scholarly journals Neither the RNA nor the Proteins of Open Reading Frames 3a and 3b of the Coronavirus Infectious Bronchitis Virus Are Essential for Replication

2006 ◽  
Vol 80 (1) ◽  
pp. 296-305 ◽  
Author(s):  
Teri Hodgson ◽  
Paul Britton ◽  
Dave Cavanagh
Keyword(s):  
Infectious Bronchitis Virus ◽  
Open Reading Frames ◽  
Wild Type Virus ◽  
Type Virus ◽  
Structural Protein ◽  
Wild Type ◽  
Infectious Bronchitis ◽  
E Protein ◽  
Initiation Of Translation ◽  
Reading Frames

ABSTRACT Gene 3 of infectious bronchitis virus is tricistronic; open reading frames (ORFs) 3a and 3b encode two small nonstructural (ns) proteins, 3a and 3b, of unknown function, and a third, structural protein E, is encoded by ORF 3c. To determine if either the 3a or the 3b protein is required for replication, we first modified their translation initiation codons to prevent translation of the 3a and 3b proteins from recombinant infectious bronchitis viruses (rIBVs). Replication in primary chick kidney (CK) cells and in chicken embryos was not affected. In chicken tracheal organ cultures (TOCs), the recombinant rIBVs reached titers similar to those of the wild-type virus, but in the case of viruses lacking the 3a protein, the titer declined reproducibly earlier. Translation of the IBV E protein is believed to be initiated by internal entry of ribosomes at a structure formed by the sequences corresponding to ORFs 3a and 3b. To assess the necessity of this mechanism, we deleted most of the sequence representing 3a and 3b to produce a gene in which ORF 3c (E) was adjacent to the gene 3 transcription-associated sequence. Western blot analysis revealed that the recombinant IBV produced fivefold less E protein. Nevertheless, titers produced in CK cells, embryos, and TOCs were similar to those of the wild-type virus, although they declined earlier in TOCs, probably due to the absence of the 3a protein. Thus, neither the tricistronic arrangement of gene 3, the internal initiation of translation of E protein, nor the 3a and 3b proteins are essential for replication per se, suggesting that these proteins are accessory proteins that may have roles in vivo.

scholarly journals Construction and Characterization of Murine Cytomegaloviruses That Contain Transposon Insertions at Open Reading Frames m09 and M83

2000 ◽  
Vol 74 (16) ◽  
pp. 7411-7421 ◽  
Author(s):  
Xiaoyan Zhan ◽  
Manfred Lee ◽  
Jianqiao Xiao ◽  
Fenyong Liu
Keyword(s):  
Viral Replication ◽  
Scid Mice ◽  
Open Reading Frames ◽  
Murine Cytomegalovirus ◽  
Wild Type Virus ◽  
Insertion Mutation ◽  
Type Virus ◽  
Wild Type ◽  
Reading Frames

ABSTRACT A transposon derived from Escherichia coliTn3 was introduced into the genome of murine cytomegalovirus (MCMV) to generate a pool of viral mutants, including two recombinant viruses that contained the transposon sequence within open reading frames m09 and M83. Our studies provide the first direct evidence to suggest that m09 is not essential for viral replication in mouse NIH 3T3 cells. Studies in cultured cells and in both BALB/c-Byj and CB17 severe combined immunodeficient (SCID) mice indicated that the transposon insertion is stable during viral propagation both in vitro and in vivo. Moreover, the virus that contained the insertion mutation in m09 exhibited a titer similar to that of the wild-type virus in the salivary glands, lungs, livers, spleens, and kidneys of both the BALB/c and SCID mice and was as virulent as the wild-type virus in killing the SCID mice when these animals were intraperitoneally infected with these viruses. These results suggest that m09 is dispensable for viral growth in these organs and that the presence of the transposon sequence in the viral genome does not significantly affect viral replication in vivo. In contrast, the virus that contained the insertion mutation in M83 exhibited a titer of at least 60-fold lower than that of the wild-type virus in the organs of the SCID mice and was attenuated in killing the SCID mice. These results demonstrate the utility of using the Tn3-based system as a mutagenesis approach for studying the function of MCMV genes in both immunocompetent and immunodeficient animals.

scholarly journals Deletion of open reading frames 9, 10 and 11 from the avian adenovirus CELO genome: effect on biodistribution and humoral responses

2005 ◽  
Vol 86 (7) ◽  
pp. 2019-2027 ◽  
Author(s):  
Frédérick Le Goff ◽  
Isabelle Méderlé-Mangeot ◽  
André Jestin ◽  
Patrick Langlois
Keyword(s):  
Open Reading Frames ◽  
Wild Type Virus ◽  
Post Inoculation ◽  
Wild Type ◽  
Recombinant Viruses ◽  
Celo Virus ◽  
The Right ◽  
Humoral Responses ◽  
Reading Frames

In this study, the in vivo effect of the 3·6 kbp deletion of the three open reading frames (ORF) 9, 10 and 11 found at the right end of the CELO genome was examined. Groups of chickens were inoculated oronasally with 105–107 p.f.u. per animal of wild-type virus and two recombinant CELO strains (rCELO) expressing luciferase and secreted alkaline phosphatase (SEAP). The tissue biodistribution, assessed by PCR, was similar for both wild-type and recombinant viruses. The infectious viral particle titre was determined by a p.f.u. counting method and the antibody responses to the CELO vector and the SEAP antigen were evaluated by ELISA. Infectious particle titres in tissues from chickens inoculated with the wild-type CELO virus increased up to 6 days post-inoculation, and declined until 11 days while titres in organs from chickens inoculated with the rCELO strain were low and only detectable at 4 days post-inoculation. Moreover, although anti-CELO antibody levels were three times lower in sera from chickens inoculated with rCELO, antibodies directed to the heterologous SEAP antigen were detected. Based on these results, no differences in tropism were observed, but the level of production of viral particles and the humoral responses appeared to decrease. Viruses replicate less efficiently with a deletion performed at the right end of the CELO genome. Nevertheless, the presence of antibodies directed to heterologous antigens makes the CELO virus an advantageous candidate for avian vaccination.

scholarly journals Rearrangement of Influenza Virus Spliced Segments for the Development of Live-Attenuated Vaccines

2016 ◽  
Vol 90 (14) ◽  
pp. 6291-6302 ◽  
Author(s):  
Aitor Nogales ◽  
Marta L. DeDiego ◽  
David J. Topham ◽  
Luis Martínez-Sobrido
Keyword(s):  
Influenza Virus ◽  
Viral Infections ◽  
Viral Proteins ◽  
Influenza Viruses ◽  
Open Reading Frames ◽  
Wild Type Virus ◽  
Wild Type ◽  
Live Attenuated Vaccines ◽  
Nonstructural Protein 1 ◽  
Reading Frames

ABSTRACTInfluenza viral infections represent a serious public health problem, with influenza virus causing a contagious respiratory disease which is most effectively prevented through vaccination. Segments 7 (M) and 8 (NS) of the influenza virus genome encode mRNA transcripts that are alternatively spliced to express two different viral proteins. This study describes the generation, using reverse genetics, of three different recombinant influenza A/Puerto Rico/8/1934 (PR8) H1N1 viruses containing M or NS viral segments individually or modified M or NS viral segments combined in which the overlapping open reading frames of matrix 1 (M1)/M2 for the modified M segment and the open reading frames of nonstructural protein 1 (NS1)/nuclear export protein (NEP) for the modified NS segment were split by using the porcine teschovirus 1 (PTV-1) 2A autoproteolytic cleavage site. Viruses with an M split segment were impaired in replication at nonpermissive high temperatures, whereas high viral titers could be obtained at permissive low temperatures (33°C). Furthermore, viruses containing the M split segment were highly attenuatedin vivo, while they retained their immunogenicity and provided protection against a lethal challenge with wild-type PR8. These results indicate that influenza viruses can be effectively attenuated by the rearrangement of spliced segments and that such attenuated viruses represent an excellent option as safe, immunogenic, and protective live-attenuated vaccines. Moreover, this is the first time in which an influenza virus containing a restructured M segment has been described. Reorganization of the M segment to encode M1 and M2 from two separate, nonoverlapping, independent open reading frames represents a useful tool to independently study mutations in the M1 and M2 viral proteins without affecting the other viral M product.IMPORTANCEVaccination represents our best therapeutic option against influenza viral infections. However, the efficacy of current influenza vaccines is suboptimal, and novel approaches are necessary for the prevention of disease caused by this important human respiratory pathogen. In this work, we describe a novel approach to generate safer and more efficient live-attenuated influenza virus vaccines (LAIVs) based on recombinant viruses whose genomes encode nonoverlapping and independent M1/M2 (split M segment [Ms]) or both M1/M2 and NS1/NEP (Ms and split NS segment [NSs]) open reading frames. Viruses containing a modified M segment were highly attenuated in mice but were able to confer, upon a single intranasal immunization, complete protection against a lethal homologous challenge with wild-type virus. Notably, the protection efficacy conferred by our viruses with split M segments was better than that conferred by the current temperature-sensitive LAIV. Altogether, these results open a new avenue for the development of safer and more protective LAIVs on the basis of the reorganization of spliced viral RNA segments in the genome.

scholarly journals Identification of Candidate Gammaherpesvirus 68 Genes Required for Virus Replication by Signature-Tagged Transposon Mutagenesis

2004 ◽  
Vol 78 (19) ◽  
pp. 10282-10290 ◽  
Author(s):  
Nathaniel J. Moorman ◽  
Chie Yu Lin ◽  
Samuel H. Speck
Keyword(s):  
Life Cycle ◽  
Virus Replication ◽  
Mammalian Cells ◽  
Open Reading Frames ◽  
Wild Type Virus ◽  
Wild Type ◽  
Transposon Insertion ◽  
Gammaherpesvirus 68 ◽  
Reading Frames

ABSTRACT Current methods for determining the role of a given gene product in the gammaherpesvirus 68 (γHV68) life cycle require generation of a specific mutation by either homologous recombination in mammalian cells or bacterial artificial chromosome-mediated mutagenesis in Escherichia coli. The mutant virus is then compared to wild-type virus, and the role of the gene in the viral life cycle is deduced from its phenotype. This process is both time-consuming and labor intensive. Here we present the use of random, transposon-mediated signature-tagged mutagenesis for the identification of candidate viral genes involved in virus replication. Pools of viral mutants, each containing a random insertion of a transposon, were generated with a transposon donor library in which each transposon contains a unique sequence identifier. These pools were transfected into mammalian cells, and the ability of each mutant to replicate was assessed by comparing the presence of virus in the output pool to that present in the input pool of viral genomes. With this approach we could rapidly screen up to 96 individual mutants simultaneously. The location of the transposon insertion was determined by sequencing individual clones with a common primer specific for the transposon end. Here we present the characterization of 53 distinct viral mutants that correspond to insertions in 29 open reading frames within the γHV68 genome. To confirm the results of the signature-tagged mutagenesis screen, we quantitated the ability of each mutant to replicate compared to wild-type γHV68. From these analyses we identified 16 γHV68 open reading frames that, when disrupted by transposon insertions, score as essential for virus replication, and six other open reading frames whose disruption led to significant attenuation of virus replication. In addition, transposon insertion in five other γHV68 open reading frames did not affect virus replication. Notably, all but one of the candidate essential replication genes identified in this screen have been shown to be essential for the replication of at least one other herpesvirus.

scholarly journals Characterization of pif, a gene required for the per os infectivity of Spodoptera littoralis nucleopolyhedrovirus

2002 ◽  
Vol 83 (12) ◽  
pp. 3013-3022 ◽  
Author(s):  
Iryna Kikhno ◽  
Serafín Gutiérrez ◽  
Liliane Croizier ◽  
Guy Croizier ◽  
Miguel López Ferber
Keyword(s):  
Spodoptera Littoralis ◽  
Restriction Analysis ◽  
Virus Genome ◽  
Biological Properties ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Virus Infectivity ◽  
Type Virus ◽  
Structural Protein ◽  
Wild Type

During plaque purification of Spodoptera littoralis nucleopolyhedrovirus in S. littoralis Sl52 cell culture, a deletion mutant virus was isolated. Analysis of the biological properties of this mutant virus revealed an absence of per os infectivity of the occluded virus. Infectivity by injection of the non-occluded (budded) virus is not different between the wild-type and the deleted virus. Restriction analysis of the mutant virus genome revealed a 4·5 kb deletion within the NotI D fragment. The observed phenotype was mapped to the deleted region by rescue experiments. The deletion was characterized and the equivalent DNA fragment on the wild-type virus was sequenced. By co-transfecting the DNA of the deleted virus with plasmids derived from the wild-type virus, it was possible to determine that ORF 7 in this fragment is responsible for the observed phenotype. ORF 7, called pif (per os infectivity factor), is homologous to ORF 119 of Autographa californica nucleopolyhedrovirus. Similar ORFs are present in all sequenced baculoviruses. The product of this gene is an occlusion body-derived virion structural protein required only for the first steps of larva infection, as viruses being produced in cells expressing the gene but not containing it in their genomes are able to produce successful infections.

The URNA Genes of Herpesvirus Saimiri (Strain C488) Are Dispensable for Transformation of Human T Cells In Vitro

1999 ◽  
Vol 73 (12) ◽  
pp. 10551-10555 ◽  
Author(s):  
Armin Ensser ◽  
André Pfinder ◽  
Ingrid Müller-Fleckenstein ◽  
Bernhard Fleckenstein
Keyword(s):  
Cell Culture ◽  
Herpesvirus Saimiri ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Human T Cell ◽  
Human T Cells ◽  
Small Nuclear Rnas ◽  
T Cell Lines

ABSTRACT The herpesvirus saimiri strain C488 genome contains five genes for small nuclear RNAs, termed herpesvirus saimiri URNAs (or HSURs). Using a cosmid-based approach, all HSURs were precisely deleted from the genome. The mutant virus replicated at levels that were similar to those of wild-type viruses in OMK cells. Although the HSURs are expressed in wild-type virus-transformed human T-cell lines, the deletion does not affect viral transformation in cell culture.

scholarly journals Kinetics of Neutralizing Antibodies of COVID-19 Patients Tested Using Clinical D614G, B.1.1.7 and B 1.351 Isolates in Microneutralization Assays

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 996
Author(s):  
Jenni Virtanen ◽  
Ruut Uusitalo ◽  
Essi M. Korhonen ◽  
Kirsi Aaltonen ◽  
Teemu Smura ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Acute Infection ◽  
Clinical Isolates ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Kinetics Of

Increasing evidence suggests that some newly emerged SARS-CoV-2 variants of concern (VoCs) resist neutralization by antibodies elicited by the early-pandemic wild-type virus. We applied neutralization tests to paired recoveree sera (n = 38) using clinical isolates representing the first wave (D614G), VoC1, and VoC2 lineages (B.1.1.7 and B 1.351). Neutralizing antibodies inhibited contemporary and VoC1 lineages, whereas inhibition of VoC2 was reduced 8-fold, with 50% of sera failing to show neutralization. These results provide evidence for the increased potential of VoC2 to reinfect previously SARS-CoV-infected individuals. The kinetics of NAbs in different patients showed similar decline against all variants, with generally low initial anti-B.1.351 responses becoming undetectable, but with anti-B.1.1.7 NAbs remaining detectable (>20) for months after acute infection.

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