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 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 In Vivo Dynamics of Reporter Flaviviridae Viruses

2019 ◽  
Vol 93 (22) ◽  
Author(s):  
Tomokazu Tamura ◽  
Manabu Igarashi ◽  
Bazarragchaa Enkhbold ◽  
Tatsuya Suzuki ◽  
Masatoshi Okamatsu ◽  
...  
Keyword(s):  
Luciferase Activity ◽  
Viral Proteins ◽  
Solvent Accessible Surface Area ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Recombinant Viruses ◽  
Diarrhea Virus ◽  
Foreign Genes

ABSTRACT Recombinant viruses possessing reporter proteins have been generated for virus research. In the case of the family Flaviviridae, we recently generated recombinant viruses, including the hepatitis C virus of the genus Hepacivirus, Japanese encephalitis virus (JEV) of the genus Flavivirus, and bovine viral diarrhea virus of the genus Pestivirus; all three viruses possess an 11-amino-acid subunit derived from NanoLuc luciferase (HiBiT). Here, we further developed the recombinant viruses and investigated their utility in vivo. Recombinant viruses harboring HiBiT in the E, NS1, or NS3 protein constructed based on the predicted secondary structure, solvent-accessible surface area, and root mean square fluctuation of the proteins exhibited comparable replication to that of the wild-type virus in vitro. The recombinant JEV carrying HiBiT in the NS1 protein exhibited propagation in mice comparable to that of the parental virus, and propagation of the recombinant was monitored by the luciferase activity. In addition, the recombinants of classical swine fever virus (CSFV) possessing HiBiT in the Erns or E2 protein also showed propagation comparable to that of the wild-type virus. The recombinant CSFV carrying HiBiT in Erns exhibited similar replication to the parental CSFV in pigs, and detection of viral propagation of this recombinant by luciferase activity was higher than that by quantitative PCR (qPCR). Taken together, these results demonstrated that the reporter Flaviviridae viruses generated herein are powerful tools for elucidating the viral life cycle and pathogeneses and provide a robust platform for the development of novel antivirals. IMPORTANCE In vivo applications of reporter viruses are necessary to understand viral pathogenesis and provide a robust platform for antiviral development. In developing such applications, determination of an ideal locus to accommodate foreign genes is important, because insertion of foreign genes into irrelevant loci can disrupt the protein functions required for viral replication. Here, we investigated the criteria to determine ideal insertion sites of foreign genes from the protein structure of viral proteins. The recombinant viruses generated by our criteria exhibited propagation comparable to that of parental viruses in vivo. Our proteomic approach based on the flexibility profile of viral proteins may provide a useful tool for constructing reporter viruses, including Flaviviridae viruses.

scholarly journals Resistance of Human Cytomegalovirus to the Benzimidazole l-Ribonucleoside Maribavir Maps to UL27

2003 ◽  
Vol 77 (21) ◽  
pp. 11499-11506 ◽  
Author(s):  
Gloria Komazin ◽  
Roger G. Ptak ◽  
Brian T. Emmer ◽  
Leroy B. Townsend ◽  
John C. Drach
Keyword(s):  
Dna Synthesis ◽  
Human Cytomegalovirus ◽  
Resistance Mutation ◽  
Open Reading Frames ◽  
Cosmid Library ◽  
Wild Type Virus ◽  
Wild Type ◽  
Nuclear Egress ◽  
The One

ABSTRACT 1-(β-d-Ribofuranosyl)-2,5,6-trichlorobenzimidazole (TCRB) and its 2-bromo analog, BDCRB, are potent and selective inhibitors of human cytomegalovirus (HCMV) DNA processing and packaging. Since they are readily metabolized in vivo, analogs were synthesized to improve biostability. One of these, 1-(β-l-ribofuranosyl)-2-isopropylamino-5,6-dichlorobenzimidazole (1263W94; maribavir), inhibits viral DNA synthesis and nuclear egress. Resistance to maribavir was mapped to UL97, and this viral kinase was shown to be a direct target of maribavir. In the present study, an HCMV strain resistant to TCRB and BDCRB was passaged in increasing concentrations of maribavir, and resistant virus was isolated. This strain (G2) grew at the same rate as the wild-type virus and was resistant to both BDCRB and maribavir. Resistance to BDCRB was expected, because the parent strain from which G2 was isolated was resistant due to known mutations in UL56 and UL89. However, no mutations were found in UL97 or other relevant open reading frames that could explain resistance to maribavir. Because sequencing of selected HCMV genes did not identify the resistance mutation, a cosmid library was made from G2, and a series of recombinant G2 wild-type viruses were constructed. Testing the recombinants for sensitivity to maribavir narrowed the locus of resistance to genes UL26 to UL32. Sequencing identified a single coding mutation in ORF UL27 (Leu335Pro) as the one responsible for resistance to maribavir. These results establish that UL27 is either directly or indirectly involved in the mechanism of action of maribavir. They also suggest that UL27 could play a role in HCMV DNA synthesis or egress of HCMV particles from the nucleus.

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 The Virology of Taterapox Virus In Vitro

Viruses ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 463 ◽  
Author(s):  
Scott Parker ◽  
Leonardo Camilo de Oliveira ◽  
Elliot Lefkowitz ◽  
Robert Hendrickson ◽  
Cláudio Bonjardim ◽  
...  
Keyword(s):  
Open Reading Frames ◽  
Wild Type ◽  
In Vitro Techniques ◽  
Cytopathic Effects ◽  
Inapparent Infection ◽  
Primate Cell ◽  
Cytoskeletal Networks ◽  
Reading Frames

Taterapox virus (TATV) is phylogenetically the closest related virus to variola—the etiological agent of smallpox. Despite the similarity, few studies have evaluated the virus. In vivo, TATV can infect several animals but produces an inapparent infection in wild-type mice; however, TATV does cause morbidity and mortality in some immunocompromised strains. We employed in vitro techniques to compare TATV to ectromelia (ECTV) and vaccinia (VACV) viruses. Both ECTV and TATV replicate efficiently in primate cell lines but TATV replicates poorly in murine cells lines. Furthermore, TATV induces cytopathic effects, but to a lesser extent than ECTV, and changes cytoskeletal networks differently than both ECTV and VACV. Bioinformatic studies revealed differences in several immunomodulator open reading frames that could contribute to the reduced virulence of TATV, which were supported by in vitro cytokine assays.

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 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 A Replication-Defective Gammaherpesvirus Efficiently Establishes Long-Term Latency in Macrophages but Not in B Cells In Vivo

2008 ◽  
Vol 82 (17) ◽  
pp. 8500-8508 ◽  
Author(s):  
Haiyan Li ◽  
Kazufumi Ikuta ◽  
John W. Sixbey ◽  
Scott A. Tibbetts
Keyword(s):  
B Cells ◽  
Viral Genome ◽  
Lytic Replication ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Virus Latency

ABSTRACT Murine gammaherpesvirus 68 (γHV68 or MHV68) is genetically related to the human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), providing a useful system for in vivo studies of the virus-host relationship. To begin to address fundamental questions about the mechanisms of the establishment of gammaherpesvirus latency, we previously generated a replication-defective γHV68 lacking the expression of the single-stranded DNA binding protein encoded by orf6. In work presented here, we demonstrate that this mutant virus established a long-term infection in vivo that was molecularly identical to wild-type virus latency. Thus, despite the absence of an acute phase of lytic replication, the mutant virus established a chronic infection in which the viral genome (i) was maintained as an episome and (ii) expressed latency-associated, but not lytic replication-associated, genes. Macrophages purified from mice infected with the replication-defective virus harbored viral genome at a frequency that was nearly identical to that of wild-type γHV68; however, the frequency of B cells harboring viral genome was greatly reduced in the absence of lytic replication. Thus, this replication-defective gammaherpesvirus efficiently established in vivo infection in macrophages that was molecularly indistinguishable from wild-type virus latency. These data point to a critical role for lytic replication or reactivation in the establishment or maintenance of latent infection in B cells.

scholarly journals Bias between the Left and Right Inverted Repeats during IS911 Targeted Insertion

2008 ◽  
Vol 190 (18) ◽  
pp. 6111-6118 ◽  
Author(s):  
P. Rousseau ◽  
C. Loot ◽  
C. Turlan ◽  
S. Nolivos ◽  
M. Chandler
Keyword(s):  
Insertion Sequence ◽  
Regulatory Protein ◽  
Open Reading Frames ◽  
Inverted Repeats ◽  
Functional Differences ◽  
Left And Right ◽  
Ordered Assembly ◽  
Overlapping Open Reading Frames ◽  
Reading Frames

ABSTRACT IS911 is a bacterial insertion sequence composed of two consecutive overlapping open reading frames (ORFs [orfA and orfB]) encoding the transposase (OrfAB) as well as a regulatory protein (OrfA). These ORFs are bordered by terminal left and right inverted repeats (IRL and IRR, respectively) with several differences in nucleotide sequence. IS911 transposition is asymmetric: each end is cleaved on one strand to generate a free 3′-OH, which is then used as the nucleophile in attacking the opposite insertion sequence (IS) end to generate a free IS circle. This will be inserted into a new target site. We show here that the ends exhibit functional differences which, in vivo, may favor the use of one compared to the other during transposition. Electromobility shift assays showed that a truncated form of the transposase [OrfAB(1-149)] exhibits higher affinity for IRR than for IRL. While there was no detectable difference in IR activities during the early steps of transposition, IRR was more efficient during the final insertion steps. We show here that the differential activities between the two IRs correlate with the different affinities of OrfAB(1-149) for the IRs during assembly of the nucleoprotein complexes leading to transposition. We conclude that the two inverted repeats are not equivalent during IS911 transposition and that this asymmetry may intervene to determine the ordered assembly of the different protein-DNA complexes involved in the reaction.

scholarly journals Genomic Sequence of Rhesus Cytomegalovirus 180.92: Insights into the Coding Potential of Rhesus Cytomegalovirus

2006 ◽  
Vol 80 (8) ◽  
pp. 4179-4182 ◽  
Author(s):  
Pierre Rivailler ◽  
Amitinder Kaur ◽  
R. Paul Johnson ◽  
Fred Wang
Keyword(s):  
Genomic Sequence ◽  
Open Reading Frames ◽  
Rhesus Cytomegalovirus ◽  
Human Cmv ◽  
Coding Capacity ◽  
Coding Potential ◽  
Reading Frames ◽  
Genetic Rearrangements

ABSTRACT A pathogenic isolate of rhesus cytomegalovirus (rhCMV 180.92) was cloned, sequenced, and annotated. Comparisons with the published rhCMV 68.1 genome revealed 8 open reading frames (ORFs) in isolate 180.92 that are absent in 68.1, 10 ORFs in 68.1 that are absent in 180.92, and 34 additional ORFs that were not previously annotated. Most of the differences appear to be due to genetic rearrangements in both isolates from a region that is frequently altered in human CMV (hCMV) during in vitro passage. These results indicate that the rhCMV ORF repertoire is larger than previously recognized. Like hCMV, understanding of the complete coding capacity of rhCMV is complicated by genomic instability and may require comparisons with additional isolates in vitro and in vivo.

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