scholarly journals An Optimized Reverse Genetics System Suitable for Efficient Recovery of Simian, Human, and Murine-Like Rotaviruses

2020 ◽  
Vol 94 (18) ◽  
Author(s):  
Liliana Sánchez-Tacuba ◽  
Ningguo Feng ◽  
Nathan J. Meade ◽  
Kenneth H. Mellits ◽  
Philippe H. Jaïs ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
High Efficiency ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Molecular Study ◽  
Expression Vectors ◽  
Group A ◽  
Improved Design

ABSTRACT An entirely plasmid-based reverse genetics (RG) system was recently developed for rotavirus (RV), opening new avenues for in-depth molecular dissection of RV biology, immunology, and pathogenesis. Several improvements to further optimize the RG efficiency have now been described. However, only a small number of individual RV strains have been recovered to date. None of the current methods have supported the recovery of murine RV, impeding the study of RV replication and pathogenesis in an in vivo suckling mouse model. Here, we describe useful modifications to the RG system that significantly improve rescue efficiency of multiple RV strains. In addition to the 11 group A RV segment-specific (+)RNAs [(+)ssRNAs], a chimeric plasmid was transfected, from which the capping enzyme NP868R of African swine fever virus (ASFV) and the T7 RNA polymerase were expressed. Second, a genetically modified MA104 cell line was used in which several components of the innate immunity were degraded. Using this RG system, we successfully recovered the simian RV RRV strain, the human RV CDC-9 strain, a reassortant between murine RV D6/2 and simian RV SA11 strains, and several reassortants and reporter RVs. All these recombinant RVs were rescued at a high efficiency (≥80% success rate) and could not be reliably rescued using several recently published RG strategies (<20%). This improved system represents an important tool and great potential for the rescue of other hard-to-recover RV strains such as low-replicating attenuated vaccine candidates or low-cell culture passage clinical isolates from humans or animals. IMPORTANCE Group A rotavirus (RV) remains as the single most important cause of severe acute gastroenteritis among infants and young children worldwide. An entirely plasmid-based reverse genetics (RG) system was recently developed, opening new ways for in-depth molecular study of RV. Despite several improvements to further optimize the RG efficiency, it has been reported that current strategies do not enable the rescue of all cultivatable RV strains. Here, we described a helpful modification to the current strategies and established a tractable RG system for the rescue of the simian RRV strain, the human CDC-9 strain, and a murine-like RV strain, which is suitable for both in vitro and in vivo studies. This improved RV reverse genetics system will facilitate study of RV biology in both in vitro and in vivo systems that will facilitate the improved design of RV vaccines, better antiviral therapies, and expression vectors.

scholarly journals Deletion of a CD2-Like Gene, 8-DR, from African Swine Fever Virus Affects Viral Infection in Domestic Swine

1998 ◽  
Vol 72 (4) ◽  
pp. 2881-2889 ◽  
Author(s):  
M. V. Borca ◽  
C. Carrillo ◽  
L. Zsak ◽  
W. W. Laegreid ◽  
G. F. Kutish ◽  
...  
Keyword(s):  
Viral Infection ◽  
Mononuclear Cells ◽  
African Swine Fever Virus ◽  
Disease Onset ◽  
African Swine Fever ◽  
Fever Virus ◽  
Parental Virus ◽  
Viral Virulence

ABSTRACT An African swine fever virus (ASFV) gene with similarity to the T-lymphocyte surface antigen CD2 has been found in the pathogenic African isolate Malawi Lil-20/1 (open reading frame [ORF] 8-DR) and a cell culture-adapted European virus, BA71V (ORF EP402R) and has been shown to be responsible for the hemadsorption phenomenon observed for ASFV-infected cells. The structural and functional similarities of the ASFV gene product to CD2, a cellular protein involved in cell-cell adhesion and T-cell-mediated immune responses, suggested a possible role for this gene in tissue tropism and/or immune evasion in the swine host. In this study, we constructed an ASFV 8-DR gene deletion mutant (Δ8-DR) and its revertant (8-DR.R) from the Malawi Lil-20/1 isolate to examine gene function in vivo. In vitro, Δ8-DR, 8-DR.R, and the parental virus exhibited indistinguishable growth characteristics on primary porcine macrophage cell cultures. In vivo,8-DR had no obvious effect on viral virulence in domestic pigs; disease onset, disease course, and mortality were similar for the mutant Δ8-DR, its revertant 8-DR.R, and the parental virus. Altered viral infection was, however, observed for pigs infected with Δ8-DR. A delay in spread to and/or replication of Δ8-DR in the draining lymph node, a delay in generalization of infection, and a 100- to 1,000-fold reduction in virus titers in lymphoid tissue and bone marrow were observed. Onset of viremia for Δ8-DR-infected animals was significantly delayed (by 2 to 5 days), and mean viremia titers were reduced approximately 10,000-fold at 5 days postinfection and 30- to 100-fold at later times; moreover, unlike in 8-DR.R-infected animals, the viremia was no longer predominantly erythrocyte associated but rather was equally distributed among erythrocyte, leukocyte, and plasma fractions. Mitogen-dependent lymphocyte proliferation of swine peripheral blood mononuclear cells in vitro was reduced by 90 to 95% following infection with 8-DR.R but remained unaltered following infection with Δ8-DR, suggesting that 8-DR has immunosuppressive activity in vitro. Together, these results suggest an immunosuppressive role for 8-DR in the swine host which facilitates early events in viral infection. This may be of most significance for ASFV infection of its highly adapted natural host, the warthog.

scholarly journals Deletion of the Gene for the Type I Interferon Inhibitor I329L from the Attenuated African Swine Fever Virus OURT88/3 Strain Reduces Protection Induced in Pigs

Vaccines ◽  
2020 ◽  
Vol 8 (2) ◽  
pp. 262 ◽  
Author(s):  
Ana Luisa Reis ◽  
Lynnette C. Goatley ◽  
Tamara Jabbar ◽  
Elisabeth Lopez ◽  
Anusyah Rathakrishnan ◽  
...  
Keyword(s):  
Type I Interferon ◽  
African Swine Fever Virus ◽  
Clinical Signs ◽  
African Swine Fever ◽  
Type I ◽  
Lethal Challenge ◽  
Host Innate Immune Response ◽  
Antibody Levels

Live attenuated vaccines are considered to be the fastest route to the development of a safe and efficacious African swine fever (ASF) vaccine. Infection with the naturally attenuated OURT88/3 strain induces protection against challenge with virulent isolates from the same or closely related genotypes. However, adverse clinical signs following immunisation have been observed. Here, we attempted to increase the OURT88/3 safety profile by deleting I329L, a gene previously shown to inhibit the host innate immune response. The resulting virus, OURT88/3ΔI329L, was tested in vitro to evaluate the replication and expression of type I interferon (IFN) and in vivo by immunisation and lethal challenge experiments in pigs. No differences were observed regarding replication; however, increased amounts of both IFN-β and IFN-α were observed in macrophages infected with the deletion mutant virus. Unexpectedly, the deletion of I329L markedly reduced protection against challenge with the virulent OURT88/1 isolate. This was associated with a decrease in both antibody levels against VP72 and the number of IFN-γ-producing cells in the blood of non-protected animals. Furthermore, a significant increase in IL-10 levels in serum was observed in pigs immunised with OURT88/3ΔI329L following challenge. Interestingly, the deletion of the I329L gene failed to attenuate the virulent Georgia/2007 isolate.

scholarly journals Development and In Vivo Evaluation of a MGF110-1L Deletion Mutant in African Swine Fever Strain Georgia

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 286
Author(s):  
Elizabeth Ramirez-Medina ◽  
Elizabeth Vuono ◽  
Sarah Pruitt ◽  
Ayushi Rai ◽  
Ediane Silva ◽  
...  
Keyword(s):  
Deletion Mutant ◽  
African Swine Fever Virus ◽  
Field Isolate ◽  
African Swine Fever ◽  
Economic Losses ◽  
Swine Industry ◽  
In Vivo Evaluation ◽  
Viral Virulence

African swine fever (ASF) is currently causing an epizootic, affecting pigs throughout Eurasia, and causing significant economic losses in the swine industry. ASF is caused by African swine fever virus (ASFV) that consists of a large dsDNA genome that encodes for more than 160 genes; few of these genes have been studied in detail. ASFV contains four multi-gene family (MGF) groups of genes that have been implicated in regulating the immune response and host specificity; however, the individual roles of most of these genes have not been well studied. Here, we describe the evaluation of the previously uncharacterized ASFV MGF110-1L open reading frame (ORF) using a deletion mutant of the ASFV currently circulating throughout Eurasia. The recombinant ASFV lacking the MGF110-1L gene (ASFV-G-ΔMGF110-1L) demonstrated in vitro that the MGF110-1L gene is non-essential, since ASFV-G-ΔMGF110-1L had similar replication kinetics in primary swine macrophage cell cultures when compared to parental highly virulent field isolate Georgia2007 (ASFV-G). Experimental infection of domestic pigs with ASFV-G-ΔMGF110-1L produced a clinical disease similar to that caused by the parental ASFV-G, confirming that deletion of the MGF110-1L gene from the ASFV genome does not affect viral virulence.

scholarly journals Clinical Indicators of Moribundity in Swine Experimentally Inoculated with African Swine Fever Virus

2020 ◽  
Author(s):  
Benjamin J Hershey ◽  
Jenna L Hagart ◽  
Karyn A Havas
Keyword(s):  
Vaccine Development ◽  
African Swine Fever Virus ◽  
Significant Risk ◽  
African Swine Fever ◽  
Fever Virus ◽  
Complex Nature ◽  
Clinical Indicators ◽  
Record Data

African swine fever virus (ASFV), the causative agent of African Swine Fever (ASF), is an infectious disease of swine that is associated with high rates of morbidity and mortality in naive populations. ASFV is challenging to work with in vitro and the in vivo immune response remains an active area of study. Vaccine development, pathogenesis, and diagnostic assay development studies often require use of live swine housed in high-containment laboratories. Studies of this type are intended to obtain key data yet must minimize the pain and distress experienced by the animals. To implement humane endpoints, pigs are ideally euthanatized by barbiturate overdose prior to death from ASFV infection, as the final stages of ASF can be clinically severe. However, due to the complex nature of ASFV pathogenesis, predicting when an infected animal will become moribund and require euthanasia is difficult. The current study was intended to aid in predicting the onset of moribundity in swine. Toward this end, we performed statistical analyses of historical health record data from 103 swine experimentally infected with ASFV. Regression analysis suggested that rectal temperature has potential utility as a marker for predicting moribundity, whereas viral strain and duration of survival after inoculation were significant risk factors for death due to disease rather than euthanasia.

scholarly journals Generation of Recombinant Rotavirus Expressing NSP3-UnaG Fusion Protein by a Simplified Reverse Genetics System

2019 ◽  
Author(s):  
Asha A. Philip ◽  
Jacob L. Perry ◽  
Heather E. Eaton ◽  
Maya Shmulevitz ◽  
Joseph M. Hyser ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Rna Virus ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Genetically Engineered ◽  
Genome Segment ◽  
Fusion Product ◽  
Infected Cells

AbstractRotavirus is a segmented double-stranded (ds)RNA virus that causes severe gastroenteritis in young children. We have established an efficient simplified rotavirus reverse genetics (RG) system that uses eleven T7 plasmids, each expressing a unique simian SA11 (+)RNA, and a CMV support plasmid for the African swine fever virus NP868R capping enzyme. With the NP868R-based system, we generated recombinant rotavirus (rSA11/NSP3-FL-UnaG) with a genetically modified 1.5-kB segment 7 dsRNA that encodes full-length NSP3 fused to UnaG, a 139-aa green fluorescent protein (FP). Analysis of rSA11/NSP3-FL-UnaG showed that the virus replicated efficiently and was genetically stable over 10 rounds of serial passage. The NSP3-UnaG fusion product was well expressed in rSA11/NSP3-FL-UnaG-infected cells, reaching levels similar to NSP3 in wildtype rSA11-infected cells. Moreover, the NSP3-UnaG protein, like functional wildtype NSP3, formed dimersin vivo. Notably, NSP3-UnaG protein was readily detected in infected cells via live cell imaging, with intensity levels ~3-fold greater than that of the NSP1-UnaG fusion product of rSA11/NSP1-FL-UnaG. Our results indicate that FP-expressing recombinant rotaviruses can be made through manipulation of the segment 7 dsRNA without deleting or interrupting any of the twelve open reading frames of the virus. Because NSP3 is expressed at levels higher than NSP1 in infected cells, rotaviruses expressing NSP3-based FPs may be a more sensitive tool for studying rotavirus biology than rotaviruses expressing NSP1-based FPs. This is the first report of a recombinant rotavirus containing a genetically engineered segment 7 dsRNA.ImportancePrevious studies have generated recombinant rotaviruses that express fluorescent proteins (FPs) by inserting reporter genes into the NSP1 open reading frame (ORF) of genome segment 5. Unfortunately, NSP1 is expressed at low levels in infected cells, making viruses expressing FP-fused NSP1 less than ideal probes of rotavirus biology. Moreover, FPs were inserted into segment 5 in such a way as to compromise NSP1, an interferon antagonist affecting viral growth and pathogenesis. We have identified an alternative approach for generating rotaviruses expressing FPs, one relying on fusing the reporter gene to the NSP3 ORF of genome segment 7. This was accomplished without interrupting any of the viral ORFs, yielding recombinant viruses likely expressing the complete set of functional viral proteins. Given that NSP3 is made at moderate levels in infected cells, rotavirus encoding NSP3-based FPs should be more sensitive probes of viral infection than rotaviruses encoding NSP1-based FPs.

scholarly journals Natural oil blend formulation as an anti-African swine fever virus agent in in vitro primary porcine alveolar macrophage culture

2021 ◽  
Vol 14 (3) ◽  
pp. 794-802
Author(s):  
Quang Lam Truong ◽  
Lan Thi Nguyen ◽  
Haig Yousef Babikian ◽  
Rajeev Kumar Jha ◽  
Hoa Thi Nguyen ◽  
...  
Keyword(s):  
Real Time ◽  
African Swine Fever Virus ◽  
Lethal Dose ◽  
African Swine Fever ◽  
Porcine Alveolar Macrophage ◽  
Ct Value ◽  
Natural Oil ◽  
Oil Blend

Background and Aim: African swine fever is one of the severe pathogens of swine. It has a significant impact on production and economics. So far, there are no known remedies, such as vaccines or drugs, reported working successfully. In the present study, the natural oil blend formulation's (NOBF) efficacy was evaluated against ASFV in vitro using porcine alveolar macrophages (PAMs) cells of swine. Materials and Methods: The capacity of NOBF against the ASFV was tested in vitro. The NOBF combines Eucalyptus globulus, Pinus sylvestris, and Lavandula latifolia. We used a 2-fold serial dilution to test the NOBF formulation dose, that is, 105 HAD50/mL, against purified lethal dose of African swine in primary PAMs cells of swine. The PAM cells survival, real-time polymerase chain reaction (PCR) test, and hemadsorption (HAD) observation were performed to check the NOBF efficacy against ASFV. Results: The in vitro trial results demonstrated that NOBF up to dilution 13 or 0.000625 mL deactivates the lethal dose 105 HAD50 of ASFV. There was no HAD (Rosetta formation) up to dilution 12 or 0.00125 mL of NOBF. The Ct value obtained by running real-time PCR of the NOBF group at 96 h post-infection was the same as the initial value or lower (25), whereas the Ct value of positive controls increased several folds (17.84). Conclusion: The in vitro trial demonstrated that NOBF could deactivate the ASFV. The NOBF has the potential to act as anti-ASFV agent in the field. The next step is to conduct in vivo level trial to determine its efficacy.

Phosphorylation of African swine fever virus proteins in vitro and in vivo

Biochimie ◽  
1988 ◽  
Vol 70 (5) ◽  
pp. 627-635 ◽  
Author(s):  
María L. Salas ◽  
José Salas ◽  
Eladio Viñuela
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Virus Proteins

scholarly journals African swine fever virus: a B cell-mitogenic virus in vivo and in vitro.

1999 ◽  
Vol 80 (6) ◽  
pp. 1453-1461 ◽  
Author(s):  
H Takamatsu ◽  
M S Denyer ◽  
C Oura ◽  
A Childerstone ◽  
J K Andersen ◽  
...  
Keyword(s):  
B Cell ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus

scholarly journals Impact of RNA-Guided Technologies for Target Identification and Deconvolution

2014 ◽  
Vol 19 (10) ◽  
pp. 1327-1337 ◽  
Author(s):  
Myles Fennell ◽  
Qing Xiang ◽  
Alexia Hwang ◽  
Chong Chen ◽  
Chun-Hao Huang ◽  
...  
Keyword(s):  
High Efficiency ◽  
Virus Production ◽  
Expression Vectors ◽  
Specific Gene ◽  
Loss Of Function ◽  
Shrna Expression ◽  
Rnai Technology ◽  
Target Effects

For well over a decade, RNA interference (RNAi) has provided a powerful tool for investigators to query specific gene targets in an easily modulated loss-of-function setting, both in vitro and in vivo. Hundreds of publications have demonstrated the utility of RNAi in arrayed and pooled-based formats, in a wide variety of cell-based systems, including clonal, stem, transformed, and primary cells. Over the years, there have been significant improvements in the design of target-specific small-interfering RNA (siRNA) and short-hairpin RNA (shRNA), expression vectors, methods for mitigating off-target effects, and accurately interpreting screening results. Recent developments in RNAi technology include the Sensor assay, high-efficiency miR-E shRNAs, improved shRNA virus production with Pasha (DRGC8) knockdown, and assessment of RNAi off-target effects by using the C9-11 method. An exciting addition to the arsenal of RNA-mediated gene modulation is the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas) system for genomic editing, allowing for gene functional knockout rather than knockdown.

Sign in / Sign up

Export Citation Format

Share Document