scholarly journals Deletion of the K145R and DP148R Genes from the Virulent ASFV Georgia 2007/1 Isolate Delays the Onset, but Does Not Reduce Severity, of Clinical Signs in Infected Pigs

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1473
Author(s):  
Anusyah Rathakrishnan ◽  
Ana L. Reis ◽  
Lynnette C. Goatley ◽  
Katy Moffat ◽  
Linda K. Dixon

African swine fever virus causes a frequently fatal disease of domestic pigs and wild boar that has a high economic impact across 3 continents. The large double-stranded DNA genome codes for approximately 160 proteins. Many of these have unknown functions and this hinders our understanding of the virus and host interactions. The purpose of the study was to evaluate the role of two virus proteins, K145R and DP148R, in virus replication in macrophages and virulence in pigs. To do this, the DP148R gene, alone or in combination with the K145R gene, was deleted from the virulent genotype II Georgia 2007/1 isolate. Neither of these deletions reduced the ability of the viruses to replicate in porcine macrophages compared to the parental wild-type virus. Pigs infected with GeorgiaΔDP148R developed clinical and post-mortem signs and high viremia, typical of acute African swine fever, and were culled on day 6 post-infection. The additional deletion of the K145R gene delayed the onset of clinical signs and viremia in pigs by 3 days, but pigs showed signs of acute African swine fever and were culled on days 10 or 13 post-infection. The results show that the deletion of DP148R did not attenuate the genotype II Georgia 2007/1 isolate, contrary to the results obtained with the genotype I Benin97/1 isolate. Additional deletion of the K145R gene delayed clinical signs, but infected pigs reached the humane endpoint. The deletion of additional genes would be required to attenuate the virus.

Viruses ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1474
Author(s):  
Elisabeth Lopez ◽  
Juanita van Heerden ◽  
Laia Bosch-Camós ◽  
Francesc Accensi ◽  
Maria Jesus Navas ◽  
...  

African swine fever (ASF) has become the major threat for the global swine industry. Furthermore, the epidemiological situation of African swine fever virus (ASFV) in some endemic regions of Sub-Saharan Africa is worse than ever, with multiple virus strains and genotypes currently circulating in a given area. Despite the recent advances on ASF vaccine development, there are no commercial vaccines yet, and most of the promising vaccine prototypes available today have been specifically designed to fight the genotype II strains currently circulating in Europe, Asia, and Oceania. Previous results from our laboratory have demonstrated the ability of BA71∆CD2, a recombinant LAV lacking CD2v, to confer protection against homologous (BA71) and heterologous genotype I (E75) and genotype II (Georgia2007/01) ASFV strains, both belonging to same clade (clade C). Here, we extend these results using BA71∆CD2 as a tool trying to understand ASFV cross-protection, using phylogenetically distant ASFV strains. We first observed that five out of six (83.3%) of the pigs immunized once with 106 PFU of BA71∆CD2 survived the tick-bite challenge using Ornithodoros sp. soft ticks naturally infected with RSA/11/2017 strain (genotype XIX, clade D). Second, only two out of six (33.3%) survived the challenge with Ken06.Bus (genotype IX, clade A), which is phylogenetically more distant to BA71∆CD2 than the RSA/11/2017 strain. On the other hand, homologous prime-boosting with BA71∆CD2 only improved the survival rate to 50% after Ken06.Bus challenge, all suffering mild ASF-compatible clinical signs, while 100% of the pigs immunized with BA71∆CD2 and boosted with the parental BA71 virulent strain survived the lethal challenge with Ken06.Bus, without almost no clinical signs of the disease. Our results confirm that cross-protection is a multifactorial phenomenon that not only depends on sequence similarity. We believe that understanding this complex phenomenon will be useful for designing future vaccines for ASF-endemic areas.


2021 ◽  
Author(s):  
Vlad Petrovan ◽  
Anusyah Rathakrishnan ◽  
Muneeb Islam ◽  
Lynnette C. Goatley ◽  
Katy Moffat ◽  
...  

The limited knowledge on the role of many of the approximately 170 proteins encoded by African swine fever virus restricts progress towards vaccine development. Previously, the DP148R gene was deleted from the genome of genotype I virulent Benin 97/1 isolate. This virus, BeninΔDP148R, induced transient moderate clinical signs after immunization and high levels of protection against challenge. However, the BeninΔDP148R virus and genome persisted in blood over a prolonged period. In the current study deletion of either EP402R or EP153R genes individually or in combination from BeninΔDP148R genome was shown not to reduce virus replication in macrophages in vitro. However, deletion of EP402R dramatically reduced the period of infectious virus persistence in blood in immunized pigs from 28 to 14 days and virus genome from 59 to 14 days, whilst maintaining high levels of protection against challenge. The additional deletion of EP153R (BeninΔDP148RΔEP153RΔEP402R) further attenuated the virus and no viremia or clinical signs were observed post-immunization. This was associated with decreased protection and detection of moderate levels of challenge virus in blood. Interestingly, the deletion of EP153R alone from BeninΔDP148R did not result in further virus attenuation and did not reduce the period of virus persistence in blood. These results show that EP402R and EP153R have a synergistic role in reducing clinical signs and levels of virus in blood. Importance: African swine fever virus (ASFV) causes a disease of domestic pigs and wild boar which results in death of almost all infected animals. The disease has a high economic impact, and no vaccine is available. We investigated the role of two ASFV proteins, called EP402R and EP153R, in determining the levels and length of time virus persists in blood from infected pigs. EP402R causes ASFV particles and infected cells to bind to red blood cells. Deletion of the EP402R gene dramatically reduced virus persistence in blood but did not reduce the level of virus. Deletion of the EP153R alone did not reduce the period or level of virus persistence in blood. However, deleting both EP153R and EP402R resulted in undetectable levels of virus in blood and no clinical signs showing the proteins act synergistically. Importantly the infected pigs were protected following infection with the wildtype virus that kills pigs.


2021 ◽  
Author(s):  
Vlad Petrovan ◽  
Anusyah Rathakrishnan ◽  
Muneeb Islam ◽  
Lynnette Goatley ◽  
Katy Moffat ◽  
...  

The limited knowledge on the role of many of the approximately 170 proteins encoded by African swine fever virus restricts progress towards vaccine development. In this study we investigated the effect of deleting combinations of different genes from a previously attenuated virus, BeninΔDP148R on: virus replication in macrophages, virus persistence and clinical signs post immunization, and induction of protection against challenge. Deletion of either EP402R or EP153R genes individually or in combination from BeninΔDP148R did not reduce virus replication in vitro. However, deletion of EP402R dramatically reduced viral persistence in vivo, whilst maintaining high levels of protection against challenge. The additional deletion of EP153R (BeninΔDP148RΔEP153RΔEP402R) further attenuated the virus and no viremia or clinical signs were observed post immunization. This was associated with decreased protection and detection of moderate levels of challenge virus in blood. Interestingly, the deletion of EP153R alone from BeninΔDP148R did not result in further virus attenuation and a slight increase in virus genome copies in blood was observed at different times post immunization when compared with BeninΔDP148R. These results show that EP402R and EP153R have a synergistic role in promoting viremia, however EP153R alone does not seem to have a major impact on virus levels in blood.


2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Rémi Pereira De Oliveira ◽  
Evelyne Hutet ◽  
Renaud Lancelot ◽  
Frédéric Paboeuf ◽  
Maxime Duhayon ◽  
...  

Abstract Background Several species of soft ticks in genus Ornithodoros are known vectors and reservoirs of African swine fever virus (ASFV). However, the underlying mechanisms of vector competence for ASFV across Ornithodoros species remain to be fully understood. To that end, this study compared ASFV replication and dissemination as well as virus vertical transmission to descendants between Ornithodorosmoubata, O. erraticus, and O. verrucosus in relation to what is known about the ability of these soft tick species to transmit ASFV to pigs. To mimic the natural situation, a more realistic model was used where soft ticks were exposed to ASFV by allowing them to engorge on viremic pigs. Methods Ornithodoros moubata ticks were infected with the ASFV strains Liv13/33 (genotype I) or Georgia2007/1 (genotype II), O. erraticus with OurT88/1 (genotype I) or Georgia2007/1 (genotype II), and O. verrucosus with Ukr12/Zapo (genotype II), resulting in five different tick–virus pairs. Quantitative PCR (qPCR) assays targeting the VP72 ASFV gene was carried out over several months on crushed ticks to study viral replication kinetics. Viral titration assays were also carried out on crushed ticks 2 months post infection to confirm virus survival in soft ticks. Ticks were dissected. and DNA was individually extracted from the following organs to study ASFV dissemination: intestine, salivary glands, and reproductive organs. DNA extracts from each organ were tested by qPCR. Lastly, larval or first nymph-stage progeny emerging from hatching eggs were tested by qPCR to assess ASFV vertical transmission. Results Comparative analyses revealed higher rates of ASFV replication and dissemination in O. moubata infected with Liv13/33, while the opposite was observed for O. erraticus infected with Georgia2007/1 and for O. verrucosus with Ukr12/Zapo. Intermediate profiles were found for O. moubata infected with Georgia2007/1 and for O. erraticus with OurT88/1. Vertical transmission occurred efficiently in O. moubata infected with Liv13/33, and at very low rates in O. erraticus infected with OurT88/1. Conclusions This study provides molecular data indicating that viral replication and dissemination in Ornithodoros ticks are major mechanisms underlying ASFV horizontal and vertical transmission. However, our results indicate that other determinants beyond viral replication also influence ASFV vector competence. Further research is required to fully understand this process in soft ticks.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Emma P. Njau ◽  
Jean-Baka Domelevo Entfellner ◽  
Eunice M. Machuka ◽  
Edwina N. Bochere ◽  
Sarah Cleaveland ◽  
...  

AbstractAfrican swine fever (ASF) caused by the African swine fever virus (ASFV) is ranked by OIE as the most important source of mortality in domestic pigs globally and is indigenous to African wild suids and soft ticks. Despite two ASFV genotypes causing economically devastating epidemics outside the continent since 1961, there have been no genome-level analyses of virus evolution in Africa. The virus was recently transported from south-eastern Africa to Georgia in 2007 and has subsequently spread to Russia, eastern Europe, China, and south-east Asia with devastating socioeconomic consequences. To date, two of the 24 currently described ASFV genotypes defined by sequencing of the p72 gene, namely genotype I and II, have been reported outside Africa, with genotype II being responsible for the ongoing pig pandemic. Multiple complete genotype II genome sequences have been reported from European, Russian and Chinese virus isolates but no complete genome sequences have yet been reported from Africa. We report herein the complete genome of a Tanzanian genotype II isolate, Tanzania/Rukwa/2017/1, collected in 2017 and determined using an Illumina short read strategy. The Tanzania/Rukwa/2017/1 sequence is 183,186 bp in length (in a single contig) and contains 188 open reading frames. Considering only un-gapped sites in the pairwise alignments, the new sequence has 99.961% identity with the updated Georgia 2007/1 reference isolate (FR682468.2), 99.960% identity with Polish isolate Pol16_29413_o23 (MG939586) and 99.957% identity with Chinese isolate ASFV-wbBS01 (MK645909.1). This represents 73 single nucleotide polymorphisms (SNPs) relative to the Polish isolate and 78 SNPs with the Chinese genome. Phylogenetic analysis indicated that Tanzania/Rukwa/2017/1 clusters most closely with Georgia 2007/1. The majority of the differences between Tanzania/Rukwa/2017/1 and Georgia 2007/1 genotype II genomes are insertions/deletions (indels) as is typical for ASFV. The indels included differences in the length and copy number of the terminal multicopy gene families, MGF 360 and 110. The Rukwa2017/1 sequence is the first complete genotype II genome from a precisely mapped locality in Africa, since the exact origin of Georgia2007/1 is unknown. It therefore provides baseline information for future analyses of the diversity and phylogeography of this globally important genetic sub-group of ASF viruses.


2020 ◽  
Author(s):  
Zhaozhong Zhu ◽  
Huiting Chen ◽  
Yang Cao ◽  
Taijiao Jiang ◽  
Yuanqiang Zou ◽  
...  

AbstractAfrican swine fever virus (ASFV) is a large and complex double-stranded DNA virus that poses serious threats to the pig industry. It is well-accepted that the multigene family (MGF) proteins are extensively distributed in ASFVs and are generally classified into five families, including MGF-100, MGF-110, MGF-300, MGF-360 and MGF-505. Most MGF proteins, however, have not been well characterized and classified within each family. To bridge this gap, this study first classified the MGF proteins into 35 groups based on protein sequence homology. A web server for classifying the MGF proteins was then established and available for free at http://www.computationalbiology.cn/MGF/home.html. Results showed that the genetic diversity of the MGF groups varied widely, mainly due to the occurrence of indels. In addition, the MGF proteins were predicted to have large structural and functional diversity, and the MGF proteins of the same MGF family tended to have similar structure, location and function. Evolutionary analysis revealed the dynamic changes of the MGF proteins in the ASFV genomes, and more than half of MGF groups were presented in all ASFV genomes, which indicated the important role of MGF proteins in ASFVs. Overall, it is expected that the work would not only provide a detailed classification for MGF proteins, but also facilitate further research on MGF proteins.


2021 ◽  
Vol 20 (03) ◽  
pp. 18-25
Author(s):  
Duy T. Do

African swine fever (ASF) is reported as a highly contagious hemorrhage lethal disease of domestic and wild swine. The causative agent of ASF is a large enveloped DNA virus with a complex structure. There are twenty-four ASFV genotypes described to date. However, in Vietnam, only genotypes II had been previously described. The genetic characterization of ASFV enhances the understanding of ASF epidemiology in terms of the extent, severity, source, and potential genetic variations among ASFV strains circulating in Southern Vietnam. Twenty ASFV strains were collected from pigs with ASFV-infected clinical signs from 10 provinces during 2019 - 2020. Partial B646L (p72) gene, complete E183L (p54), and CP204L (p30) genes were amplified, purified, and sequenced. Phylogenetic analysis confirmed the circulation of genotype II by both the partial B646L (p72) and full-length E183 (p54) gene sequencing. Analysis of the p72, p54, and p30 regions did not indicate any change in the nucleotide and amino acid sequences among these strains. The results of this study revealed that these ASFVs shared high homology to ASFV isolates detected in Northern Vietnam and China.


Pathogens ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 757
Author(s):  
Sandra Barroso-Arévalo ◽  
Jose A. Barasona ◽  
Estefanía Cadenas-Fernández ◽  
José M. Sánchez-Vizcaíno

African swine fever virus (ASFv) is one of the most challenging pathogens to affect both domestic and wild pigs. The disease has now spread to Europe and Asia, causing great damage to the pig industry. Although no commercial vaccine with which to control the disease is, as yet, available, some potential vaccine candidates have shown good results in terms of protection. However, little is known about the host immune mechanisms underlying that protection, especially in wild boar, which is the main reservoir of the disease in Europe. Here, we study the role played by two cytokines (IL-10 and IFN-γ) in wild boar orally inoculated with the attenuated vaccine candidate Lv17/WB/Rie1 and challenged with a virulent ASFv genotype II isolate. A group of naïve wild boar challenged with the latter isolate was also established as a control group. Our results showed that both cytokines play a key role in protecting the host against the challenge virus. While high levels of IL-10 in serum may trigger an immune system malfunctioning in challenged animals, the provision of stable levels of this cytokine over time may help to control the disease. This, together with high and timely induction of IFN-γ by the vaccine candidate, could help protect animals from fatal outcomes. Further studies should be conducted in order to support these preliminary results and confirm the role of these two cytokines as potential markers of the evolution of ASFV infection.


Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 177
Author(s):  
Jutta Pikalo ◽  
Paul Deutschmann ◽  
Melina Fischer ◽  
Hanna Roszyk ◽  
Martin Beer ◽  
...  

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.


2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Hu Suk Lee ◽  
Vuong Nghia Bui ◽  
Duy Tung Dao ◽  
Ngoc Anh Bui ◽  
Thanh Duy Le ◽  
...  

Abstract Background African swine fever (ASF), caused by the ASF virus (ASFV), was first reported in Vietnam in 2019 and spread rapidly thereafter. Better insights into ASFV characteristics and early detection by surveillance could help control its spread. However, the pathogenicity and methods for early detection of ASFV isolates from Vietnam have not been established. Therefore, we investigated the pathogenicity of ASFV and explored alternative sampling methods for early detection. Results Ten pigs were intramuscularly inoculated with an ASFV strain from Vietnam (titer, 103.5 HAD50/mL), and their temperature, clinical signs, and virus excretion patterns were recorded. In addition, herd and environmental samples were collected daily. The pigs died 5–8 days-post-inoculation (dpi), and the incubation period was 3.7 ± 0.5 dpi. ASFV genome was first detected in the blood (2.2 ± 0.8) and then in rectal (3.1 ± 0.7), nasal (3.2 ± 0.4), and oral (3.6 ± 0.7 dpi) swab samples. ASFV was detected in oral fluid samples collected using a chewed rope from 3 dpi. The liver showed the highest viral loads, and ear tissue also exhibited high viral loads among 11 tissues obtained from dead pigs. Overall, ASFV from Vietnam was classified as peracute to acute form. The rope-based oral fluid collection method could be useful for early ASFV detection and allows successful ASF surveillance in large pig farms. Furthermore, ear tissue samples might be a simple alternative specimen for diagnosing ASF infection in dead pigs. Conclusions Our data provide valuable insights into the characteristics of a typical ASFV strain isolated in Vietnam and suggest an alternative, non-invasive specimen collection strategy for early detection.


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