scholarly journals Rearranged Genomic RNA Segments Offer a New Approach to the Reverse Genetics of Rotaviruses

2010 ◽  
Vol 84 (13) ◽  
pp. 6711-6719 ◽  
Author(s):  
Cécile Troupin ◽  
Axelle Dehée ◽  
Aurélie Schnuriger ◽  
Patrice Vende ◽  
Didier Poncet ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Reverse Genetics ◽  
Nonstructural Protein ◽  
Helper Virus ◽  
Wild Type ◽  
Human Rotavirus ◽  
Protein Functions ◽  
Group A Rotaviruses ◽  
Group A

ABSTRACT Group A rotaviruses (RV), members of the Reoviridae family, are a major cause of infantile acute gastroenteritis. The RV genome consists of 11 double-stranded RNA segments. In some cases, an RNA segment is replaced by a rearranged RNA segment, which is derived from its standard counterpart by partial sequence duplication. We report here a reverse genetics system for RV based on the preferential packaging of rearranged RNA segments. Using this system, wild-type or in vitro-engineered forms of rearranged segment 7 from a human rotavirus (encoding the NSP3 protein), derived from cloned cDNAs and transcribed in the cytoplasm of COS-7 cells with the help of T7 RNA polymerase, replaced the wild-type segment 7 of a bovine helper virus (strain RF). Recombinant RF viruses (i.e., engineered monoreassortant RF viruses) containing an exogenous rearranged RNA were recovered by propagating the viral progeny in MA-104 cells, with no need for additional selective pressure. Our findings offer the possibility to extend RV reverse genetics to segments encoding nonstructural or structural proteins for which no potent selective tools, such as neutralizing antibodies, are available. In addition, the system described here is the first to enable the introduction of a mutated gene expressing a modified nonstructural protein into an infectious RV. This reverse genetics system offers new perspectives for investigating RV protein functions and developing recombinant live RV vaccines containing specific changes targeted for attenuation.

scholarly journals The Porcine Reproductive and Respiratory Syndrome Virus nsp2 Cysteine Protease Domain Possesses both trans- and cis-Cleavage Activities

2009 ◽  
Vol 83 (18) ◽  
pp. 9449-9463 ◽  
Author(s):  
Jun Han ◽  
Mark S. Rutherford ◽  
Kay S. Faaberg
Keyword(s):  
Cysteine Protease ◽  
Reverse Genetics ◽  
Nonstructural Protein ◽  
Point Mutations ◽  
Respiratory Syndrome Virus ◽  
Protease Domain ◽  
Nonstructural Protein 2 ◽  
Cleavage Activity ◽  
Cysteine Protease Domain

ABSTRACT The N terminus of the replicase nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) contains a putative cysteine protease domain (PL2). Previously, we demonstrated that deletion of either the PL2 core domain (amino acids [aa] 47 to 180) or the immediate downstream region (aa 181 to 323) is lethal to the virus. In this study, the PL2 domain was found to encode an active enzyme that mediates efficient processing of nsp2-3 in CHO cells. The PL2 protease possessed both trans- and cis-cleavage activities, which were distinguished by individual point mutations in the protease domain. The minimal size required to maintain these two enzymatic activities included nsp2 aa 47 to 240 (Tyr47 to Cys240) and aa 47 to 323 (Tyr47 to Leu323), respectively. Introduction of targeted amino acid mutations in the protease domain confirmed the importance of the putative Cys55- His124 catalytic motif for nsp2/3 proteolysis in vitro, as were three additional conserved cysteine residues (Cys111, Cys142, and Cys147). The conserved aspartic acids (e.g., Asp89) were essential for the PL2 protease trans-cleavage activity. Reverse genetics revealed that the PL2 trans-cleavage activity played an important role in the PRRSV replication cycle in that mutations that impaired the PL2 protease trans function, but not the cis activity, were detrimental to viral viability. Lastly, the potential nsp2/3 cleavage site was probed. Mutations with the largest impact on in vitro cleavage were at or near the G1196|G1197 dipeptide.

scholarly journals Murine monoclonal antibodies against RBD of SARS-CoV-2 neutralize authentic wild type SARS-CoV-2 as well as B.1.1.7 and B.1.351 viruses and protect in vivo in a mouse model in a neutralization dependent manner

2021 ◽  
Author(s):  
Fatima Amanat ◽  
Shirin Strohmeier ◽  
Wen-Hsin Lee ◽  
Sandhya Bangaru ◽  
Andrew B Ward ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Severe Acute Respiratory Syndrome ◽  
Mouse Model ◽  
Neutralizing Antibodies ◽  
Dependent Manner ◽  
Receptor Binding Domain ◽  
Wild Type ◽  
Murine Monoclonal Antibodies

After first emerging in December 2019 in China, severe acute respiratory syndrome 2 (SARS-CoV-2) has since caused a pandemic leading to millions of infections and deaths worldwide. Vaccines have been developed and authorized but supply of these vaccines is currently limited. With new variants of the virus now emerging and spreading globally, it is essential to develop therapeutics that are broadly protective and bind conserved epitopes in the receptor binding domain (RBD) or the whole spike of SARS-CoV-2. In this study, we have generated mouse monoclonal antibodies (mAbs) against different epitopes on the RBD and assessed binding and neutralization against authentic SARS-CoV-2. We have demonstrated that antibodies with neutralizing activity, but not non-neutralizing antibodies, lower viral titers in the lungs when administered in a prophylactic setting in vivo in a mouse challenge model. In addition, most of the mAbs cross-neutralize the B.1.351 as well as the B.1.1.7 variants in vitro.

scholarly journals Effect of Intragenic Rearrangement and Changes in the 3′ Consensus Sequence on NSP1 Expression and Rotavirus Replication

2001 ◽  
Vol 75 (5) ◽  
pp. 2076-2086 ◽  
Author(s):  
John T. Patton ◽  
Zenobia Taraporewala ◽  
Dayue Chen ◽  
Vladimir Chizhikov ◽  
Melinda Jones ◽  
...  
Keyword(s):  
Cell Culture ◽  
Consensus Sequence ◽  
Active Role ◽  
Serial Passage ◽  
Selective Advantage ◽  
Wild Type ◽  
Reading Frame ◽  
Group A Rotaviruses ◽  
Group A ◽  
Dsrna Genome

ABSTRACT The nonpolyadenylated mRNAs of rotavirus are templates for the synthesis of protein and the segmented double-stranded RNA (dsRNA) genome. During serial passage of simian SA11 rotaviruses in cell culture, two variants emerged with gene 5 dsRNAs containing large (1.1 and 0.5 kb) sequence duplications within the open reading frame (ORF) for NSP1. Due to the sequence rearrangements, both variants encoded only C-truncated forms of NSP1. Comparison of these and other variants encoding defective NSP1 with their corresponding wild-type viruses indicated that the inability to encode authentic NSP1 results in a small-plaque phenotype. Thus, although nonessential, NSP1 probably plays an active role in rotavirus replication in cell culture. In determining the sequences of the gene 5 dsRNAs of the SA11 variants and wild-type viruses, it was unexpectedly found that their 3′ termini ended with 5′-UGAACC-3′ instead of the 3′ consensus sequence 5′-UGACC-3′, which is present on the mRNAs of nearly all other group A rotaviruses. Cell-free assays indicated that the A insertion into the 3′ consensus sequence interfered with its ability to promote dsRNA synthesis and to function as a translation enhancer. The results provide evidence that the 3′ consensus sequence of the gene 5 dsRNAs of SA11 rotaviruses has undergone a mutation causing it to operate suboptimally in RNA replication and in the expression of NSP1 during the virus life cycle. Indeed, just as rotavirus variants which encode defective NSP1 appear to have a selective advantage over those encoding wild-type NSP1 in cell culture, it may be that the atypical 3′ end of SA11 gene 5 has been selected for because it promotes the expression of lower levels of NSP1 than the 3′ consensus sequence.

scholarly journals Murine Coronavirus Ubiquitin-Like Domain Is Important for Papain-Like Protease Stability and Viral Pathogenesis

2015 ◽  
Vol 89 (9) ◽  
pp. 4907-4917 ◽  
Author(s):  
Anna M. Mielech ◽  
Xufang Deng ◽  
Yafang Chen ◽  
Eveline Kindler ◽  
Dorthea L. Wheeler ◽  
...  
Keyword(s):  
Viral Replication ◽  
Protease Activity ◽  
Reverse Genetics ◽  
Hepatitis Virus ◽  
Nonstructural Protein ◽  
Viral Pathogenesis ◽  
Wild Type ◽  
Viral Protease ◽  
Novel Approach ◽  
Protease Stability

ABSTRACTUbiquitin-like domains (Ubls) now are recognized as common elements adjacent to viral and cellular proteases; however, their function is unclear. Structural studies of the papain-like protease (PLP) domains of coronaviruses (CoVs) revealed an adjacent Ubl domain in severe acute respiratory syndrome CoV, Middle East respiratory syndrome CoV, and the murine CoV, mouse hepatitis virus (MHV). Here, we tested the effect of altering the Ubl adjacent to PLP2 of MHV on enzyme activity, viral replication, and pathogenesis. Using deletion and substitution approaches, we identified sites within the Ubl domain, residues 785 to 787 of nonstructural protein 3, which negatively affect protease activity, and valine residues 785 and 787, which negatively affect deubiquitinating activity. Using reverse genetics, we engineered Ubl mutant viruses and found that AM2 (V787S) and AM3 (V785S) viruses replicate efficiently at 37°C but generate smaller plaques than wild-type (WT) virus, and AM2 is defective for replication at higher temperatures. To evaluate the effect of the mutation on protease activity, we purified WT and Ubl mutant PLP2 and found that the proteases exhibit similar specific activities at 25°C. However, the thermal stability of the Ubl mutant PLP2 was significantly reduced at 30°C, thereby reducing the total enzymatic activity. To determine if the destabilizing mutation affects viral pathogenesis, we infected C57BL/6 mice with WT or AM2 virus and found that the mutant virus is highly attenuated, yet it replicates sufficiently to elicit protective immunity. These studies revealed that modulating the Ubl domain adjacent to the PLP reduces protease stability and viral pathogenesis, revealing a novel approach to coronavirus attenuation.IMPORTANCEIntroducing mutations into a protein or virus can have either direct or indirect effects on function. We asked if changes in the Ubl domain, a conserved domain adjacent to the coronavirus papain-like protease, altered the viral protease activity or affected viral replication or pathogenesis. Our studies using purified wild-type and Ubl mutant proteases revealed that mutations in the viral Ubl domain destabilize and inactivate the adjacent viral protease. Furthermore, we show that a CoV encoding the mutant Ubl domain is unable to replicate at high temperature or cause lethal disease in mice. Our results identify the coronavirus Ubl domain as a novel modulator of viral protease stability and reveal manipulating the Ubl domain as a new approach for attenuating coronavirus replication and pathogenesis.

scholarly journals Mutations in the NS1 Protein of Swine Influenza Virus Impair Anti-Interferon Activity and Confer Attenuation in Pigs

2005 ◽  
Vol 79 (12) ◽  
pp. 7535-7543 ◽  
Author(s):  
Alicia Solórzano ◽  
Richard J. Webby ◽  
Kelly M. Lager ◽  
Bruce H. Janke ◽  
Adolfo García-Sastre ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Nonstructural Protein ◽  
Swine Influenza Virus ◽  
Swine Influenza ◽  
Influenza Viruses ◽  
Model Systems ◽  
Ns1 Protein ◽  
Wild Type ◽  
Virulence Attenuation

ABSTRACT It has been shown previously that the nonstructural protein NS1 of influenza virus is an alpha/beta interferon (IFN-α/β) antagonist, both in vitro and in experimental animal model systems. However, evidence of this function in a natural host has not yet been obtained. Here we investigated the role of the NS1 protein in the virulence of a swine influenza virus (SIV) isolate in pigs by using reverse genetics. The virulent wild-type A/Swine/Texas/4199-2/98 (TX/98) virus and various mutants encoding carboxy-truncated NS1 proteins were rescued. Growth properties of TX/98 viruses with mutated NS1, induction of IFN in tissue culture, and virulence-attenuation in pigs were analyzed and compared to those of the recombinant wild-type TX/98 virus. Our results indicate that deletions in the NS1 protein decrease the ability of the TX/98 virus to prevent IFN-α/β synthesis in pig cells. Moreover, all NS1 mutant viruses were attenuated in pigs, and this correlated with the amount of IFN-α/β induced in vitro. These data suggest that the NS1 protein of SIV is a virulence factor. Due to their attenuation, NS1-mutated swine influenza viruses might have a great potential as live attenuated vaccine candidates against SIV infections of pigs.

scholarly journals Molecular characterization of group A rotaviruses detected in children with gastroenteritis in Ireland in 2006–2009

2011 ◽  
Vol 140 (2) ◽  
pp. 247-259 ◽  
Author(s):  
O. CASHMAN ◽  
P. J. COLLINS ◽  
G. LENNON ◽  
B. CRYAN ◽  
V. MARTELLA ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Irish Population ◽  
Surveillance Activity ◽  
Human Rotavirus ◽  
Group A Rotaviruses ◽  
Group A ◽  
Hospital Acquired ◽  
Rotavirus Infections ◽  
Pcr Genotyping

SUMMARYCommunity and hospital-acquired cases of human rotavirus are responsible for millions of gastroenteritis cases in children worldwide, chiefly in developing countries, and vaccines are now available. During surveillance activity for human rotavirus infections in Ireland, between 2006 and 2009, a total of 420 rotavirus strains were collected and analysed. Upon either PCR genotyping and sequence analysis, a variety of VP7 (G1–G4 and G9) and VP4 (P[4], P[6], P[8] and P[9]) genotypes were detected. Strains G1P[8] were found to be predominant throughout the period 2006–2008, with slight fluctuations seen in the very limited samples available in 2008–2009. Upon either PCR genotyping and sequence analysis of selected strains, the G1, G3 and G9 viruses were found to contain E1 (Wa-like) NSP4 and I1 VP6 genotypes, while the analysed G2 strains possessed E2 NSP4 and I2 VP6 genotypes, a genetic make-up which is highly conserved in the major human rotavirus genogroups Wa- and Kun-like, respectively. Upon sequence analysis of the most common VP4 genotype, P[8], at least two distinct lineages were identified, both unrelated to P[8] Irish rotaviruses circulating in previous years, and more closely related to recent European humans rotaviruses. Moreover, sequence analysis of the VP7 of G1 rotaviruses revealed the onset of a G1 variant, previously unseen in the Irish population.

scholarly journals A Universal Dengue Vaccine Elicits Neutralizing Antibodies Against Strains from All Four Dengue Serotypes

2020 ◽  
Author(s):  
Naoko Uno ◽  
Ted M. Ross
Keyword(s):  
Dengue Virus ◽  
Neutralizing Antibodies ◽  
Protective Immunity ◽  
Rhesus Macaques ◽  
Effective Vaccine ◽  
Wild Type ◽  
Monovalent Vaccine ◽  
Viral Particles ◽  
Tropical Areas

Any potential dengue virus (DENV) vaccine needs to elicit protective immunity against strains from all four serotypes to avoid potential antibody dependent enhancement (ADE). In this study, four independent DENV envelope (E) glycoproteins were generated using wild-type E sequences from viruses isolated between 1943 to 2006 using computationally-optimized broadly reactive antigen (COBRA) methodology. COBRA and wild-type E antigens were expressed on the surface of subvirion viral particles (SVPs). Four separate wild-type E antigens were used for each serotype. Mice vaccinated with wild-type DENV SVPs had anti-E IgG antibodies that neutralized serotype specific viruses. COBRA DENV SVPs elicited a broader breadth of antibodies that neutralized strains across all four serotypes. Two COBRA DENV vaccine candidates that elicited the broadest breadth of neutralizing antibodies in mice were used to vaccinate rhesus macaques (Macca mulata) that were either immunologically naïve to any DENV serotype or were had pre-existing antibodies to DENV. Antibodies elicited by COBRA DENV E immunogens neutralized all 12 strains of DENV in vitro, which was comparable to antibodies elicited by a tetravalent wild-type E SVP vaccination mixture. Therefore, using a single DENV COBRA E protein can elicit neutralizing antibodies against strains representing all four serotypes of DENV in both naïve and dengue pre-immune populations. Importance Dengue virus infects millions of people living in the tropical areas of the world. Dengue induced diseases can range from mild to severe with death. An effective vaccine will need to neutralize viruses from all four serotypes of dengue without induced enhanced disease. A dengue E vaccine candidate generated by computationally optimized broadly reactive antigen algorithms elicits broadly neutralizing protection for current circulating strains from all four serotypes regardless of immune status. Most Dengue vaccines in development formulate four separate components based on prM-E from a wild type strain representing each serotype. Designing a monovalent vaccine that elicits protective immunity against all four serotypes is an effective and economical strategy

scholarly journals Human, porcine and bovine rotaviruses in Slovenia: evidence of interspecies transmission and genome reassortment

2008 ◽  
Vol 89 (7) ◽  
pp. 1690-1698 ◽  
Author(s):  
Andrej Steyer ◽  
Mateja Poljšak-Prijatelj ◽  
Darja Barlič-Maganja ◽  
Jožica Marin
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Zoonotic Transmission ◽  
Interspecies Transmission ◽  
Genotype Combination ◽  
Human Rotavirus ◽  
Group A Rotaviruses ◽  
Group A ◽  
Stool Samples ◽  
Genome Reassortment

A surveillance of human, porcine and bovine rotaviruses was carried out in Slovenia in 2004 and 2005. Stool samples were collected from a total of 406 pigs (373 from asymptomatic animals), 132 cattle (126 from asymptomatic animals) and 241 humans (all with diarrhoea), tested for group A rotaviruses using RT-PCR and analysed by sequencing. The aims of the study were to determine the incidence of asymptomatic rotavirus infection in animals, to look for evidence of zoonotic transmission and to detect reassortment among rotaviruses. The rates of asymptomatic shedding of rotaviruses in pigs and cattle were 18.0 % (67/373) and 4.0 % (5/126), respectively. Evidence for zoonotic transmission was detected in one human rotavirus strain, SI-MB6, with the G3P[6] genotype combination, as the nucleotide and predicted amino acid sequences of the VP6, VP7, VP8* and NSP4 genes of strain SI-MB6 and of porcine strains showed high nucleotide and amino acid sequence identity. Two porcine rotavirus strains carried VP7 of probable human origin, suggesting an interspecies reassortment event in the past.

scholarly journals Molecular Characterization of a Subgroup Specificity Associated with the Rotavirus Inner Capsid Protein VP2

2008 ◽  
Vol 82 (6) ◽  
pp. 2752-2764 ◽  
Author(s):  
Sarah M. McDonald ◽  
John T. Patton
Keyword(s):  
Monoclonal Antibody ◽  
Capsid Protein ◽  
Neutralizing Antibodies ◽  
Bovine Rotavirus ◽  
Single Region ◽  
Molecular Determinants ◽  
High Resolution Structure ◽  
Group A Rotaviruses ◽  
Group A

ABSTRACT Group A rotaviruses are classified into serotypes, based on the reactivity pattern of neutralizing antibodies to VP4 and VP7, as well as into subgroups (SGs), based on non-neutralizing antibodies directed against VP6. The inner capsid protein (VP2) has also been described as a SG antigen; however, little is known regarding the molecular determinants of VP2 SG specificity. In this study, we characterize VP2 SGs by correlating genetic markers with the immunoreactivity of the SG-specific monoclonal antibody (YO-60). Our results show that VP2 proteins similar in sequence to that of the prototypic human strain Wa are recognized by YO-60, classifying them as VP2 SG-II. In contrast, proteins not bound by YO-60 are similar to those of human strains DS-1 or AU-1 and represent VP2 SG-I. Using a mutagenesis approach, we identified residues that determine recognition by either YO-60 or the group A-specific VP2 monoclonal antibody (6E8). We found that YO-60 binds to a conformationally dependent epitope that includes Wa VP2 residue M328. The epitope for 6E8 is also contingent upon VP2 conformation and resides within a single region of the protein (Wa VP2 residues A440 to T530). Using a high-resolution structure of bovine rotavirus double-layered particles, we predicted these epitopes to be spatially distinct from each other and located on opposite surfaces of VP2. This study reveals the extent of genetic variation among group A rotavirus VP2 proteins and illuminates the molecular basis for a previously described SG specificity associated with the rotavirus inner capsid protein.

scholarly journals Antibodies against a Synthetic Peptide of SagA Neutralize the Cytolytic Activity of Streptolysin S from Group A Streptococci

2002 ◽  
Vol 70 (4) ◽  
pp. 2166-2170 ◽  
Author(s):  
James B. Dale ◽  
Edna Y. Chiang ◽  
David L. Hasty ◽  
Harry S. Courtney
Keyword(s):  
Synthetic Peptide ◽  
Neutralizing Antibodies ◽  
Natural Infection ◽  
Convincing Evidence ◽  
Cytolytic Activity ◽  
M Protein ◽  
Group A Streptococci ◽  
Streptolysin S ◽  
Group A

ABSTRACT Virtually all group A streptococci (GAS) produce streptolysin S (SLS), a cytolytic toxin that is responsible for the beta-hemolysis surrounding colonies of the organisms grown on blood agar. SLS is an important virulence determinant of GAS, and recent studies have identified a nine-gene locus that is responsible for synthesis and transport of the toxin. SLS is not immunogenic; thus, no neutralizing antibodies are evoked during the course of natural infection. In the present study, we show that a synthetic peptide containing amino acid residues 10 to 30 of the putative SLS (SagA) propeptide [SLS(10-30)] coupled to keyhole limpet hemocyanin evoked antibodies in rabbits that completely neutralized the hemolytic activity of the toxin in vitro. Inhibition of hemolysis was reversed by preincubation of the immune serum with soluble, unconjugated peptide, indicating the specificity of the antibodies. In addition, antibodies that were affinity purified over an SLS(10-30) peptide column completely inhibited SLS-mediated hemolysis. The SLS(10-30) antisera did not opsonize group A streptococci; however, when combined with type-specific M protein antisera, the SLS antibodies significantly enhanced phagocytosis mediated by M protein antibodies. Thus, we have shown for the first time that it is possible to raise neutralizing antibodies against one of the most potent bacterial cytolytic toxins known. Our data also provide convincing evidence that the sagA gene actually encodes the SLS peptide of GAS. The synthetic peptide may prove to be an important component of vaccines designed to prevent GAS infections.

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