full-length genome revealed that all samples were infected with the same PV type. The whole virus genome was 7946 bp in length and possessed the common PV genomic organization. The virus was identified as a novel PV type and designated Bos grunniens papillomavirus type 1 (BgPV-1) based on the nucleotide sequence alignment of the L1 ORF. It is classified in the Delta-4 species of the genus Deltapapillomavirus based on phylogenetic analysis of the L1 ORF. Identification of this novel PV type provides further information about the pathology, development of diagnostic methods and evolutionary studies of the family Papillomaviridae.

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