scholarly journals A Novel Approach To Display Structural Proteins of Hepatitis C Virus Quasispecies in Patients Reveals a Key Role of E2 HVR1 in Viral Evolution

2020 ◽  
Vol 94 (17) ◽  
Author(s):  
Yimin Tong ◽  
Qingchao Li ◽  
Rui Li ◽  
Yongfen Xu ◽  
Yu Pan ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Vaccine Development ◽  
Viral Evolution ◽  
Hcv Infection ◽  
Clinical Isolates ◽  
Structural Proteins ◽  
Structural Protein ◽  
Protein Coding ◽  
Viral Structural Proteins

ABSTRACT Hepatitis C virus (HCV) infection remains a major worldwide health problem despite development of highly effective direct-acting antivirals. HCV rapidly evolves upon acute infection and generates multiple viral variants (quasispecies), leading to immune evasion and persistent viral infection. Identification of epitopes of broadly neutralizing anti-HCV antibodies (nAbs) is critical to guide HCV vaccine development. In this study, we developed a new reverse genetics system for HCV infection based on trans-complementation of viral structural proteins. The HCV genome (JFH1 strain) lacking the structural protein-coding sequence can be efficiently rescued by ectopic expression of core-E1-E2-p7-NS2 (core-NS2) or core-E1-E2-p7 (core-p7) in trans, leading to production of single-round infectious virions designated HCVΔS. JFH1-based HCVΔS can be also rescued by expressing core-NS2 of other HCV genotypes, rendering it an efficient tool to display the structural proteins of HCV strains of interests. Furthermore, we successfully rescued HCVΔS with structural proteins from clinical isolates. Multiple viral structural proteins with different sensitivities to nAbs were identified from a same patient serum, demonstrating the genetic diversity of HCV quasispecies in vivo. Interestingly, the structural protein-coding sequences of highly divergent viral quasispecies from the same patient can be clustered based on their hypervariable region 1 (HVR1) in viral envelope protein E2, which critically dictates the sensitivity to neutralizing antibodies. In summary, we developed a novel reverse genetics system that efficiently displays viral structural proteins from HCV clinical isolates, and analysis of quasispecies from the same patient using this system demonstrated that E2 HVR1 is the major determinant of viral evolution in vivo. IMPORTANCE A cell culture model that can recapitulate the diversity of HCV quasispecies in patients is important for analysis of neutralizing epitopes and HCV vaccine development. In this study, we developed a new reverse genetics system for HCV infection based on trans-complementation of viral structural proteins (HCVΔS). This system can be used to display structural proteins of HCV strains of multiple genotypes as well as clinical isolates. By using this system, we showed that multiple different HCV structural proteins from a same patient were displayed on HCVΔS. Interestingly, these variant structural proteins within the same patient can be classified according to the sequence of HVR1in E2, which dictates viral sensitivity to nAbs and viral evolution in vivo. Our work provided a new tool to study highly divergent HCV quasispecies and shed light on underlying mechanisms driving HCV evolution.

scholarly journals Rotavirus NSP1 contributes to intestinal viral replication, pathogenesis, and transmission

2021 ◽  
Author(s):  
Gaopeng Hou ◽  
Qiru Zeng ◽  
Jelle Matthijnssens ◽  
Harry B. Greenberg ◽  
Siyuan Ding
Keyword(s):  
Reverse Genetics ◽  
Vaccine Development ◽  
Gene Segment ◽  
Competent Cell ◽  
Structural Protein ◽  
Mesenteric Lymph Nodes ◽  
Independent Manner ◽  
Systemic Spread

Rotavirus (RV)-encoded non-structural protein 1 (NSP1), the product of gene segment 5, effectively antagonizes host interferon (IFN) signaling via multiple mechanisms. Recent studies with the newly established RV reverse genetics system indicate that NSP1 is not essential for the replication of simian RV SA11 strain in cell culture. However, the role of NSP1 in RV infection in vivo remains poorly characterized due to the limited replication of heterologous simian RVs in the suckling mouse model. Here, we used an optimized reverse genetics system and successfully recovered recombinant murine RVs with or without NSP1 expression. While the NSP1-null virus replicated comparably with the parental murine RV in IFN-deficient and IFN-competent cell lines in vitro, it was highly attenuated in 5-day-old wild-type suckling pups. In the absence of NSP1 expression, murine RV had significantly reduced replication in the ileum, systemic spread to mesenteric lymph nodes, fecal shedding, diarrhea occurrence, and transmission to uninoculated littermates. Of interest, the replication and pathogenesis defects of NSP1-null RV were only minimally rescued in Stat1knockout pups, suggesting that NSP1 facilitates RV replication in an IFN-independent manner. Our findings highlight a pivotal function of NSP1 during homologous RV infections in vivo and identify NSP1 as an ideal viral protein for targeted attenuation for future vaccine development.

scholarly journals Identification of a Novel Virulence Factor in Recombinant Pneumonia Virus of Mice

2007 ◽  
Vol 81 (17) ◽  
pp. 9490-9501 ◽  
Author(s):  
Christine D. Krempl ◽  
Anna Wnekowicz ◽  
Elaine W. Lamirande ◽  
Giw Nayebagha ◽  
Peter L. Collins ◽  
...  
Keyword(s):  
Virulence Factor ◽  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Virulent Strain ◽  
Consensus Sequence ◽  
Cytoplasmic Tail ◽  
Syncytial Virus

ABSTRACT Pneumonia virus of mice (PVM) is a murine relative of human respiratory syncytial virus (HRSV). Here we developed a reverse genetics system for PVM based on a consensus sequence for virulent strain 15. Recombinant PVM and a version engineered to express green fluorescent protein replicated as efficiently as the biological parent in vitro but were 4- and 12.5-fold attenuated in vivo, respectively. The G proteins of HRSV and PVM have been suggested to contribute to viral pathogenesis, but this had not been possible to study in a defined manner in a fully permissive host. As a first step, we evaluated recombinant mutants bearing a deletion of the entire G gene (ΔG) or expressing a G protein lacking its cytoplasmic tail (Gt). Both G mutants replicated as efficiently in vitro as their recombinant parent, but both were nonpathogenic in mice at doses that would otherwise be lethal. We could not detect replication of the ΔG mutant in mice, indicating that its attenuation is based on a severe reduction in the virus load. In contrast, the Gt mutant appeared to replicate as efficiently in mice as its recombinant parent. Thus, the reduction in virulence associated with the Gt mutant could not be accounted for by a reduction in viral replication. These results identified the cytoplasmic tail of G as a virulence factor whose effect is not mediated solely by the viral load. In addition to its intrinsic interest, a recombinant virus that replicates with wild-type-like efficiency but does not cause disease defines optimal properties for vaccine development.

scholarly journals In Vivo Study of the HC-TN Strain of Hepatitis C Virus Recovered from a Patient with Fulminant Hepatitis: RNA Transcripts of a Molecular Clone (pHC-TN) Are Infectious in Chimpanzees but Not in Huh7.5 Cells

2007 ◽  
Vol 81 (13) ◽  
pp. 7208-7219 ◽  
Author(s):  
Akito Sakai ◽  
Shingo Takikawa ◽  
Robert Thimme ◽  
Jean-Christophe Meunier ◽  
Hans Christian Spangenberg ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Immune Responses ◽  
Disease Severity ◽  
Neutralizing Antibodies ◽  
Viral Evolution ◽  
Acute Infection ◽  
Hcv Infection ◽  
Rna Transcripts

ABSTRACT Both viral and host factors are thought to influence the pathogenesis of hepatitis C virus (HCV) infection. We studied strain HC-TN (genotype 1a), which caused fulminant hepatic failure in a patient and, subsequently, severe hepatitis in a chimpanzee (CH1422), to analyze the relationship between disease severity, host immune response, viral evolution, and outcome. A second chimpanzee (CH1581) was infected from CH1422 plasma, and a third chimpanzee (CH1579) was infected from RNA transcripts of a consensus cDNA of HC-TN (pHC-TN). RNA transcripts of pHC-TN did not replicate in Huh7.5 cells, which were recently found to be susceptible to infection with another fulminant HCV strain (JFH1). The courses of viremia were similar in the three animals. However, CH1581 and CH1579 developed a less severe acute hepatitis than CH1422. CH1579 and CH1422 resolved the infection, whereas CH1581 became persistently infected. CH1579 and CH1581, despite their differing outcomes, both developed significant intrahepatic cellular immune responses, but not antibodies to the envelope glycoproteins or neutralizing antibodies, during the acute infection. We analyzed the polyprotein sequences of virus recovered at five and nine time points from CH1579 and CH1581, respectively, during the first year of follow-up. High mutation rates and high proportions of nonsynonymous mutations suggested immune pressure and positive selection in both animals. Changes were not selected until after the initial decrease in virus titers and after the development of immune responses and hepatitis. Subsequently, however, mutations emerged repeatedly in both animals. Overall, our results indicate that disease severity and outcome of acute HCV infection depend primarily on the host response.

scholarly journals Immunological and mutational analysis of SARS-CoV-2 structural proteins from Asian countries

2021 ◽  
Vol 8 (5) ◽  
pp. 4367-4390
Author(s):  
Deepak Kumar Jha ◽  
Niti Yashvardhini ◽  
Amit Kumar
Keyword(s):  
Envelope Protein ◽  
Mutational Analysis ◽  
Viral Evolution ◽  
Point Mutations ◽  
High Rate ◽  
World Health ◽  
Structural Proteins ◽  
Structural Protein ◽  
Health Crisis ◽  
Novel Coronavirus

Introduction: The emergence of a novel coronavirus, SARS-CoV-2, an etiolating agent of coronavirus disease (COVID-19), has become a pandemic of global concern. Considering the huge number of morbidity and mortality worldwide, the World Health Organization, on 11th March 2020, has announced an unprecedented public health crisis. This virus is a member of plus sense RNA viruses that can show a high rate of mutations. The ongoing multiple mutations in the structural proteins of coronavirus drive viral evolution, enabling them to evade the host immunity and rapidly acquire drug resistance against COVID-19. In the present study, we focused mainly on the prevalence of mutations in the four types of structural proteins like S (spike), E (envelope), M (membrane), and N (nucleocapsid) that are required for the assembly of a complete virion particle. Further, we estimated the antigenicity and allergenicity of these structural proteins to design and develop a potentially good candidate vaccine against SARS-CoV-2. Methods: In the present in silico study, envelope protein was found highly antigenic followed by nucleocapsid, membrane, and spike protein of SARS-CoV-2. Results: Consequently, in this study, we detected 987 mutations from 729 sequences of Asia in October 2020 and compared them with China's 1st Wuhan isolate sequence as a reference. Spike showed the highest mutations with 807 point mutations among the four structural proteins, followed by nucleocapsid with 151 mutations, while envelope showed 19 and membrane only 10 point mutations. Conclusion: Taken together, our study revealed, variation occurring in the structural protein of SARS-CoV-2 might be altering their structure and functions, and envelope protein appears to be a promising vaccine candidate to curb coronavirus infections.

scholarly journals A Tick-Borne Langat Virus Mutant That Is Temperature Sensitive and Host Range Restricted in Neuroblastoma Cells and Lacks Neuroinvasiveness for Immunodeficient Mice

2006 ◽  
Vol 80 (3) ◽  
pp. 1427-1439 ◽  
Author(s):  
Alexander A. Rumyantsev ◽  
Brian R. Murphy ◽  
Alexander G. Pletnev
Keyword(s):  
Reverse Genetics ◽  
Neuroblastoma Cells ◽  
Scid Mice ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Structural Protein ◽  
Human Neuroblastoma ◽  
Immunodeficient Mice ◽  
Langat Virus

ABSTRACT Langat virus (LGT), the naturally attenuated member of the tick-borne encephalitis virus (TBEV) complex, was tested extensively in clinical trials as a live TBEV vaccine and was found to induce a protective, durable immune response; however, it retained a low residual neuroinvasiveness in mice and humans. In order to ablate or reduce this property, LGT mutants that produced a small plaque size or temperature-sensitive (ts) phenotype in Vero cells were generated using 5-fluorouracil. One of these ts mutants, clone E5-104, exhibited a more than 103-fold reduction in replication at the permissive temperature in both mouse and human neuroblastoma cells and lacked detectable neuroinvasiveness for highly sensitive immunodeficient mice. The E5-104 mutant possessed five amino acid substitutions in the structural protein E and one change in each of the nonstructural proteins NS3 and NS5. Using reverse genetics, we demonstrated that a Lys46→Glu substitution in NS3 as well as a single Lys315→Glu change in E significantly impaired the growth of LGT in neuroblastoma cells and reduced its peripheral neurovirulence for SCID mice. This study and our previous experience with chimeric flaviviruses indicated that a decrease in viral replication in neuroblastoma cells might serve as a predictor of in vivo attenuation of the neurotropic flaviviruses. The combination of seven mutations identified in the nonneuroinvasive E5-104 mutant provided a useful foundation for further development of a live attenuated TBEV vaccine. An evaluation of the complete sequence of virus recovered from brain of SCID mice inoculated with LGT mutants identified sites in the LGT genome that promoted neurovirulence/neuroinvasiveness.

scholarly journals In Vitro and In Vivo Characterization of MicroRNA-Targeted Alphavirus Replicon and Helper RNAs

2010 ◽  
Vol 84 (15) ◽  
pp. 7713-7725 ◽  
Author(s):  
Kurt I. Kamrud ◽  
V. McNeil Coffield ◽  
Gary Owens ◽  
Christin Goodman ◽  
Kim Alterson ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Rna Replication ◽  
Structural Proteins ◽  
Expression Vectors ◽  
Cellular Mirnas ◽  
Rna Regulation ◽  
Oncolytic Adenoviruses ◽  
Vector Systems

ABSTRACT Alphavirus-based replicon vector systems (family Togaviridae) have been developed as expression vectors with demonstrated potential in vaccine development against both infectious diseases and cancer. The single-cycle nature of virus-like replicon particles (VRP), generated by supplying the structural proteins from separate replicable helper RNAs, is an attractive safety component of these systems. MicroRNAs (miRNAs) have emerged as important cellular RNA regulation elements. Recently, miRNAs have been employed as a mechanism to attenuate or restrict cellular tropism of replication-competent viruses, such as oncolytic adenoviruses, vesicular stomatitis virus, and picornaviruses as well as nonreplicating lentiviral and adenoviral vectors. Here, we describe the incorporation of miRNA-specific target sequences into replicable alphavirus helper RNAs that are used in trans to provide the structural proteins required for VRP production. VRP were found to be efficiently produced using miRNA-targeted helper RNAs if miRNA-specific inhibitors were introduced into cells during VRP production. In the absence of such inhibitors, cellular miRNAs were capable of downregulating helper RNA replication in vitro. When miRNA targets were incorporated into a replicon RNA, cellular miRNAs were capable of downregulating replicon RNA replication upon delivery of VRP into animals, demonstrating activity in vivo. These data provide the first example of miRNA-specific repression of alphavirus replicon and helper RNA replication and demonstrate the feasibility of miRNA targeting of expression vector helper functions that are provided in trans.

scholarly journals Establishment of a stable transfection method in Babesia microti and identification of a novel bidirectional promoter of Babesia microti

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Dabbu Kumar Jaijyan ◽  
Kavitha Govindasamy ◽  
Jyoti Singh ◽  
Shreya Bhattacharya ◽  
Agam Prasad Singh
Keyword(s):  
Drug Targets ◽  
Reverse Genetics ◽  
Developmental Stages ◽  
Vaccine Development ◽  
Genetic Manipulation ◽  
Babesia Microti ◽  
Stable Transfection ◽  
Growth Curve Analysis ◽  
Transfection Method

Abstract Babesia microti, an emerging human pathogen, is primarily transmitted through a bite of an infected tick and blood transfusions in human. Stable transfection technique has been reported in many protozoan parasites over the past few years. However, in vivo transient and stable transfection method has not been established for Babesia microti. Here, for the first time, we present a method of transient as well as stable transfection of the Babesia microti (B. microti) in the in vivo conditions. We have identified a novel promoter of B. microti. We also demonstrated that Plasmodium berghei DHFR promoter is recognized and functional in B. microti. We show that BM-CTQ41297 promoter control the expression of two genes, which are present on either side and thus represents a bi-functional promoter in B. microti. The predicted promoter activity values using Promoter 2.0 program is higher for BM- CTQ41297 promoter than strong promoters such as β-actin, ef-1β, and many other promoters. Furthermore, we discovered a non-essential locus for the genetic manipulation of the parasite, allowing us to stably integrate foreign genes; GFP, mCherry, into the B. microti. The transfection using an electroporation method and genetic manipulation of B. microti is now achievable and it is possible to obtain transfected viable parasites under in vivo growing conditions. The growth curve analysis of transfected and WT B. microti are similar indicating no defects in the transgenic parasites. This study will enable other researchers in understanding the B. microti biology, host modulation and diverse parasite developmental stages using reverse genetics and holds great potential to identify novel drug targets and vaccine development.

scholarly journals Recent advances in understanding dengue

F1000Research ◽  
2019 ◽  
Vol 8 ◽  
pp. 1279 ◽  
Author(s):  
Scott Halstead
Keyword(s):  
Vascular Permeability ◽  
Vaccine Development ◽  
Severe Disease ◽  
Denv Infection ◽  
Severe Dengue ◽  
Structural Protein ◽  
Control Programs ◽  
Past Half Century

This is a selective review of recent publications on dengue clinical features, epidemiology, pathogenesis, and vaccine development placed in a context of observations made over the past half century. Four dengue viruses (DENVs) are transmitted by urban cycle mosquitoes causing diseases whose nature and severity are influenced by interacting factors such as virus, age, immune status of the host, and human genetic variability. A phenomenon that controls the kinetics of DENV infection, antibody-dependent enhancement, best explains the correlation of the vascular permeability syndrome with second heterotypic DENV infections and infection in the presence of passively acquired antibodies. Based on growing evidence in vivo and in vitro, the tissue-damaging DENV non-structural protein 1 (NS1) is responsible for most of the pathophysiological features of severe dengue. This review considers the contribution of hemophagocytic histiocytosis syndrome to cases of severe dengue, the role of movement of humans in dengue epidemiology, and modeling and planning control programs and describes a country-wide survey for dengue infections in Bangladesh and efforts to learn what controls the clinical outcome of dengue infections. Progress and problems with three tetravalent live-attenuated vaccines are reviewed. Several research mysteries remain: why is the risk of severe disease during second heterotypic DENV infection so low, why is the onset of vascular permeability correlated with defervescence, and what are the crucial components of protective immunity?

scholarly journals Rescue of SARS-CoV-2 from a Single Bacterial Artificial Chromosome

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Chengjin Ye ◽  
Kevin Chiem ◽  
Jun-Gyu Park ◽  
Fatai Oladunni ◽  
Roy Nelson Platt ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Severe Acute Respiratory Syndrome ◽  
Reverse Genetics ◽  
Vaccine Development ◽  
Cultured Cells ◽  
Artificial Chromosome ◽  
Natural Isolate ◽  
Growth Properties

ABSTRACT Infectious coronavirus (CoV) disease 2019 (COVID-19) emerged in the city of Wuhan (China) in December 2019, causing a pandemic that has dramatically impacted public health and socioeconomic activities worldwide. A previously unknown coronavirus, severe acute respiratory syndrome CoV-2 (SARS-CoV-2), has been identified as the causative agent of COVID-19. To date, there are no U.S. Food and Drug Administration (FDA)-approved vaccines or therapeutics available for the prevention or treatment of SARS-CoV-2 infection and/or associated COVID-19 disease, which has triggered a large influx of scientific efforts to develop countermeasures to control SARS-CoV-2 spread. To contribute to these efforts, we have developed an infectious cDNA clone of the SARS-CoV-2 USA-WA1/2020 strain based on the use of a bacterial artificial chromosome (BAC). Recombinant SARS-CoV-2 (rSARS-CoV-2) was readily rescued by transfection of the BAC into Vero E6 cells. Importantly, BAC-derived rSARS-CoV-2 exhibited growth properties and plaque sizes in cultured cells comparable to those of the natural SARS-CoV-2 isolate. Likewise, rSARS-CoV-2 showed levels of replication similar to those of the natural isolate in nasal turbinates and lungs of infected golden Syrian hamsters. This is, to our knowledge, the first BAC-based reverse genetics system for the generation of infectious rSARS-CoV-2 that displays features in vivo similar to those of a natural viral isolate. This SARS-CoV-2 BAC-based reverse genetics will facilitate studies addressing several important questions in the biology of SARS-CoV-2, as well as the identification of antivirals and development of vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19 disease. IMPORTANCE The pandemic coronavirus (CoV) disease 2019 (COVID-19) caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a major threat to global human health. To date, there are no approved prophylactics or therapeutics available for COVID-19. Reverse genetics is a powerful approach to understand factors involved in viral pathogenesis, antiviral screening, and vaccine development. In this study, we describe the feasibility of generating recombinant SARS-CoV-2 (rSARS-CoV-2) by transfection of a single bacterial artificial chromosome (BAC). Importantly, rSARS-CoV-2 possesses the same phenotype as the natural isolate in vitro and in vivo. This is the first description of a BAC-based reverse genetics system for SARS-CoV-2 and the first time that an rSARS-CoV-2 isolate has been shown to be phenotypically identical to a natural isolate in a validated animal model of SARS-CoV-2 infection. The BAC-based reverse genetics approach will facilitate the study of SARS-CoV-2 and the development of prophylactics and therapeutics for the treatment of COVID-19.

scholarly journals Prevalence of, Antibody Response to, and Immunity Induced by Haemophilus ducreyi Hemolysin

1999 ◽  
Vol 67 (7) ◽  
pp. 3317-3328 ◽  
Author(s):  
Susan M. Dutro ◽  
Gwendolyn E. Wood ◽  
Patricia A. Totten
Keyword(s):  
Vaccine Development ◽  
Rabbit Model ◽  
Etiologic Agent ◽  
Haemophilus Ducreyi ◽  
Structural Protein ◽  
Wild Type ◽  
Ulcer Disease ◽  
A Cell

ABSTRACT Haemophilus ducreyi, the etiologic agent of chancroid, a genital ulcer disease, produces a cell-associated hemolysin whose role in virulence is not well defined. Hemolysin is encoded by two genes, hhdA and hhdB, which, based on their homology to Serratia marcescens shlA and shlBgenes, are believed to encode the hemolysin structural protein and a protein required for secretion and modification of this protein, respectively. In this study, we determined the prevalence and expression of the hemolysin genes in 90 H. ducreyi isolates obtained from diverse geographic locations from 1952 to 1996 and found that all strains contained DNA homologous to the hhdB andhhdA genes. In addition, all strains expressed a hemolytic activity. We also determined that hemolysin is expressed in vivo and is immunogenic, as indicated by the induction of antibodies to hemolysin in both the primate and rabbit disease models as well as in human patients with naturally acquired chancroid. Wild-type strain 35000 and isogenic hemolysin-negative mutants showed no difference in lesion development in the temperature-dependent rabbit model. However, immunization of rabbits with the purified hemolysin protein reduced the recovery of wild-type H. ducreyi, but not hemolysin-negative mutants, from lesions. Our study indicates that hemolysin is a possible candidate for vaccine development due to its immunogenicity, expression in vitro and in vivo by most, if not all, strains, and the effect of immunization on reducing the recovery of viable H. ducreyi in experimental disease in rabbits.

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