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Journal articles on the topic 'Capsid coding region'
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1
Chinsangaram, Jarasvech, Clayton Beard, Peter W. Mason, Marla K. Zellner, Gordon Ward, and Marvin J. Grubman. "Antibody Response in Mice Inoculated with DNA Expressing Foot-and-Mouth Disease Virus Capsid Proteins." Journal of Virology 72, no. 5 (May 1, 1998): 4454–57. http://dx.doi.org/10.1128/jvi.72.5.4454-4457.1998.
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ABSTRACT Candidate foot-and-mouth disease (FMD) DNA vaccines designed to produce viral capsids lacking infectious viral nucleic acid were evaluated. Plasmid DNAs containing a portion of the FMDV genome coding for the capsid precursor protein (P1-2A) and wild-type or mutant viral proteinase 3C (plasmids P12X3C or P12X3C-mut, respectively) were constructed. Cell-free translation reactions programmed with pP12X3C (wild-type 3C) and pP12X3C-mut produced a capsid precursor, but only the reactions programmed with the plasmid encoding the functional proteinase resulted in P1-2A processing and capsid formation. Baby hamster kidney (BHK) cells also produced viral capsid proteins when transfected with these plasmids. Plasmid P12X3C was administered to mice by intramuscular, intradermal, and epithelial (gene gun) inoculations. Anti-FMD virus (FMDV) antibodies were detected by radioimmunoprecipitation (RIP) and plaque reduction neutralization assays only in sera of mice inoculated by using a gene gun. When pP12X3C and pP12X3C-mut were inoculated into mice by using a gene gun, both plasmids elicited an antibody response detectable by RIP but only pP12X3C elicited a neutralizing antibody response. These results suggest that capsid formation in situ is required for effective immunization. Expression and stimulation of an immune response was enhanced by addition of an intron sequence upstream of the coding region, while addition of the FMDV internal ribosome entry site or leader proteinase (L) coding region either had no effect or reduced the immune response.
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Jia, Xi-Yu, Marc Van Eden, Marc G. Busch, Ellie Ehrenfeld, and Donald F. Summers. "trans-Encapsidation of a Poliovirus Replicon by Different Picornavirus Capsid Proteins." Journal of Virology 72, no. 10 (October 1, 1998): 7972–77. http://dx.doi.org/10.1128/jvi.72.10.7972-7977.1998.
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ABSTRACT A trans-encapsidation assay was established to study the specificity of picornavirus RNA encapsidation. A poliovirus replicon with the luciferase gene replacing the capsid protein-coding region was coexpressed in transfected HeLa cells with capsid proteins from homologous or heterologous virus. Successfultrans-encapsidation resulted in assembly and production of virions whose replication, upon subsequent infection of HeLa cells, was accompanied by expression of luciferase activity. The amount of luciferase activity was proportional to the amount oftrans-encapsidated virus produced from the cotransfection. When poliovirus capsid proteins were supplied in trans, >2 × 106 infectious particles/ml were produced. When coxsackievirus B3, human rhinovirus 14, mengovirus, or hepatitis A virus (HAV) capsid proteins were supplied in trans, all but HAV showed some encapsidation of the replicon. The overall encapsidation efficiency of the replicon RNA by heterologous capsid proteins was significantly lower than when poliovirus capsid was used.trans-encapsidated particles could be completely neutralized with specific antisera against each of the donor virus capsids. The results indicate that encapsidation is regulated by specific viral nucleic acid and protein sequences.
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Bouslama, Lamjed, Dorsaf Nasri, Lionel Chollet, Khaoula Belguith, Thomas Bourlet, Mahjoub Aouni, Bruno Pozzetto, and Sylvie Pillet. "Natural Recombination Event within the Capsid Genomic Region Leading to a Chimeric Strain of Human Enterovirus B." Journal of Virology 81, no. 17 (May 30, 2007): 8944–52. http://dx.doi.org/10.1128/jvi.00180-07.
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ABSTRACT Recombination between two strains is a known phenomenon for enteroviruses replicating within a single cell. We describe a recombinant strain recovered from human stools, typed as coxsackievirus B4 (CV-B4) and CV-B3 after partial sequencing of the VP1 and VP2 coding regions, respectively. The strain was neutralized by a polyclonal CV-B3-specific antiserum but not by a CV-B4-specific antiserum. The nucleotide sequence analysis of the whole structural genomic region showed the occurrence of a recombination event at position 1950 within the VP3 capsid gene, in a region coding for the 2b antigenic site previously described for CV-B3. This observation evidences for the first time the occurrence of an interserotypic recombination within the VP2-VP3-VP1 capsid region between two nonpoliovirus enterovirus strains. The neutralization pattern suggests that the major antigenic site is located within the VP2 protein.
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Déjardin, Jérôme, Guillaume Bompard-Maréchal, Muriel Audit, Thomas J. Hope, Marc Sitbon, and Marylène Mougel. "A Novel Subgenomic Murine Leukemia Virus RNA Transcript Results from Alternative Splicing." Journal of Virology 74, no. 8 (April 15, 2000): 3709–14. http://dx.doi.org/10.1128/jvi.74.8.3709-3714.2000.
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ABSTRACT Here we show the existence of a novel subgenomic 4.4-kb RNA in cells infected with the prototypic replication-competent Friend or Moloney murine leukemia viruses (MuLV). This RNA derives by splicing from an alternative donor site (SD′) within the capsid-coding region to the canonical envelope splice acceptor site. The position and the sequence of SD′ was highly conserved among mammalian type C and D oncoviruses. Point mutations used to inactivate SD′ without changing the capsid-coding ability affected viral RNA splicing and reduced viral replication in infected cells.
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Sasaki, Jun, and Nobuhiko Nakashima. "Translation Initiation at the CUU Codon Is Mediated by the Internal Ribosome Entry Site of an Insect Picorna-Like Virus In Vitro." Journal of Virology 73, no. 2 (February 1, 1999): 1219–26. http://dx.doi.org/10.1128/jvi.73.2.1219-1226.1999.
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ABSTRACT AUG-unrelated translation initiation was found in an insect picorna-like virus, Plautia stali intestine virus (PSIV). The positive-strand RNA genome of the virus contains two nonoverlapping open reading frames (ORFs). The capsid protein gene is located in the 3′-proximal ORF and lacks an AUG initiation codon. We examined the translation mechanism and the initiation codon of the capsid protein gene by using various dicistronic and monocistronic RNAs in vitro. The capsid protein gene was translated cap independently in the presence of the upstream cistron, indicating that the gene is translated by internal ribosome entry. Deletion analysis showed that the internal ribosome entry site (IRES) consisted of approximately 250 bases and that its 3′ boundary extended slightly into the capsid-coding region. The initiation codon for the IRES-mediated translation was identified as the CUU codon, which is located just upstream of the 5′ terminus of the capsid-coding region by site-directed mutagenesis. In vitro translation assays of monocistronic RNAs lacking the 5′ part of the IRES showed that this CUU codon was not recognized by scanning ribosomes. This suggests that the PSIV IRES can effectively direct translation initiation without stable codon-anticodon pairing between the initiation codon and the initiator methionyl-tRNA.
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Dahourou, George, Sophie Guillot, Olivier Le Gall, and Radu Crainic. "Genetic recombination in wild-type poliovirus." Journal of General Virology 83, no. 12 (December 1, 2002): 3103–10. http://dx.doi.org/10.1099/0022-1317-83-12-3103.
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Poliovirus isolates were screened for recombinants by combined analysis of two distant polymorphic segments of the poliovirus genome (one in the capsid and the other in the polymerase-coding region). Using a restriction fragment length polymorphism (RFLP) assay, a high number of recombinant genomes was found among vaccine-derived strains excreted by poliovirus vaccine vaccinees or vaccine-associated paralytic poliomyelitis cases. Some of these subjects carried a wild-type poliovirus (non-vaccine-specific) nucleotide sequence in the 3′ part of the genome. Using a similar approach, a collection of wild-type poliovirus strains isolated in South India between 1985 and 1993 was screened for recombinants. Genotypes were defined by the parallel application of RFLP assays and genomic sequencing of the capsid protein VP1 and the 3D polymerase polypeptide. Analyses revealed several instances where the position of an isolate on the phylogenic tree for the capsid protein-coding segment did not agree with its position on the tree for the polymerase-coding region. In this way, several wild-type/wild-type and wild-type/vaccine recombinants could be identified, indicating that recombination is encountered commonly in the natural evolution of poliovirus strains.
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Savolainen, Carita, Pia Laine, Mick N. Mulders, and Tapani Hovi. "Sequence analysis of human rhinoviruses in the RNA-dependent RNA polymerase coding region reveals large within-species variation." Journal of General Virology 85, no. 8 (August 1, 2004): 2271–77. http://dx.doi.org/10.1099/vir.0.79897-0.
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Human rhinoviruses (HRVs; family Picornaviridae), the most frequent causative agents of respiratory infections, comprise more than 100 distinct serotypes. According to previous phylogenetic analysis of the VP4/VP2-coding sequences, all but one of the HRV prototype strains distribute between the two established species, Human rhinovirus A (HRV-A) and Human rhinovirus B (HRV-B). Here, partial sequences of the RNA-dependent RNA polymerase (3D polymerase)-coding gene of 48 HRV prototype strains and 12 field isolates were analysed. The designated division of the HRV strains into the species HRV-A and HRV-B was also seen in the 3D-coding region. Phylogenetically, HRV-B clustered closer to human enterovirus (HEV) species HEV-B, HEV-C and poliovirus than to HRV-A. Intraspecies variation within both HRV-A and HRV-B was greater in the 3D-coding region than in the VP4/VP2-coding region, with the difference maxima reaching 48 % at the nucleotide level and 36 % at the amino acid level in HRV-A and 53 and 35 %, respectively, in HRV-B. Within both species, a few strains formed a separate cluster differing from the majority of strains as much as HEV-B from HEV-C. Furthermore, the tree topology within HRV-A differed from that for VP4/VP2, suggesting possible recombination events in the evolutionary history of the strains. However, all 12 field isolates clustered similarly, as in the capsid region. These results showed that the within-species variation in the 3D region is greater in HRV than in HEV. Furthermore, HRV variation in the 3D region exceeds that in the capsid-coding region.
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Harvala, Heli, Hannu Kalimo, Leif Dahllund, Juhana Santti, Pamela Hughes, Timo Hyypiä, and Glyn Stanway. "Mapping of tissue tropism determinants in coxsackievirus genomes." Journal of General Virology 83, no. 7 (July 1, 2002): 1697–706. http://dx.doi.org/10.1099/0022-1317-83-7-1697.
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Genomic regions responsible for the different tissue tropisms of coxsackievirus A9 (CAV9) and coxsackievirus B3 (CBV3) in newborn mice were investigated using recombinant viruses. Infectious cDNA clones of CAV9, a virus known to infect striated muscle, and CBV3, affecting the central nervous system, pancreas, liver, brown fat and striated muscle, were used to generate chimeric viruses. In situ hybridization analysis of different tissues from mice infected with the recombinant viruses, constructed by exchanging the 5′ non-coding region (5′NCR), structural and non-structural genes, demonstrated that the pancreo- and liver tropism map predominantly to CBV3 sequences within the capsid genes, evidently due to receptor recognition. Although the major neurotropism determinant in the CBV3 genome was in the capsid region, viruses containing the CAV9 capsid were also able to initiate infection in the central nervous system provided they contained the CBV3 5′NCR. The presence of the 5′NCR of CAV9 clearly enhanced muscle tissue tropism.
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Shibuya, Norihiro, Takashi Nishiyama, Yasushi Kanamori, Hitoshi Saito, and Nobuhiko Nakashima. "Conditional Rather than Absolute Requirements of the Capsid Coding Sequence for Initiation of Methionine-Independent Translation in Plautia stali Intestine Virus." Journal of Virology 77, no. 22 (November 15, 2003): 12002–10. http://dx.doi.org/10.1128/jvi.77.22.12002-12010.2003.
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ABSTRACT The positive-stranded RNA genome of Plautia stali intestine virus (PSIV) has an internal ribosome entry site (IRES) in an intergenic region (IGR). The IGR-IRES of PSIV initiates translation of the capsid protein by using CAA, the codon for glutamine. It was previously reported (J. Sasaki and N. Nakashima, J. Virol. 73:1219-1226, 1999) that IGR-IRES extended by several nucleotides into the capsid open reading frame (ORF). Despite the fact that the secondary structure model of the IGR-IRES is highly conserved, we were unable to find structural similarities in the 5′ region of the capsid ORFs in related viruses. Therefore, we reevaluated the role of the capsid ORF in IGR-IRES-mediated translation in PSIV. Mutation of the CAA codon with various triplets did not inhibit IGR-IRES-mediated translation. N-terminal amino acid analyses of mutated products showed that the IGR-IRES could initiate translation by using various elongator tRNAs. By replacement of the capsid ORF with exogenous coding sequences having AUG deleted, translation products were produced in most cases, but capsid-exogenous fusion proteins were produced more efficiently than were the translation products. These data indicate that the 5′ part of the capsid ORF is not an absolute requirement for the IGR-IRES-mediated translation. RNA structure probing analyses showed that the 5′ part of the capsid ORF was a single strand, while that of exogenous reading frames was structured. Exogenous sequences also caused structural distortion in the 3′ part of the IGR-IRES. We hypothesize that the single-stranded capsid ORF helps to form the tertiary structure of the IGR-IRES and facilitates precise positioning of ribosomes.
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Audit, Muriel, Jérôme Déjardin, Barbara Hohl, Christine Sidobre, Thomas J. Hope, Marylène Mougel, and Marc Sitbon. "Introduction of a cis-Acting Mutation in the Capsid-Coding Gene of Moloney Murine Leukemia Virus Extends Its Leukemogenic Properties." Journal of Virology 73, no. 12 (December 1, 1999): 10472–79. http://dx.doi.org/10.1128/jvi.73.12.10472-10479.1999.
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ABSTRACT Inoculation of newborn mice with the retrovirus Moloney murine leukemia virus (MuLV) results in the exclusive development of T lymphomas with gross thymic enlargement. The T-cell leukemogenic property of Moloney MuLV has been mapped to the U3 enhancer region of the viral promoter. However, we now describe a mutant Moloney MuLV which can induce the rapid development of a uniquely broad panel of leukemic cell types. This mutant Moloney MuLV with synonymous differences (MSD1) was obtained by introduction of nucleotide substitutions at positions 1598, 1599, and 1601 in the capsid gene which maintained the wild-type (WT) coding potential. Leukemias were observed in all MSD1-inoculated animals after a latency period that was shorter than or similar to that of WT Moloney MuLV. Importantly, though, only 56% of MSD1-induced leukemias demonstrated the characteristic thymoma phenotype observed in all WT Moloney MuLV leukemias. The remainder of MSD1-inoculated animals presented either with bona fide clonal erythroid or myelomonocytic leukemias or, alternatively, with other severe erythroid and unidentified disorders. Amplification and sequencing of U3 and capsid-coding regions showed that the inoculated parental MSD1 sequences were conserved in the leukemic spleens. This is the first report of a replication-competent MuLV lacking oncogenes which can rapidly lead to the development of such a broad range of leukemic cell types. Moreover, the ability of MSD1 to transform erythroid and myelomonocytic lineages is not due to changes in the U3 viral enhancer region but rather is the result of acis-acting effect of the capsid-coding gagsequence.
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Martín, Javier, Elena Samoilovich, Glynis Dunn, Angie Lackenby, Esphir Feldman, Alan Heath, Ekaterina Svirchevskaya, Gill Cooper, Marina Yermalovich, and Philip D. Minor. "Isolation of an Intertypic Poliovirus Capsid Recombinant from a Child with Vaccine-Associated Paralytic Poliomyelitis." Journal of Virology 76, no. 21 (November 1, 2002): 10921–28. http://dx.doi.org/10.1128/jvi.76.21.10921-10928.2002.
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ABSTRACT The isolation of a capsid intertypic poliovirus recombinant from a child with vaccine-associated paralytic poliomyelitis is described. Virus 31043 had a Sabin-derived type 3-type 2-type 1 recombinant genome with a 5′-end crossover point within the capsid coding region. The result was a poliovirus chimera containing the entire coding sequence for antigenic site 3a derived from the Sabin type 2 strain. The recombinant virus showed altered antigenic properties but did not acquire type 2 antigenic characteristics. The significance of the presence in nature of such poliovirus chimeras and the consequences for the current efforts to detect potentially dangerous vaccine-derived poliovirus strains are discussed in the context of the global polio eradication initiative.
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Yong, Xin Ee, V. Raghuvamsi Palur, Ganesh S. Anand, Thorsten Wohland, and Kamal K. Sharma. "Dengue virus 2 capsid protein chaperones the strand displacement of 5′-3′ cyclization sequences." Nucleic Acids Research 49, no. 10 (May 25, 2021): 5832–44. http://dx.doi.org/10.1093/nar/gkab379.
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Abstract By virtue of its chaperone activity, the capsid protein of dengue virus strain 2 (DENV2C) promotes nucleic acid structural rearrangements. However, the role of DENV2C during the interaction of RNA elements involved in stabilizing the 5′-3′ panhandle structure of DENV RNA is still unclear. Therefore, we determined how DENV2C affects structural functionality of the capsid-coding region hairpin element (cHP) during annealing and strand displacement of the 9-nt cyclization sequence (5CS) and its complementary 3CS. cHP has two distinct functions: a role in translation start codon selection and a role in RNA synthesis. Our results showed that cHP impedes annealing between 5CS and 3CS. Although DENV2C does not modulate structural functionality of cHP, it accelerates annealing and specifically promotes strand displacement of 3CS during 5′-3′ panhandle formation. Furthermore, DENV2C exerts its chaperone activity by favouring one of the active conformations of cHP. Based on our results, we propose mechanisms for annealing and strand displacement involving cHP. Thus, our results provide mechanistic insights into how DENV2C regulates RNA synthesis by modulating essential RNA elements in the capsid-coding region, that in turn allow for DENV replication.
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Brown, Betty, M. Steven Oberste, Kaija Maher, and Mark A. Pallansch. "Complete Genomic Sequencing Shows that Polioviruses and Members of Human Enterovirus Species C Are Closely Related in the Noncapsid Coding Region." Journal of Virology 77, no. 16 (August 15, 2003): 8973–84. http://dx.doi.org/10.1128/jvi.77.16.8973-8984.2003.
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ABSTRACT The 65 human enterovirus serotypes are currently classified into five species: Poliovirus (3 serotypes), Human enterovirus A (HEV-A) (12 serotypes), HEV-B (37 serotypes), HEV-C (11 serotypes), and HEV-D (2 serotypes). Coxsackie A virus (CAV) serotypes 1, 11, 13, 15, 17, 18, 19, 20, 21, 22, and 24 constitute HEV-C. We have determined the complete genome sequences for the remaining nine HEV-C serotypes and compared them with the complete sequences of CAV21, CAV24, and the polioviruses. The viruses were most diverse in the capsid region (4 to 36% amino acid difference). A high degree of capsid sequence conservation (96% amino acid identity) suggests that CAV15 and CAV18 should be classified as strains of CAV11 and CAV13, respectively. In the 3CD region, CAV1, CAV19, and CAV22 differed from one another by only 1.2 to 1.4% and CAV11, CAV13, CAV17, CAV20, CAV21, CAV24, and the polioviruses differed from one another by only 1.2 to 3.6%. The two groups, however, differed from one another by 14.6 to 16.2%. The polioviruses as a group were monophyletic only in the capsid region. Only one group of serotypes (CAV1, CAV19, and CAV22) was consistently monophyletic in multiple genome regions. Incongruities among phylogenetic trees based on different genome regions strongly suggest that recombination has occurred between the polioviruses, CAV11, CAV13, CAV17, and CAV20. The close relationship among the polioviruses and CAV11, CAV13, CAV17, CAV20, CAV21, and CAV24 and the uniqueness of CAV1, CAV19, and CAV22 suggest that revisions should be made to the classification of these viruses.
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Blomqvist, Soile, Carita Savolainen-Kopra, Anja Paananen, Tapani Hovi, and Merja Roivainen. "Molecular characterization of human rhinovirus field strains isolated during surveillance of enteroviruses." Journal of General Virology 90, no. 6 (June 1, 2009): 1371–81. http://dx.doi.org/10.1099/vir.0.008508-0.
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Human rhinoviruses (HRVs), which are the most frequent causative agents of acute upper respiratory tract infections, are abundant worldwide. We have identified HRV strains in environmental specimens collected in Finland, Latvia and Slovakia during the surveillance of polio- and other enteroviruses. These acid-sensitive HRV strains were isolated under conditions optimized for growth of most of the enteroviruses, i.e. in stationary human rhabdomyosarcoma cells incubated at 36 °C. Phylogenetic analysis of the sequences derived from the partial 5′ non-coding region and the capsid region coding for proteins VP4/VP2 and VP1 showed that the HRV field strains clustered together with prototype strains of the HRV minor receptor group. Partial sequences of the 3D polymerase coding region generally followed this pattern, with the exception of a set of three HRV field strains that formed a subcluster not close to any of the established HRV-A types, suggesting that recombination may have occurred during evolution of these HRV strains. Phylogenetic analysis of the VP4/VP2 capsid protein coding region showed that the ‘environmental’ HRV field strains were practically identical to HRV strains recently sequenced by others in Australia, the United States and Japan. Analysis of amino acids corresponding to the intercellular adhesion molecule-1 receptor footprint in major receptor group HRVs and also in the low-density lipoprotein receptor footprint of minor receptor group HRVs showed conservation of the ‘minor receptor group-like’ amino acids, indicating that the field strains may have maintained their minor receptor group specificity.
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Santti, Juhana, Timo Hyypiä, Leena Kinnunen, and Mika Salminen. "Evidence of Recombination among Enteroviruses." Journal of Virology 73, no. 10 (October 1, 1999): 8741–49. http://dx.doi.org/10.1128/jvi.73.10.8741-8749.1999.
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ABSTRACT Human enteroviruses consist of more than 60 serotypes, reflecting a wide range of evolutionary divergence. They have been genetically classified into four clusters on the basis of sequence homology in the coding region of the single-stranded RNA genome. To explore further the genetic relationships between human enteroviruses and to characterize the evolutionary mechanisms responsible for variation, previously sequenced genomes were subjected to detailed comparison. Bootstrap and genetic similarity analyses were used to systematically scan the alignments of complete genomic sequences. Bootstrap analysis provided evidence from an early recombination event at the junction of the 5′ noncoding and coding regions of the progenitors of the current clusters. Analysis within the genetic clusters indicated that enterovirus prototype strains include intraspecies recombinants. Recombination breakpoints were detected in all genomic regions except the capsid protein coding region. Our results suggest that recombination is a significant and relatively frequent mechanism in the evolution of enterovirus genomes.
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McInerney, Gerald M., Andrew M. Q. King, Natalie Ross-Smith, and Graham J. Belsham. "Replication-competent foot-and-mouth disease virus RNAs lacking capsid coding sequences." Microbiology 81, no. 7 (July 1, 2000): 1699–702. http://dx.doi.org/10.1099/0022-1317-81-7-1699.
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RNA transcripts were prepared from plasmids encoding an infectious cDNA of foot-and-mouth disease virus (FMDV) or derivatives in which the leader (Lab and Lb) and capsid protein coding sequences were deleted or replaced by sequences encoding chloramphenicol acetyltransferase (CAT). The transcripts were electroporated into BHK cells and the expression of CAT and the FMDV 3C protease was monitored. Detection of CAT and 3C was dependent on the ability of the transcript to replicate. All of the Lb coding sequence and 94% of P1 (the capsid protein precursor) coding sequence could be deleted without any apparent effect on the ability of the RNA to replicate. Thus, no cis-acting replication element is present within this region of the FMDV genome. Trans-encapsidation of these FMDV replicons was very inefficient, which may explain the lack of production of defective-interfering particles in FMDV-infected cells.
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Clyde, Karen, and Eva Harris. "RNA Secondary Structure in the Coding Region of Dengue Virus Type 2 Directs Translation Start Codon Selection and Is Required for Viral Replication." Journal of Virology 80, no. 5 (March 1, 2006): 2170–82. http://dx.doi.org/10.1128/jvi.80.5.2170-2182.2006.
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ABSTRACT Dengue virus is a positive-strand RNA virus and a member of the genus Flavivirus, which includes West Nile, yellow fever, and tick-borne encephalitis viruses. Flavivirus genomes are translated as a single polyprotein that is subsequently cleaved into 10 proteins, the first of which is the viral capsid (C) protein. Dengue virus type 2 (DENV2) and other mosquito-borne flaviviruses initiate translation of C from a start codon in a suboptimal context and have multiple in-frame AUGs downstream. Here, we show that an RNA hairpin structure in the capsid coding region (cHP) directs translation start site selection in human and mosquito cells. The ability of the cHP to direct initiation from the first start codon is proportional to its thermodynamic stability, is position dependent, and is sequence independent, consistent with a mechanism in which the scanning initiation complex stalls momentarily over the first AUG as it begins to unwind the cHP. The cHP of tick-borne flaviviruses is not maintained in a position to influence start codon selection, which suggests that this coding region cis element may serve another function in the flavivirus life cycle. Here, we demonstrate that the DENV2 cHP and both the first and second AUGs of C are necessary for efficient viral replication in human and mosquito cells. While numerous regulatory elements have been identified in the untranslated regions of RNA viral genomes, we show that the cHP is a coding-region RNA element that directs start codon selection and is required for viral replication.
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López-Bueno, Alberto, Noelia Valle, Jesús M. Gallego, Joel Pérez, and José M. Almendral. "Enhanced Cytoplasmic Sequestration of the Nuclear Export Receptor CRM1 by NS2 Mutations Developed in the Host Regulates Parvovirus Fitness." Journal of Virology 78, no. 19 (October 1, 2004): 10674–84. http://dx.doi.org/10.1128/jvi.78.19.10674-10684.2004.
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ABSTRACT To investigate whether a DNA virus can evade passive immunotherapy with a polyclonal antiserum, we analyzed the protection of a neutralizing capsid antiserum against a lethal infection of the immunosuppressive strain of the parvovirus minute virus of mice (MVMi) in 42 immunodeficient mice over a period of 200 days. A few mice were effectively protected, but most developed a delayed lethal leukopenic syndrome during the treatment or weeks afterwards. Unexpectedly, viruses isolated from treated but also from control leukopenic mice showed no amino acid changes throughout the entire capsid coding region, although the viral populations were genetically heterogeneous, mainly in the second exon of the coding sequence of the NS2 nonstructural protein. The NS2 point amino acid changes (T88A, K96E, L103P, and L153 M) that were consistently selected in several mice clustered within the nuclear exportin CRM1 binding domain, in a reading frame that did not alter the overlapping NS1 coding region. These mutations endowed emerging viruses with an increased fitness that was demonstrable by their relative resistance to the neutralizing capsid antiserum in a postentry plaque-forming assay, the rapid overgrowth of a competing wild-type (wt) population in culture, and a larger yield of infectious particles. Mutant NS2 proteins interacted with a higher affinity and sequestered CRM1 in the perinuclear region of the cytoplasm more efficiently than the wt. Correspondingly this phenomenon, as well as the following timely ordered release of the NS1 nonstructural protein and the empty capsid from the nucleus to the cytoplasm, occurred markedly earlier in the infection cycle of the mutant viruses. We hypothesize that the enhanced cytoplasmic sequestration of CRM1 by the NS2 mutations selected in mice may trigger pleiotropic effects leading to an accelerated MVMi life cycle and thus to increased fitness. These results strengthen our earlier report on the rapid evolutionary capacity of this mammalian-specific DNA virus in vivo and indicate that the NS2-CRM1 interaction is an important determinant of parvovirus virulence that can be modulated in nature, hampering the effectiveness of passive antibody therapies in the long term.
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Arita, Minetaro, David R. Kilpatrick, Tomofumi Nakamura, Cara C. Burns, David Bukbuk, Soji B. Oderinde, M. Steven Oberste, Olen M. Kew, Mark A. Pallansch, and Hiroyuki Shimizu. "Development of an Efficient Entire-Capsid-Coding-Region Amplification Method for Direct Detection of Poliovirus from Stool Extracts." Journal of Clinical Microbiology 53, no. 1 (October 22, 2014): 73–78. http://dx.doi.org/10.1128/jcm.02384-14.
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Laboratory diagnosis has played a critical role in the Global Polio Eradication Initiative since 1988, by isolating and identifying poliovirus (PV) from stool specimens by using cell culture as a highly sensitive system to detect PV. In the present study, we aimed to develop a molecular method to detect PV directly from stool extracts, with a high efficiency comparable to that of cell culture. We developed a method to efficiently amplify the entire capsid coding region of human enteroviruses (EVs) including PV. cDNAs of the entire capsid coding region (3.9 kb) were obtained from as few as 50 copies of PV genomes. PV was detected from the cDNAs with an improved PV-specific real-time reverse transcription-PCR system and nucleotide sequence analysis of the VP1 coding region. For assay validation, we analyzed 84 stool extracts that were positive for PV in cell culture and detected PV genomes from 100% of the extracts (84/84 samples) with this method in combination with a PV-specific extraction method. PV could be detected in 2/4 stool extract samples that were negative for PV in cell culture. In PV-positive samples, EV species C viruses were also detected with high frequency (27% [23/86 samples]). This method would be useful for direct detection of PV from stool extracts without using cell culture.
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Honkimaa, Anni, Bryn Kimura, Amir-Babak Sioofy-Khojine, Jake Lin, Jutta Laiho, Sami Oikarinen, and Heikki Hyöty. "Genetic Adaptation of Coxsackievirus B1 during Persistent Infection in Pancreatic Cells." Microorganisms 8, no. 11 (November 15, 2020): 1790. http://dx.doi.org/10.3390/microorganisms8111790.
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Coxsackie B (CVB) viruses have been associated with type 1 diabetes. We have recently observed that CVB1 was linked to the initiation of the autoimmune process leading to type 1 diabetes in Finnish children. Viral persistency in the pancreas is currently considered as one possible mechanism. In the current study persistent infection was established in pancreatic ductal and beta cell lines (PANC-1 and 1.1B4) using four different CVB1 strains, including the prototype strain and three clinical isolates. We sequenced 5′ untranslated region (UTR) and regions coding for structural and non-structural proteins and the second single open reading frame (ORF) protein of all persisting CVB1 strains using next generation sequencing to identify mutations that are common for all of these strains. One mutation, K257R in VP1, was found from all persisting CVB1 strains. The mutations were mainly accumulated in viral structural proteins, especially at BC, DE, EF loops and C-terminus of viral capsid protein 1 (VP1), the puff region of VP2, the knob region of VP3 and infection-enhancing epitope of VP4. This showed that the capsid region of the viruses sustains various changes during persistency some of which could be hallmark(s) of persistency.
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Haldeman-Cahill, Ruth, José-Antonio Daròs, and James C. Carrington. "Secondary Structures in the Capsid Protein Coding Sequence and 3′ Nontranslated Region Involved in Amplification of the Tobacco Etch Virus Genome." Journal of Virology 72, no. 5 (May 1, 1998): 4072–79. http://dx.doi.org/10.1128/jvi.72.5.4072-4079.1998.
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ABSTRACT The 3′-terminal 350 nucleotides of the tobacco etch potyvirus (TEV) genome span the end of the capsid protein (CP)-coding sequence and the 3′ nontranslated region (NTR). The CP-coding sequence within this region contains a 105-nucleotide cis-active element required for genome replication (S. Mahajan, V. V. Dolja, and J. C. Carrington, J. Virol. 70:4370–4379, 1996). To investigate the sequence and secondary structure requirements within the CP cis-active region and the 3′ NTR, a systematic linker-scanning mutagenesis analysis was done. Forty-six mutations, each with two to six nucleotide substitutions, were introduced at consecutive hexanucleotide positions in the genome of a recombinant TEV strain expressing a reporter protein (β-glucuronidase). Genome amplification activity of each mutant in the protoplast cell culture system was measured. Mutations that severely debilitated genome amplification were identified throughout the CP-codingcis-active sequence and at several distinct locations within the 3′ NTR. However, based on a computer model of RNA folding, mutations that had the most severe effects mapped to regions that were predicted to form base-paired secondary structures. Linker-scanning mutations predicted to affect either strand of a base-paired structure within the CP-coding cis-active sequence, a base-paired structure between two segments of the CP-coding cis-active sequence and a contiguous 14-nucleotide segment of the 3′ NTR, and a base-paired structure near the 3′ terminus of the 3′ NTR inactivated genome amplification. Compensatory mutations that restored base pair interactions in each of these regions restored amplification activity, although to differing levels depending on the structure restored. These data reveal that the 3′ terminus of the TEV genome consists of a series of functionally discrete sequences and secondary structures and that the CP-coding sequence and 3′ NTR are coadapted for genome amplification function through a requirement for base pair interactions.
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Sokolowski, Marcus, Wei Tan, Marianne Jellne, and Stefan Schwartz. "mRNA Instability Elements in the Human Papillomavirus Type 16 L2 Coding Region." Journal of Virology 72, no. 2 (February 1, 1998): 1504–15. http://dx.doi.org/10.1128/jvi.72.2.1504-1515.1998.
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ABSTRACT Human papillomavirus capsid proteins L1 and L2 are detected only in terminally differentiated cells, indicating that expression of the L1 and L2 genes is blocked in dividing cells. The results presented here establish that the human papillomavirus type 16 L2 coding region contains cis-acting inhibitory sequences. When placed downstream of a reporter gene, the human papillomavirus type 16 L2 sequence reduced both mRNA and protein levels in an orientation-dependent manner. Deletion analysis revealed that the L2 sequence contains two cis-acting inhibitory RNA regions. We identified an inhibitory region in the 5′-most 845 nucleotides of L2 that acted by reducing cytoplasmic mRNA stability and a second, weaker inhibitory region in the 3′ end of L2. In contrast, human papillomavirus type 1 L1 and L2 genes did not encode strong inhibitory sequences. This result is consistent with observations of high virus production in human papillomavirus type 1-infected tissue, whereas only low levels of human papillomavirus type 16 virions are detectable in infected epithelium. The presence of inhibitory sequences in the L1 and L2 mRNAs may aid the virus in avoiding the host immunosurveillance and in establishing persistent infections.
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Mueller, Steffen, Dimitris Papamichail, J. Robert Coleman, Steven Skiena, and Eckard Wimmer. "Reduction of the Rate of Poliovirus Protein Synthesis through Large-Scale Codon Deoptimization Causes Attenuation of Viral Virulence by Lowering Specific Infectivity." Journal of Virology 80, no. 19 (October 1, 2006): 9687–96. http://dx.doi.org/10.1128/jvi.00738-06.
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ABSTRACT Exploring the utility of de novo gene synthesis with the aim of designing stably attenuated polioviruses (PV), we followed two strategies to construct PV variants containing synthetic replacements of the capsid coding sequences either by deoptimizing synonymous codon usage (PV-AB) or by maximizing synonymous codon position changes of the existing wild-type (wt) poliovirus codons (PV-SD). Despite 934 nucleotide changes in the capsid coding region, PV-SD RNA produced virus with wild-type characteristics. In contrast, no viable virus was recovered from PV-AB RNA carrying 680 silent mutations, due to a reduction of genome translation and replication below a critical level. After subcloning of smaller portions of the AB capsid coding sequence into the wt background, several viable viruses were obtained with a wide range of phenotypes corresponding to their efficiency of directing genome translation. Surprisingly, when inoculated with equal infectious doses (PFU), even the most replication-deficient viruses appeared to be as pathogenic in PV-sensitive CD155tg (transgenic) mice as the PV(M) wild type. However, infection with equal amounts of virus particles revealed a neuroattenuated phenotype over 100-fold. Direct analysis indicated a striking reduction of the specific infectivity of PV-AB-type virus particles. Due to the distribution effect of many silent mutations over large genome segments, codon-deoptimized viruses should have genetically stable phenotypes, and they may prove suitable as attenuated substrates for the production of poliovirus vaccines.
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Martín, Javier, Kofi Odoom, Gráinne Tuite, Glynis Dunn, Nicola Hopewell, Gill Cooper, Catherine Fitzharris, Karina Butler, William W. Hall, and Philip D. Minor. "Long-Term Excretion of Vaccine-Derived Poliovirus by a Healthy Child." Journal of Virology 78, no. 24 (December 15, 2004): 13839–47. http://dx.doi.org/10.1128/jvi.78.24.13839-13847.2004.
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ABSTRACT A child was found to be excreting type 1 vaccine-derived poliovirus (VDPV) with a 1.1% sequence drift from Sabin type 1 vaccine strain in the VP1 coding region 6 months after he was immunized with oral live polio vaccine. Seventeen type 1 poliovirus isolates were recovered from stools taken from this child during the following 4 months. Contrary to expectation, the child was not deficient in humoral immunity and showed high levels of serum neutralization against poliovirus. Selected virus isolates were characterized in terms of their antigenic properties, virulence in transgenic mice, sensitivity for growth at high temperatures, and differences in nucleotide sequence from the Sabin type 1 strain. The VDPV isolates showed mutations at key nucleotide positions that correlated with the observed reversion to biological properties typical of wild polioviruses. A number of capsid mutations mapped at known antigenic sites leading to changes in the viral antigenic structure. Estimates of sequence evolution based on the accumulation of nucleotide changes in the VP1 coding region detected a “defective” molecular clock running at an apparent faster speed of 2.05% nucleotide changes per year versus 1% shown in previous studies. Remarkably, when compared to several type 1 VDPV strains of different origins, isolates from this child showed a much higher proportion of nonsynonymous versus synonymous nucleotide changes in the capsid coding region. This anomaly could explain the high VP1 sequence drift found and the ability of these virus strains to replicate in the gut for a longer period than expected.
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Fasina, Olufemi O., Yanming Dong, and David J. Pintel. "NP1 Protein of the Bocaparvovirus Minute Virus of Canines Controls Access to the Viral Capsid Genes via Its Role in RNA Processing." Journal of Virology 90, no. 4 (December 4, 2015): 1718–28. http://dx.doi.org/10.1128/jvi.02618-15.
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ABSTRACTMinute virus of canines (MVC) is an autonomous parvovirus in the genusBocaparvovirus. It has a single promoter that generates a single pre-mRNA processed via alternative splicing and alternative polyadenylation to produce at least 8 mRNA transcripts. MVC contains two polyadenylation sites, one at the right-hand end of the genome, (pA)d, and another complex site, (pA)p, within the capsid-coding region. During viral infection, the mRNAs must extend through (pA)p and undergo additional splicing of the immediately upstream 3D∕3A intron to access the capsid gene. MVC NP1 is a 22-kDa nuclear phosphoprotein unique to the genusBocaparvovirusof theParvovirinaewhich we have shown governs suppression of (pA)p independently of viral genome replication. We show here that in addition to suppression of (pA)p, NP1 is also required for the excision of the MVC 3D∕3A intron, independently of its effect on alternative polyadenylation. Mutations of the arginine∕serine (SR) di-repeats within the intrinsically disordered amino terminus of NP1 are required for splicing of the capsid transcript but not suppression of polyadenylation at (pA)p. 3′-end processing of MVC mRNAs at (pA)p is critical for viral genome replication and the optimal expression of NP1 and NS1. Thus, a finely tuned balance between (pA)p suppression and usage is necessary for efficient virus replication. NP1 is the first parvovirus protein implicated in RNA processing. Its characterization reveals another way that parvoviruses govern access to their capsid protein genes, namely, at the RNA level, by regulating the essential splicing of an intron and the suppression of an internal polyadenylation site.IMPORTANCETheParvovirinaeare small nonenveloped icosahedral viruses that are important pathogens in many animal species, including humans. Although parvoviruses have only subtle early-to-late expression shifts, they all regulate access to their capsid genes. Minute virus of canines (MVC) is an autonomous parvovirus in the genusBocaparvovirus. It has a single promoter generating a single pre-mRNA which is processed via alternative splicing and alternative polyadenylation to generate at least 8 mRNA transcripts. MVC contains two polyadenylation sites, one at the right-hand end of the genome, (pA)d, and another, (pA)p, within the capsid-coding region. It had not been clear how the potent internal polyadenylation motif is suppressed to allow processing, export, and accumulation of the spliced capsid protein-encoding mRNAs. We show here that MVC NP1, the first parvovirus protein to be implicated in RNA processing, governs access to the MVC capsid gene by facilitating splicing and suppressing internal polyadenylation of MVC pre-mRNAs.
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Burns, Cara C., Ray Campagnoli, Jing Shaw, Annelet Vincent, Jaume Jorba, and Olen Kew. "Genetic Inactivation of Poliovirus Infectivity by Increasing the Frequencies of CpG and UpA Dinucleotides within and across Synonymous Capsid Region Codons." Journal of Virology 83, no. 19 (July 15, 2009): 9957–69. http://dx.doi.org/10.1128/jvi.00508-09.
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ABSTRACT Replicative fitness of poliovirus can be modulated systematically by replacement of preferred capsid region codons with synonymous unpreferred codons. To determine the key genetic contributors to fitness reduction, we introduced different sets of synonymous codons into the capsid coding region of an infectious clone derived from the type 2 prototype strain MEF-1. Replicative fitness in HeLa cells, measured by plaque areas and virus yields in single-step growth experiments, decreased sharply with increased frequencies of the dinucleotides CpG (suppressed in higher eukaryotes and most RNA viruses) and UpA (suppressed nearly universally). Replacement of MEF-1 capsid codons with the corresponding codons from another type 2 prototype strain (Lansing), a randomization of MEF-1 synonymous codons, increased the %G+C without increasing CpG, and reductions in the effective number of codons used had much smaller individual effects on fitness. Poliovirus fitness was reduced to the threshold of viability when CpG and UpA dinucleotides were saturated within and across synonymous codons of a capsid region interval representing only ∼9% of the total genome. Codon replacements were associated with moderate decreases in total virion production but large decreases in the specific infectivities of intact poliovirions and viral RNAs. Replication of codon replacement viruses, but not MEF-1, was temperature sensitive at 39.5°C. Synthesis and processing of viral intracellular proteins were largely unaltered in most codon replacement constructs. Replacement of natural codons with synonymous codons with increased frequencies of CpG and UpA dinucleotides may offer a general approach to the development of attenuated vaccines with well-defined antigenicities and very high genetic stabilities.
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Gallinella, G., S. Venturoli, G. Gentilomi, M. Musiani, and M. Zerbini. "Extent of sequence variability in a genomic region coding for capsid proteins of B19 parvovirus." Archives of Virology 140, no. 6 (June 1995): 1119–25. http://dx.doi.org/10.1007/bf01315420.
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Rao, Qing, Zhen Zhang, Hongchao Jiang, Meifen Wang, Rong Huang, Tingyi Du, Xiaomei Liu, et al. "Comparison of coxsackievirus A12 genome isolated from patients with different symptoms in Yunnan, Southwest China." Future Virology 15, no. 10 (October 2020): 683–91. http://dx.doi.org/10.2217/fvl-2020-0103.
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Aim: Coxsackievirus A12 (CVA12) mainly causes hand–foot–mouth disease (HFMD) or herpangina (HA). Thus, the genomic characteristics of CVA12 isolates from Southwest China, especially the discrepancy between CVA12-HFMD and CVA12-HA were analyzed. Patients & methods: The full-length genome sequences of CVA12-HFMD and CVA12-HA were obtained and phylogenetic, nucleotide mutation, amino acid substitution and recombinant analyses were performed. Results: All CVA12 were closest to the Netherlands (2013) and have possibly recombined in the capsid coding region (P1) with other HEV-A. In the coding region, 54 base mutation result in 11 nonsynonymous mutations and four of them were identical mutations between CVA12-HFMD and CVA12-HA. Conclusion: Whether the presence of four consistent nonsynonymous mutation sites affect the virulence of the CVA12 deserves further study.
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Bøtner, Anette, Naresh K. Kakker, Cyril Barbezange, Stephen Berryman, Terry Jackson, and Graham J. Belsham. "Capsid proteins from field strains of foot-and-mouth disease virus confer a pathogenic phenotype in cattle on an attenuated, cell-culture-adapted virus." Journal of General Virology 92, no. 5 (May 1, 2011): 1141–51. http://dx.doi.org/10.1099/vir.0.029710-0.
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Chimeric foot-and-mouth disease viruses (FMDVs) have been generated from plasmids containing full-length FMDV cDNAs and characterized. The parental virus cDNA was derived from the cell-culture-adapted O1Kaufbeuren B64 (O1K B64) strain. Chimeric viruses, containing capsid coding sequences derived from the O/UKG/34/2001 or A/Turkey 2/2006 field viruses, were constructed using the backbone from the O1K B64 cDNA, and viable viruses (O1K/O-UKG and O1K/A-Tur, respectively) were successfully rescued in each case. These viruses grew well in primary bovine thyroid cells but grew less efficiently in BHK cells than the rescued parental O1K B64 virus. The two chimeric viruses displayed the expected antigenicity in serotype-specific antigen ELISAs. Following inoculation of each virus into cattle, the rescued O1K B64 strain proved to be attenuated whereas, with each chimeric virus, typical clinical signs of foot-and-mouth disease were observed, which then spread to in-contact animals. Thus, the surface-exposed capsid proteins of the O1K B64 strain are responsible for its attenuation in cattle. Consequently, there is no evidence for any adaptation, acquired during cell culture, outside the capsid coding region within the O1K B64 strain that inhibits replication in cattle. These chimeric infectious cDNA plasmids provide a basis for the analysis of FMDV pathogenicity and characterization of receptor utilization in vivo.
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Garlapati, Srinivas, and Ching C. Wang. "Structural Elements in the 5′-Untranslated Region of Giardiavirus Transcript Essential for Internal Ribosome Entry Site-Mediated Translation Initiation." Eukaryotic Cell 4, no. 4 (April 2005): 742–54. http://dx.doi.org/10.1128/ec.4.4.742-754.2005.
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ABSTRACT Translation of uncapped giardiavirus (GLV) mRNA in Giardia lamblia requires the presence of a 5′-untranslated region (5′-UTR) and a viral capsid coding region. We used dicistronic viral constructs to show that the downstream 253 nucleotides (nt) of the 5′-UTR plus the initial 264-nt capsid coding region constitute an internal ribosome entry site (IRES). Predicted secondary structures in the 253-nt 5′-UTR include stem-loops U3, U4a, U4b, U4c, and U5. Chemical and enzymatic probing analysis confirmed the presence of all predicted stem-loops except U4a. Disruption of stem-loop structures U3 and U5 by site-directed mutagenesis resulted in a drastic reduction in translation of a monocistronic viral transcript, which could be restored by compensatory sequence changes. Mutations disrupting stem-loops U4b and U4c do not exert an appreciable effect on translation, but certain sequences in the U4a region and in U4b do appear to play important roles in the IRES. Structural analysis also suggests that an 8-nt U3 loop sequence (nt 147 to 154) pairs with an 8-nt downstream sequence (nt 168 to 175) to form a pseudoknot. Disruption of this pseudoknot by mutagenesis resulted in a drastic reduction in translation, which could be restored by compensatory sequence changes. This study has defined the secondary structure in the 5′-UTR of the IRES. Together with the previous results, we have now completed analysis of the entire structure of GLV IRES and fully defined the functionally essential structural elements in it.
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Belsham, Graham J. "Influence of the Leader protein coding region of foot-and-mouth disease virus on virus replication." Journal of General Virology 94, no. 7 (July 1, 2013): 1486–95. http://dx.doi.org/10.1099/vir.0.052126-0.
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The foot-and-mouth disease virus (FMDV) Leader (L) protein is produced in two forms, Lab and Lb, differing only at their amino-termini, due to the use of separate initiation codons, usually 84 nt apart. It has been shown previously, and confirmed here, that precise deletion of the Lab coding sequence is lethal for the virus, whereas loss of the Lb coding sequence results in a virus that is viable in BHK cells. In addition, it is now shown that deletion of the ‘spacer’ region between these two initiation codons can be tolerated. Growth of the virus precisely lacking just the Lb coding sequence resulted in a previously undetected accumulation of frameshift mutations within the ‘spacer’ region. These mutations block the inappropriate fusion of amino acid sequences to the amino-terminus of the capsid protein precursor. Modification, by site-directed mutagenesis, of the Lab initiation codon, in the context of the virus lacking the Lb coding region, was also tolerated by the virus within BHK cells. However, precise loss of the Lb coding sequence alone blocked FMDV replication in primary bovine thyroid cells. Thus, the requirement for the Leader protein coding sequences is highly dependent on the nature and extent of the residual Leader protein sequences and on the host cell system used. FMDVs precisely lacking Lb and with the Lab initiation codon modified may represent safer seed viruses for vaccine production.
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Jamal, Syed M., Giancarlo Ferrari, Safia Ahmed, Preben Normann, Stephen Curry, and Graham J. Belsham. "Evolutionary analysis of serotype A foot-and-mouth disease viruses circulating in Pakistan and Afghanistan during 2002–2009." Journal of General Virology 92, no. 12 (December 1, 2011): 2849–64. http://dx.doi.org/10.1099/vir.0.035626-0.
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Foot-and-mouth disease (FMD) is endemic in Pakistan and Afghanistan. Three different serotypes of the virus, namely O, A and Asia-1, are responsible for the outbreaks of this disease in these countries. In the present study, the nucleotide-coding sequences for the VP1 capsid protein (69 samples) or for all four capsid proteins (P1, seven representative samples) of the serotype A FMD viruses circulating in Pakistan and Afghanistan were determined. Phylogenetic analysis of the foot-and-mouth disease virus (FMDV) VP1-coding sequences from these countries collected between 2002 and 2009 revealed the presence of at least four lineages within two distinct genotypes, all belonging to the Asia topotype, within serotype A. The predominant lineage observed was A-Iran05 but three other lineages (a new one is named here A-Pak09) were also identified. The A-Iran05 lineage is still evolving as revealed by the presence of seven distinct variants, the dominant being the A-Iran05AFG-07 and A-Iran05BAR-08 sublineages. The rate of evolution of the A-Iran05 lineage was found to be about 1.2×10−2 substitutions per nucleotide per year. This high rate of change is consistent with the rapid appearance of new variants of FMDV serotype A in the region. The A22/Iraq FMDV vaccine is antigenically distinct from the A-Iran05BAR-08 viruses. Mapping of the amino acid changes between the capsid proteins of the A22/Iraq vaccine strain and the A-Iran05BAR-08 viruses onto the A22/Iraq capsid structure identified candidate amino acid substitutions, exposed on the virus surface, which may explain this antigenic difference.
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de Borba, Luana, Sergio M. Villordo, Nestor G. Iglesias, Claudia V. Filomatori, Leopoldo G. Gebhard, and Andrea V. Gamarnik. "Overlapping Local and Long-Range RNA-RNA Interactions Modulate Dengue Virus Genome Cyclization and Replication." Journal of Virology 89, no. 6 (January 14, 2015): 3430–37. http://dx.doi.org/10.1128/jvi.02677-14.
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The dengue virus genome is a dynamic molecule that adopts different conformations in the infected cell. Here, using RNA folding predictions, chemical probing analysis, RNA binding assays, and functional studies, we identified newcis-acting elements present in the capsid coding sequence that facilitate cyclization of the viral RNA by hybridization with a sequence involved in a local dumbbell structure at the viral 3′ untranslated region (UTR). The identified interaction differentially enhances viral replication in mosquito and mammalian cells.
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Cumming, Sarah A., Thanaporn Cheun-Im, Stephen G. Milligan, and Sheila V. Graham. "Human papillomavirus type 16 late gene expression is regulated by cellular RNA processing factors in response to epithelial differentiation." Biochemical Society Transactions 36, no. 3 (May 21, 2008): 522–24. http://dx.doi.org/10.1042/bst0360522.
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HPV16 (human papillomavirus type 16) is a 7.9 kb double-stranded DNA virus that infects anogenital mucosal epithelia. In some rare cases, in women, infection can progress to cervical cancer. HPV16 gene expression is regulated through use of multiple promoters and alternative splicing and polyadenylation. The virus genome can be divided into an early and a late coding region. The late coding region contains the L1 and L2 genes. These encode the virus capsid proteins L1 and L2; protein expression is confined to the upper epithelial layers and is regulated post-transcriptionally in response to epithelial differentiation. A 79 nt RNA regulatory element, the LRE (late regulatory element), involved in this regulation is sited at the 3′-end of the L1 gene and extends into the late 3′-UTR (3′-untranslated region). This element represses late gene expression in differentiated epithelial cells and may activate it in differentiated cells. The present paper describes our current knowledge of LRE RNA–protein interaction and their possible functions.
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Rector, Annabel, Koenraad Van Doorslaer, Mads Bertelsen, Ian K. Barker, Rolf-Arne Olberg, Philippe Lemey, John P. Sundberg, and Marc Van Ranst. "Isolation and cloning of the raccoon (Procyon lotor) papillomavirus type 1 by using degenerate papillomavirus-specific primers." Journal of General Virology 86, no. 7 (July 1, 2005): 2029–33. http://dx.doi.org/10.1099/vir.0.80874-0.
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Partial sequences of a novel papillomavirus were amplified from a cutaneous lesion biopsy of a raccoon (Procyon lotor), by using PCR with degenerate papillomavirus-specific primers. The Procyon lotor papillomavirus type 1 (PlPV-1) DNA was amplified with long template PCR in two overlapping fragments, together encompassing the entire genome, and the complete PlPV-1 genomic sequence was determined. The PlPV-1 genome consists of 8170 bp, and contains the typical papillomaviral open reading frames, encoding five early proteins and two late capsid proteins. Besides the classical non-coding region (NCR1) between the end of L1 and the start of E6, PlPV-1 contains an additional non-coding region (NCR2) of 1065 bp between the early and late protein region, which has previously also been described for the canine oral papillomavirus (COPV) and the Felis domesticus papillomavirus (FdPV-1). Phylogenetic analysis places PlPV-1 together with COPV and FdPV-1 in a monophyletic branch which encompasses the Lambda papillomavirus genus.
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Ching, Karen Z., Tatsunori Nakano, Louisa E. Chapman, Austin Demby, and Betty H. Robertson. "Genetic characterization of wild-type genotype VII hepatitis A virus." Journal of General Virology 83, no. 1 (January 1, 2002): 53–60. http://dx.doi.org/10.1099/0022-1317-83-1-53.
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The complete genome sequence of the only identified genotype VII hepatitis A virus (HAV), strain SLF88, was obtained from PCR amplicons generated by a modified long PCR approach. There was 90% nucleotide identity in the 5′ untranslated region compared to other known HAV sequences. In the remainder of the genome containing the long open reading frame, there was about 85% nucleotide identity to human HAV genotypes IA and IB and 80% identity to simian HAV genotype V. Compared to HAV strain HM-175, the capsid amino acids were highly conserved, with only four homologous amino acid changes, while an increasing number of amino acid differences was seen in the P2 and P3 genome regions. While nucleotide variability within the three functional coding regions did not differ, the P3D region was found to have the largest number of amino acid changes compared to HM-175.
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Tosh, Chakradhar, Divakar Hemadri, and Aniket Sanyal. "Evidence of recombination in the capsid-coding region of type A foot-and-mouth disease virus." Journal of General Virology 83, no. 10 (October 1, 2002): 2455–60. http://dx.doi.org/10.1099/0022-1317-83-10-2455.
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Caro, Valérie, Sophie Guillot, Francis Delpeyroux, and Radu Crainic. "Molecular strategy for ‘serotyping’ of human enteroviruses." Journal of General Virology 82, no. 1 (January 1, 2001): 79–91. http://dx.doi.org/10.1099/0022-1317-82-1-79.
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To explore further the phylogenetic relationships between human enteroviruses and to develop new diagnostic approaches, we designed a pair of generic primers in order to study a 1452 bp genomic fragment (relative to the poliovirus Mahoney genome), including the 3′ end of the VP1-coding region, the 2A- and 2B-coding regions, and the 5′ moiety of the 2C-coding region. Fifty-nine of the 64 prototype strains and 45 field isolates of various origins, involving 21 serotypes and 6 strains untypeable by standard immunological techniques, were successfully amplified with these primers. By determining the nucleotide sequence of the genomic fragment encoding the C-terminal third of the VP1 capsid protein we developed a molecular typing method based on RT–PCR and sequencing. If field isolate sequences were compared to human enterovirus VP1 sequences available in databases, nucleotide identity score was, in each case, highest with the homotypic prototype (74.8 to 89.4%). Phylogenetic trees were generated from alignments of partial VP1 sequences with several phylogeny algorithms. In all cases, the new classification of enteroviruses into five identified species was confirmed and strains of the same serotype were always monophyletic. Analysis of the results confirmed that the 3′ third of the VP1-coding sequence contains serotype-specific information and can be used as the basis of an effective and rapid molecular typing method. Furthermore, the amplification of such a long genomic fragment, including non-structural regions, is straightforward and could be used to investigate genome variability and to identify recombination breakpoints or specific attributes of pathogenicity.
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Collier, Brian, Daniel Öberg, Xiaomin Zhao, and Stefan Schwartz. "Specific Inactivation of Inhibitory Sequences in the 5′ End of the Human Papillomavirus Type 16 L1 Open Reading Frame Results in Production of High Levels of L1 Protein in Human Epithelial Cells." Journal of Virology 76, no. 6 (March 15, 2002): 2739–52. http://dx.doi.org/10.1128/jvi.76.6.2739-2752.2002.
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ABSTRACT The expression of human papillomavirus type 16 late genes encoding virus capsid proteins L1 and L2 is restricted to terminally differentiated epithelial cells in the superficial layers of the squamous epithelium. We wish to understand the molecular mechanisms that determine the levels of expression of the human papillomavirus type 16 late genes. We have previously shown that the L1 coding region contains inhibitory sequences. Here we extend previous findings to show that the 5′ end of the L1 gene contains strong inhibitory sequences but that the 3′ end does not. We show that the first 514 nucleotides of the L1 coding region contain multiple inhibitory elements that act independently of one another and that the major inhibitory element is located within the first 129 nucleotides of the L1 gene. Introduction of point mutations in the inhibitory elements in the 5′ end of the L1 gene which altered the RNA sequence without affecting the protein sequence specifically inactivated the inhibitory elements and resulted in production of high levels of human papillomavirus type 16 L1 mRNA and protein in human epithelial cells. Furthermore, we show that inhibitory sequences are present in the L1 coding regions of multiple human papillomavirus types, demonstrating that these elements are conserved among the human papillomaviruses, and suggest that they have an important function in the viral life cycle.
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Lai, Vicky C. H., Weidong Zhong, Angela Skelton, Paul Ingravallo, Venteislav Vassilev, Ruben O. Donis, Zhi Hong, and Johnson Y. N. Lau. "Generation and Characterization of a Hepatitis C Virus NS3 Protease-Dependent Bovine Viral Diarrhea Virus." Journal of Virology 74, no. 14 (July 15, 2000): 6339–47. http://dx.doi.org/10.1128/jvi.74.14.6339-6347.2000.
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ABSTRACT Unique to pestiviruses, the N-terminal protein encoded by the bovine viral diarrhea virus (BVDV) genome is a cysteine protease (Npro) responsible for a self-cleavage that releases the N terminus of the core protein (C). This unique protease is dispensable for viral replication, and its coding region can be replaced by a ubiquitin gene directly fused in frame to the core. To develop an antiviral assay that allows the assessment of anti-hepatitis C virus (HCV) NS3 protease inhibitors, a chimeric BVDV in which the coding region of Npro was replaced by that of an NS4A cofactor-tethered HCV NS3 protease domain was generated. This cofactor-tethered HCV protease domain was linked in frame to the core protein of BVDV through an HCV NS5A-NS5B junction site and mimicked the proteolytic function of Npro in the release of BVDV core for capsid assembly. A similar chimeric construct was built with an inactive HCV NS3 protease to serve as a control. Genomic RNA transcripts derived from both chimeric clones, PH/B(wild-type HCV NS3 protease) and PH/B(S139A) (mutant HCV NS3 protease) were then transfected into bovine cells (MDBK). Only the RNA transcripts from the PH/B clone yielded viable viruses, whereas the mutant clone, PH/B(S139A), failed to produce any signs of infection, suggesting that the unprocessed fusion protein rendered the BVDV core protein defective in capsid assembly. Like the wild-type BVDV (NADL), the chimeric virus was cytopathic and formed plaques on the cell monolayer. Sequence and biochemical analyses confirmed the identity of the chimeric virus and further revealed variant viruses due to growth adaptation. Growth analysis revealed comparable replication kinetics between the wild-type and the chimeric BVDVs. Finally, to assess the genetic stability of the chimeric virus, an Npro-null BVDV (BVDV−Npro in which the entire Npro coding region was deleted) was produced. Although cytopathic, BVDV−Npro was highly defective in viral replication and growth, a finding consistent with the observed stability of the chimeric virus after serial passages.
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Zheng, Huai-Ying, Tomokazu Takasaka, Kazuyuki Noda, Akira Kanazawa, Hideo Mori, Tomoyuki Kabuki, Kohsuke Joh, et al. "New sequence polymorphisms in the outer loops of the JC polyomavirus major capsid protein (VP1) possibly associated with progressive multifocal leukoencephalopathy." Journal of General Virology 86, no. 7 (July 1, 2005): 2035–45. http://dx.doi.org/10.1099/vir.0.80863-0.
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JC polyomavirus (JCPyV) causes progressive multifocal leukoencephalopathy (PML) in patients with decreased immune competence. To elucidate genetic changes in JCPyV associated with the pathogenesis of PML, multiple complete JCPyV DNA clones originating from the brains of three PML cases were established and sequenced. Although unique rearranged control regions occurred in all clones, a low level of nucleotide variation was also found in the coding region. In each case, a parental coding sequence was identified, from which variant coding sequences with nucleotide substitutions would have been generated. A comparison between the parental and variant coding sequences demonstrated that all 12 detected nucleotide substitutions gave rise to amino acid changes. Interestingly, seven of these changes were located in the surface loops of the major capsid protein (VP1). Finally, 16 reported VP1 sequences of PML-type JCPyV (i.e. derived from the brain or cerebrospinal fluid of PML patients) were compared with their genotypic prototypes, generated as consensus sequences of representative archetypal isolates belonging to the same genotypes; 13 VP1 proteins had amino acid changes in the surface loops. In contrast, VP1 proteins from isolates from the urine of immunocompetent and immunosuppressed patients rarely underwent mutations in the VP1 loops. The present findings suggest that PML-type JCPyV frequently undergoes amino acid substitutions in the VP1 loops. These polymorphisms should serve as a new marker for the identification of JCPyV isolates associated with PML. The biological significance of these mutations, however, remains unclear.
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Brunet, Sylvie, Cécile Sigoillot-Claude, Daniel Pialot, and Hervé Poulet. "Multiple Correspondence Analysis on Amino Acid Properties within the Variable Region of the Capsid Protein Shows Differences between Classical and Virulent Systemic Feline Calicivirus Strains." Viruses 11, no. 12 (November 23, 2019): 1090. http://dx.doi.org/10.3390/v11121090.
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Feline calicivirus (FCV) is a widespread and highly prevalent pathogen of domestic cats, responsible for mild upper respiratory tract disease. Outbreaks of severe virulent systemic disease (VSD) associated with FCV infection have been reported worldwide. VSD FCV strains have a broader tropism and cause a systemic vascular compromise. Despite clear differences in the pathogenesis of VSD and oral respiratory infections, attempts to identify specific molecular markers of VSD strains on the major capsid protein VP1 have failed. Region E of VP1 is responsible for the interaction with the cell receptor Junctional Adhesion Molecule JAM-1 (FeJAM-1) and with VP2 minor capsid protein during the entry of the virus. We carried out an original analysis on the sequences from region E of VSD and classical strains. A Multiple Correspondence Analysis was performed on a Boolean matrix built by coding sequences on the basis of their amino acid properties. For the first time, this approach was able to differentiate VSD and classical FCV. Seven remarkable residue positions were shown to be statistically significant for pathotype differentiation, mainly located in the N-terminal hypervariable part of region E. As structural analysis suggested an interaction of these residues with FeJAM-1 or VP2, post-binding events, and specific conformational changes may explain the difference of pathogenesis between pathotypes.
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Abouhaidar, M. G. "Nucleotide Sequence of the Capsid Protein Gene and 3' Non-coding Region of Papaya Mosaic Virus RNA." Journal of General Virology 69, no. 1 (January 1, 1988): 219–26. http://dx.doi.org/10.1099/0022-1317-69-1-219.
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Jia, Xi-Yu, Donald F. Summers, and Ellie Ehrenfeld. "Primary Cleavage of the HAV Capsid Protein Precursor in the Middle of the Proposed 2A Coding Region." Virology 193, no. 1 (March 1993): 515–19. http://dx.doi.org/10.1006/viro.1993.1157.
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Du, Yanjie, Teng Liu, Yifeng Qin, Qinting Dong, Ying Chen, Kang Ouyang, Zuzhang Wei, and Weijian Huang. "Insertion of Exogenous Genes within the ORF1a Coding Region of Porcine Astrovirus." Viruses 13, no. 11 (October 21, 2021): 2119. http://dx.doi.org/10.3390/v13112119.
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A tagged or reporter astrovirus can be a valuable tool for the analysis of various aspects of the virus life cycle, and to aid in the development of genetically engineered astroviruses as vectors. Here, transposon-mediated insertion mutagenesis was used to insert a 15-nucleotide (nt) sequence into random sites of open reading frame 1a (ORF1a) based on an infectious full-length cDNA clone of porcine astrovirus (PAstV). Five sites in the predicted coiled-coil structures (CC), genome-linked protein (VPg), and hypervariable region (HVR) in ORF1a of the PAstV genome were identified that could tolerate random 15 nt insertions. Incorporation of the commonly used epitope tags, His, Flag, and HA, into four of the five insertion sites permitted the production of infectious viruses and allowed recognition by specifically tagged monoclonal antibodies. The results of immuno-fluorescent assays showed that Flag-tagged ORF1a protein overlapped partially with capsid and ORF2b proteins in the cytoplasm. Improved light-oxygen-voltage (iLOV) gene was also introduced at the insertion sites of CC, VPg, and HVR. Only one viable recombinant reporter PAstV expressing iLOV inserted in HVR was recovered. Biological analysis of the reporter virus showed that it displayed similar growth characteristics, and yet produced less infectious virus particles, when compared with the parental virus. The recombinant virus carrying the iLOV fused with the HVR of ORF1a protein maintained its stability and showed green fluorescence after 15 passages in cell cultures. The resultant fluorescently tagged virus could provide a promising tool for the rapid screening of antiviral drugs as well as allowing the visualization of PAstV infection and replication in living cells.
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Khalifeh, Anthony, Simona Kraberger, Daria Dziewulska, Arvind Varsani, and Tomasz Stenzel. "A Pilot Study Investigating the Dynamics of Pigeon Circovirus Recombination in Domesticated Pigeons Housed in a Single Loft." Viruses 13, no. 6 (May 22, 2021): 964. http://dx.doi.org/10.3390/v13060964.
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Pigeon circovirus (PiCV) infects pigeon populations worldwide and has been associated with immunosuppression in younger pigeons. Recombination is a common mechanism of evolution that has previously been shown in various members of the Circoviridae family, including PiCV. In this study, three groups of pigeons acquired from separate lofts were screened for PiCV, and their genome sequence was determined. Following this, they were housed in a single loft for 22 days, during which blood and cloacal swab samples were taken. From these blood and cloacal swabs, PiCV genomes were determined with the aim to study the spread and recombination dynamics of PiCV in the birds. Genome sequences of PiCV were determined from seven pigeons (seven tested PiCV positive) before they were housed together in a loft (n = 58 sequences) and thereafter from the ten pigeons from blood and cloacal swabs (n = 120). These 178 PiCV genome sequences represent seven genotypes (98% pairwise identity genotype demarcation), and they share >88% genome-wide pairwise identity. Recombination analysis revealed 13 recombination events, and a recombination hotspot spanning the 3′ prime region, the replication-associated protein (rep) gene and the intergenic region. A cold spot in the capsid protein-coding region of the genome was also identified. The majority of the recombinant regions were identified in the rep coding region. This study provides insights into the evolutionary dynamics of PiCV in pigeons kept under closed rearing systems.
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Chehadeh, Wassim, Sanaa Abdulkader Moalim Ali, and Syeda Mubeen Maimoona. "Molecular characterization of three enteroviral strains isolated in Kuwait from young children with serious conditions." Journal of Infection in Developing Countries 11, no. 08 (September 5, 2017): 626–39. http://dx.doi.org/10.3855/jidc.9065.
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Introduction: Human enteroviruses are single stranded RNA viruses associated with many serious diseases such as encephalitis and myocarditis. They consist of up to 100 immunologically and genetically distinct types. Three enteroviral isolates, 2104, 3936 and 3988, were previously isolated from patients with neurological disorders or sepsis-like illness. In this study, the molecular characterization of the three isolates was investigated.
Methodology: A full genome sequencing was performed by Sanger method, followed by phylogenetic and bootscanning analyses. A detailed analysis of genetic differences between the clinical and prototype isolates were investigated by mapping polymorphisms at nucleotide and amino acid levels, and by comparing RNA secondary structure in the noncoding regions.
Results: Based on the phylogenetic analysis of the VP1 gene and complete genome, 2104 was typed as coxsackievirus B1, 3936 as coxsackievirus B5, and 3988 as echovirus 7. Similarity and bootscan plots provided support for intra- and intertypic recombination crossover points occurring mainly along the nonstructural coding regions of the isolates. A sequence divergence of 12 to 14% was detected in the 5’-noncoding region between the clinical isolates and their corresponding prototype strains. Synonymous and nonsynonymous substitutions could be also mapped to different coding regions of the isolates, including those coding for the Puff, Knob and the hydrophobic pocket of the capsid. Examination of relative frequencies of synonymous and nonsynonymous substitutions in different coding regions of enteroviral isolates showed no evidence for selective pressure.
Conclusion: The results provided a better understanding of the genetic variations, evolution and adaptation of enteroviruses in Kuwait.
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Kao, J., L. Jia, T. Tian, L. Rubio, and B. W. Falk. "First Report of Cucurbit Yellow Stunting Disorder Virus (Genus Crinivirus) in North America." Plant Disease 84, no. 1 (January 2000): 101. http://dx.doi.org/10.1094/pdis.2000.84.1.101c.
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In late summer 1999, field- and greenhouse-grown melon plants (Cucumis melo) showing severe stunting and yellowing symptoms were observed near Donna in southern Texas and near the town of Reynosa in northern Mexico. Symptoms were typical of those caused by viruses in the genus Crinivirus, family Closteroviridae. High populations of Bemisia spp. whiteflies were associated with these plantings, with many plants showing heavy infestation. Laboratory analyses showed that melon plants from both locations were infected by the whitefly-transmitted Cucurbit yellow stunting disorder virus (CYSDV). Positive hybridization reactions with digoxigenin-UTP-labeled transcript probes corresponding to the CYSDV heat shock protein 70 (HSP70) homolog coding region (1) were obtained for RNAs extracted from symptomatic plants. Similar probes for the related Lettuce infectious yellows virus (LIYV) and Beet pseudo-yellows virus (BPYV), two whitefly-transmitted viruses previously reported from North America (2), did not hybridize with the RNAs. Definitive confirmation of CYSDV was obtained by performing reverse-transcription polymerase chain reaction (RT-PCR) analyses for two distinct CYSDV coding regions. RT-PCR with primers corresponding to CYSDV, but not LIYV or BPYV HSP70 homolog coding regions, gave specific (≈500 bp) products from corresponding test plants. RNAs from healthy control plants gave no RT-PCR product. Because the HSP70 coding region is highly conserved (2), we also performed RT-PCR with primers designed for the Spanish CYSDV capsid protein gene (GenBank accession AJ243000). Positive RT-PCR products of ≈700 bp were obtained only from the Texas and Mexico melon plants. CYSDV is a widespread and damaging virus of cucurbits in southern Europe and the Middle East (2). This is the first report of this important virus in North America. References: (1) Tian et al. Phytopathology 86:1167, 1996. (2) Rubio et al. Phytopathology 89:707, 1999.
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Ali, Hashim, Aleksei Lulla, Alex S. Nicholson, Jack Hankinson, Elizabeth B. Wignall-Fleming, Rhian L. O’Connor, Diem-Lan Vu, et al. "Attenuation hotspots in neurotropic human astroviruses." PLOS Biology 21, no. 7 (July 17, 2023): e3001815. http://dx.doi.org/10.1371/journal.pbio.3001815.
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During the last decade, the detection of neurotropic astroviruses has increased dramatically. The MLB genogroup of astroviruses represents a genetically distinct group of zoonotic astroviruses associated with gastroenteritis and severe neurological complications in young children, the immunocompromised, and the elderly. Using different virus evolution approaches, we identified dispensable regions in the 3′ end of the capsid-coding region responsible for attenuation of MLB astroviruses in susceptible cell lines. To create recombinant viruses with identified deletions, MLB reverse genetics (RG) and replicon systems were developed. Recombinant truncated MLB viruses resulted in imbalanced RNA synthesis and strong attenuation in iPSC-derived neuronal cultures confirming the location of neurotropism determinants. This approach can be used for the development of vaccine candidates using attenuated astroviruses that infect humans, livestock animals, and poultry.
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Harvala, Heli, Hannu Kalimo, Jeffrey Bergelson, Glyn Stanway, and Timo Hyypiä. "Tissue tropism of recombinant coxsackieviruses in an adult mouse model." Journal of General Virology 86, no. 7 (July 1, 2005): 1897–907. http://dx.doi.org/10.1099/vir.0.80603-0.
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Recombinant viruses, constructed by exchanging the 5′ non-coding region (5′NCR), structural and non-structural protein coding sequences were used to investigate determinants responsible for differences between coxsackievirus A9 (CAV9) and coxsackievirus B3 (CBV3) infections in adult mice and two cell lines. Plaque assay titration of recombinant and parental viruses from different tissues from adult BALB/c mice demonstrated that the structural region of CBV3 determined tropism to the liver tissue due to receptor recognition, and the 5′NCR of CBV3 enhanced viral multiplication in the mouse pancreas. Infection with a chimeric virus, containing the structural region from CBV3 and the rest of the genome from CAV9, and the parental CBV3 strain, caused high levels of viraemia in adult mice. The ability of these viruses to infect the central nervous system suggested that neurotropism is associated with high replication levels and the presence of the CBV3 capsid proteins, which also enhanced formation of neutralizing antibodies. Moreover, the appearance of neutralizing antibodies correlated directly with the clearance of the viruses from the tissues. These results demonstrate potential pathogenicity of intraspecies recombinant coxsackieviruses, and the complexity of the genetic determinants underlying tissue tropism.