scholarly journals Deletion of the SH gene from avian metapneumovirus has a greater impact on virus production and immunogenicity in turkeys than deletion of the G gene or M2-2 open reading frame

2008 ◽  
Vol 89 (2) ◽  
pp. 525-533 ◽  
Author(s):  
Roger Ling ◽  
Sabrina Sinkovic ◽  
Didier Toquin ◽  
Olivier Guionie ◽  
Nicolas Eterradossi ◽  
...  
Keyword(s):  
Cell Culture ◽  
Antibody Response ◽  
Antibody Production ◽  
Reverse Genetics ◽  
Open Reading Frame ◽  
Virus Growth ◽  
Reading Frame ◽  
Viral Yield

Subgroup A avian metapneumoviruses lacking either the SH or G gene or the M2-2 open reading frame were generated by using a reverse-genetics approach. The growth properties of these viruses were studied in vitro and in vivo in their natural host. Deletion of the SH gene alone resulted in the generation of a syncytial-plaque phenotype and this was reversed by the introduction of the SH gene from a subgroup B, but not a subgroup C, virus. Infected turkeys were assessed for antibody production and the presence of viral genomic RNA in tracheal swabs. The virus with a deleted SH gene also showed the greatest impairment of replication both in cell culture and in infected turkeys. This contrasts with the situation with other pneumoviruses in culture and in model animals, where deletion of the SH gene results in little effect upon viral yield and a good antibody response. Replication of the G- and M2-2-deleted viruses was impaired more severely in turkeys than in cell culture, with only some animals showing evidence of virus growth and antibody production. There was no correlation between virus replication and antibody response, suggesting that replication sites other than the trachea may be important for induction of antibody responses.

scholarly journals Murine cytomegalovirus open reading frame m29.1 augments virus replication both in vitro and in vivo

2007 ◽  
Vol 88 (11) ◽  
pp. 2941-2951 ◽  
Author(s):  
Mohammad M. Ahasan ◽  
Clive Sweet
Keyword(s):  
Virus Replication ◽  
Post Infection ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Open Reading Frame ◽  
Murine Cytomegalovirus ◽  
Reading Frame ◽  
Codon Mutation

Murine cytomegalovirus mutant Rc29, with a premature stop codon mutation in the m29 open reading frame (ORF), produced no apparent phenotype in cell culture or following infection of BALB/c mice. In contrast, a similar mutant virus, Rc29.1, with a premature stop codon mutation in its m29.1 ORF, showed reduced virus yields (2–3 log10 p.f.u. ml−1) in tissue culture. Mutant virus yields in BALB/c mice were delayed, reduced (∼1 log10 p.f.u. per tissue) and persisted less well in salivary glands compared with wild-type (wt) and revertant (Rv29.1) virus. In severe combined immunodeficiency mice, Rc29.1 virus showed delayed and reduced replication initially in all tissues (liver, spleen, kidneys, heart, lung and salivary glands). This delayed death until 31 days post-infection (p.i.) compared with wt (23 days p.i.) but at death virus yields were similar to wt. m29 gene transcription was initiated at early times post-infection, while production of a transcript from ORF m29.1 in the presence of cycloheximide indicated that it was an immediate-early gene. ORFs m29.1 and M28 are expressed from a bicistronic message, which is spliced infrequently. However, it is likely that each ORF expresses its own protein, as antiserum derived in rabbits to the m29.1 protein expressed in bacteria from the m29.1 ORF detected only one protein in Western blot analysis of the size predicted for the m29.1 protein. Our results suggest that neither ORF is essential for virus replication but m29.1 is important for optimal viral growth in vitro and in vivo.

scholarly journals Characterization of the Glycoprotein Stable Signal Peptide in Mediating Pichinde Virus Replication and Virulence

2016 ◽  
Vol 90 (22) ◽  
pp. 10390-10397 ◽  
Author(s):  
Junjie Shao ◽  
Xiaoying Liu ◽  
Hinh Ly ◽  
Yuying Liang
Keyword(s):  
Cell Culture ◽  
Membrane Fusion ◽  
Viral Entry ◽  
Reverse Genetics ◽  
Cell Entry ◽  
Pichinde Virus ◽  
Viral Virulence ◽  
Stable Signal

ABSTRACTArenaviruses can cause lethal hemorrhagic fevers in humans with few preventative and therapeutic measures. The arenaviral glycoprotein stable signal peptide (SSP) is unique among signal peptides in that it is an integral component of the mature glycoprotein complex (GPC) and plays important roles not only in GPC expression and processing but also in the membrane fusion process during viral entry. Using the Pichinde virus (PICV) reverse genetics system, we analyzed the effects of alanine substitutions at many conserved residues within the SSP on viral replication in cell culture and in a guinea pig infection model. Our data showed that the K33A, F49A, and C57A mutations abolished GPC-mediated cell entry and therefore could not allow for the generation of viable recombinant viruses, demonstrating that these residues are essential for the PICV life cycle. The G2A mutation caused a marked reduction of cell entry at the membrane fusion step, and while this mutant virus was viable, it was significantly attenuatedin vitroandin vivo. The N20A mutation also reduced membrane fusion activity and viral virulence in guinea pigs, but it did not significantly affect cell entry or viral growth in cell culture. Two other mutations (N37A and R55A) did not affect membrane fusion or viral growthin vitrobut significantly reduced viral virulencein vivo. Taken together, our data suggest that the GPC SSP plays an essential role in mediating viral entry and also contributes to viral virulencein vivo.IMPORTANCESeveral arenaviruses, such as Lassa fever virus, can cause severe and lethal hemorrhagic fever diseases with high mortality and morbidity, and no FDA-approved vaccines or therapies are currently available. Viral entry into cells is mediated by arenavirus GPC that consists of an SSP, the receptor-binding GP1, and transmembrane GP2 protein subunits. Using a reverse genetics system of a prototypic arenavirus, Pichinde virus (PICV), we have shown for the first time in the context of virus infections of cell culture and of guinea pigs that the SSP plays an essential role in mediating the membrane fusion step as well as in other yet-to-be-determined processes during viral infection. Our study provides important insights into the biological roles of GPC SSP and implicates it as a good target for the development of antivirals against deadly human arenavirus pathogens.

scholarly journals STUDIES ON ANTIBODY PRODUCTION

1963 ◽  
Vol 117 (6) ◽  
pp. 1053-1062 ◽  
Author(s):  
Thomas F. O'Brien ◽  
Maria C. Michaelides ◽  
Albert H. Coons
Keyword(s):  
Lymph Node ◽  
Bovine Serum Albumin ◽  
Antibody Response ◽  
Antibody Production ◽  
Bovine Serum ◽  
Time Course ◽  
Antibody Synthesis ◽  
Anamnestic Response

The in vitro anamnestic antibody response of popliteal lymph node fragments to additions of antigen closely resembles the in vivo anamnestic antibody response in its sensitivity to antigen, in the time course of antibody production, and in the sequence of appearance and the morphology of the antibody containing cells. Most of the cells responsible for antibody synthesis remain in the explant and do not migrate, although a few can be found in the outgrowing sheet of cells. The smallest concentration of bovine serum albumin which stimulates an anamnestic response in vitro is about 1 x 10–9 gm/ml.

scholarly journals Ilarviruses Encode a Cucumovirus-Like 2b Gene That Is Absent in Other Genera within the Bromoviridae

1998 ◽  
Vol 72 (8) ◽  
pp. 6956-6959 ◽  
Author(s):  
Hong-Wu Xin ◽  
Liang-Hui Ji ◽  
Simon W. Scott ◽  
Robert H. Symons ◽  
Shou-Wei Ding
Keyword(s):  
Open Reading Frame ◽  
Latent Virus ◽  
Evolutionary Origin ◽  
Protein Species ◽  
Subgenomic Rna ◽  
Reading Frame ◽  
2B Protein ◽  
Sequence Similarities

ABSTRACT We found that RNA 2 of the four ilarviruses sequenced to date encodes an additional conserved open reading frame (ORF), 2b, that overlaps the 3′ end of the previously known ORF, 2a. A novel RNA species of 851 nucleotides was found to accumulate to high levels in plants infected with spinach latent virus (SpLV). Further analysis showed that RNA 4A is a subgenomic RNA of RNA 2 and encodes all of ORF 2b. Moreover, a protein species of the size expected for SpLV ORF 2b was translated in vitro from the RNA 4A-containing virion RNAs. The data support the suggestion that the SpLV 2b protein is translated in vivo. The 2b gene of ilarviruses, which is not encoded by alfamoviruses and bromoviruses, shares several features with the previously reported cucumovirus 2b gene; however, their encoded proteins share no detectable sequence similarities. The evolutionary origin of the 2b gene is discussed.

Molecular biology and pathogenesis of hepatitis E virus

1999 ◽  
Vol 1 (18) ◽  
pp. 1-16 ◽  
Author(s):  
Shahid Jameel
Keyword(s):  
Viral Genome ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Host Interactions ◽  
Processing Enzymes ◽  
Open Reading Frame 2

Hepatitis E virus (HEV) infection results in hepatitis E, an acute and self-limited disease. The virus is transmitted in a faecal–oral manner and is a major cause of viral hepatitis in much of the developing world, where it causes rampant sporadic infections and large epidemics. A curious feature of hepatitis E is the unusually high rates of mortality that are observed in pregnant women, in whom the disease is exacerbated by the development of fulminant liver disease. In the absence of viable in vitro propagation systems, several geographical isolates of HEV have been maintained in vivo in nonhuman primates and, subsequently, the viral genome has been cloned and sequenced. HEV has been classified provisionally into a separate family known as the HEV-like viruses, which has at least four recognised genotypes, but has only a single serotype. The viral genome is a positive-stranded (+)RNA of ~7.5 kb and encodes at least three proteins. Open reading frame 1 (ORF1) encodes the viral nonstructural polyprotein, which has domains that are homologous to some of the replication and processing enzymes found in other +RNA viruses. The HEV protein itself remains poorly characterised. The protein encoded by open reading frame 2 (ORF2) is the major HEV capsid protein, and the protein encoded by open reading frame 3 (ORF3) appears to be involved in virus–host interactions. Several questions related to the biology, epidemiology and pathogenesis of HEV remain unanswered; the progress of a few of these is reviewed here.

scholarly journals Analysis of in vitro and in vivo products of the TMV 30kDa open reading frame using antisera raised against a synthetic peptide

FEBS Letters ◽  
1983 ◽  
Vol 164 (2) ◽  
pp. 355-360 ◽  
Author(s):  
Paula A. Kiberstis ◽  
Antonello Pessi ◽  
Eric Atherton ◽  
Richard Jackson ◽  
Tony Hunter ◽  
...  
Keyword(s):  
Synthetic Peptide ◽  
Open Reading Frame ◽  
Reading Frame

scholarly journals Murine Cytomegalovirus Open Reading Frame M27 Plays an Important Role in Growth and Virulence in Mice

2001 ◽  
Vol 75 (4) ◽  
pp. 1697-1707 ◽  
Author(s):  
Gerardo Abenes ◽  
Manfred Lee ◽  
Erik Haghjoo ◽  
Tuong Tong ◽  
Xiaoyan Zhan ◽  
...  
Keyword(s):  
Scid Mice ◽  
Open Reading Frame ◽  
Murine Cytomegalovirus ◽  
Wild Type Virus ◽  
Wild Type ◽  
Reading Frame ◽  
Viral Virulence ◽  
Carboxyl Terminal

ABSTRACT Using a Tn3-based transposon mutagenesis approach, we have generated a pool of murine cytomegalovirus (MCMV) mutants. In this study, one of the mutants, RvM27, which contained the transposon sequence at open reading frame M27, was characterized both in tissue culture and in immunocompetent BALB/c mice and immunodeficient SCID mice. Our results suggest that the M27 carboxyl-terminal sequence is dispensable for viral replication in vitro. Compared to the wild-type strain and a rescued virus that restored the M27 region, RvM27 was attenuated in growth in both BALB/c and SCID mice that were intraperitoneally infected with the viruses. Specifically, the titers of RvM27 in the salivary glands, lungs, spleens, livers, and kidneys of the infected SCID mice at 21 days postinfection were 50- to 500-fold lower than those of the wild-type virus and the rescued virus. Moreover, the virulence of the mutant virus appeared to be attenuated, because no deaths occurred among SCID mice infected with RvM27 for up to 37 days postinfection, while all the animals infected with the wild-type and rescued viruses died within 27 days postinfection. Our observations provide the first direct evidence to suggest that a disruption of M27 expression results in reduced viral growth and attenuated viral virulence in vivo in infected animals. Moreover, these results suggest that M27 is a viral determinant required for optimal MCMV growth and virulence in vivo and provide insight into the functions of the M27 homologues found in other animal and human CMVs as well as in other betaherpesviruses.

scholarly journals Reverse Genetics for Type I Feline Coronavirus Field Isolate To Study the Molecular Pathogenesis of Feline Infectious Peritonitis

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Rosina Ehmann ◽  
Claudia Kristen-Burmann ◽  
Barbara Bank-Wolf ◽  
Matthias König ◽  
Christiane Herden ◽  
...  
Keyword(s):  
Cell Culture ◽  
Reverse Genetics ◽  
Rna Viruses ◽  
High Titer ◽  
Field Isolate ◽  
Molecular Pathogenesis ◽  
Feline Infectious Peritonitis ◽  
Persistent Infections

ABSTRACTFeline infectious peritonitis (FIP), one of the most important lethal infections of cats, is caused by feline infectious peritonitis virus (FIPV), the high-virulence biotype of feline coronaviruses (FCoVs). FIPVs are suggested to emerge from feline enteric coronaviruses (FECVs) by acquiring mutations in specific genes in the course of persistent infections. Although numerous studies identified mutations predicted to be responsible for the FECV-FIPV biotype switch, the presumed roles of specific genetic changes in FIP pathogenesis have not been confirmed experimentally. Reverse genetics systems established previously for serotype I and the less common serotype II FCoVs were based on cell culture-adapted FIPV strains which, however, were shown to be unsuitable for FIP pathogenesis studiesin vivo. To date, systems to produce and manipulate recombinant serotype I field viruses have not been developed, mainly because these viruses cannot be grownin vitro. Here, we report the first reverse genetics system based on a serotype I FECV field isolate that is suitable to produce high-titer stocks of recombinant FECVs. We demonstrate that these recombinant viruses cause productive persistent infections in cats that are similar to what is observed in natural infections. The system provides an excellent tool for studying FCoVs that do not grow in standard cell culture systems and will greatly facilitate studies into the molecular pathogenesis of FIP. Importantly, the system could also be adapted for studies of other RNA viruses with large genomes whose production and characterizationin vivoare currently hampered by the lack ofin vitropropagation systems.IMPORTANCEThe availability of recombinant serotype I FCoV field isolates that are amenable to genetic manipulation is key to studying the molecular pathogenesis of FIP, especially since previous studies using cell culture-adapted FIPVs had proven unsuccessful. To our knowledge, we report the first serotype I FECV field isolate-based reverse genetics system that allows the production of high-titer recombinant virus stocks that can be used for subsequentin vivostudies in cats. The system represents a milestone in FCoV research. It provides an essential tool for studying the molecular pathogenesis of FIP and, more specifically, the functions of specific gene products in causing a fundamentally different progression of disease following acquisition of specific mutations. The system developed in this study will also be useful for studying other coronaviruses or more distantly related RNA viruses with large genomes for which suitablein vitroculture systems are not available.

scholarly journals Murine Cytomegalovirus Containing a Mutation at Open Reading Frame M37 Is Severely Attenuated in Growth and Virulence In Vivo

2000 ◽  
Vol 74 (23) ◽  
pp. 11099-11107 ◽  
Author(s):  
Manfred Lee ◽  
Jianqiao Xiao ◽  
Erik Haghjoo ◽  
Xiaoyan Zhan ◽  
Gerry Abenes ◽  
...  
Keyword(s):  
Scid Mice ◽  
Open Reading Frame ◽  
Murine Cytomegalovirus ◽  
Wild Type ◽  
Reading Frame ◽  
Viral Mutant ◽  
Intraperitoneal Infection ◽  
Nih 3T3

ABSTRACT A pool of murine cytomegalovirus (MCMV) mutants was generated by using a Tn3-based transposon mutagenesis procedure. One of the mutants, RvM37, which contained the transposon sequence at open reading frame M37, was characterized both in tissue culture and in immunocompetent BALB/c and immunodeficient SCID mice. Our results provide the first direct evidence to suggest that M37 is not essential for viral replication in vitro in NIH 3T3 cells. Compared to the wild-type strain and a rescued virus that restored the M37 region, the viral mutant was severely attenuated in growth in both BALB/c and SCID mice after intraperitoneal infection. Specifically, titers of the Smith strain and rescued virus in the salivary glands, lungs, spleens, livers, and kidneys of the SCID mice at 21 days postinfection were about 5 × 105, 2 × 105, 5 × 104, 5 × 103, and 1 × 104 PFU/ml of organ homogenate, respectively; in contrast, titers of RvM37 in these organs were less than 102 PFU/ml of organ homogenate. Moreover, the virulence of the mutant virus appeared to be significantly attenuated because none of the SCID mice infected with RvM37 had died by 120 days postinfection, while all animals infected with the wild-type and rescued viruses had died by 26 days postinfection. Our results suggest that M37 probably encodes a virulence factor and is required for MCMV virulence in SCID mice and for optimal viral growth in vivo.

scholarly journals In Vitro and In Vivo Characterization of a Murine Cytomegalovirus with a Transposon Insertional Mutation at Open Reading Frame M43

2000 ◽  
Vol 74 (20) ◽  
pp. 9488-9497 ◽  
Author(s):  
Jianqiao Xiao ◽  
Tuong Tong ◽  
Xiaoyan Zhan ◽  
Erik Haghjoo ◽  
Fenyong Liu
Keyword(s):  
Salivary Glands ◽  
Scid Mice ◽  
Open Reading Frame ◽  
Murine Cytomegalovirus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Reading Frame

ABSTRACT We have recently generated a pool of murine cytomegalovirus (MCMV) mutants by using a Tn3-based transposon mutagenesis approach. In this study, one of the MCMV mutants, RvM43, which contained the transposon inserted in open reading frame M43, was characterized. Our results provide the first direct evidence to suggest that M43 is not essential for viral replication in vitro in NIH 3T3 cells. Moreover, RvM43 exhibited a titer similar to that of the wild-type virus in the lungs, livers, spleens, and kidneys of both BALB/c and SCID mice and was as virulent as the wild-type virus in killing SCID mice that had been intraperitoneally infected with the viruses. In contrast, titers of the mutant virus in the salivary glands of the infected animals at 21 days postinfection were significantly (100 to 1,000-fold) lower than those of the wild-type virus and a rescued virus that restored the M43 region and its expression. Thus, M43 appears to be not essential for viral growth in vivo in the lungs, livers, spleens, and kidneys of infected animals and is also dispensable for virulence in killing SCID mice. Moreover, our results suggest that M43 is an MCMV determinant for growth in the salivary glands. Studies of viral genes required for replication in the salivary glands are important in understanding the mechanism of viral tropism for the salivary glands and shedding in saliva, which is believed to be one of the major routes of CMV transmission among healthy human populations.

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