The molecular determinants and consequences of recombination in the evolution of human enteroviruses

Recombination is an important biological process in a diverse range of viruses, particularly those with single-stranded ribonucleic acid (RNA) genomes including the enteroviruses. Mutations caused by the error-prone RNA-dependent RNA polymerase (RdRp) and the vast population size of these virus populations are evolutionary mechanisms that generate genetic diversity – this allows viruses to survive under changing environmental pressures (e.g. adaptive host immunity). Ribonucleic acid recombination has been identified as another contributing mechanism involved in diversification, by removing interfering or lethal mutations from a virus genome, and by establishing new viruses. Virus RNA recombination is well documented and is identified in several virus families including picornaviruses. However, recombination is a rare event and the study of the molecular mechanisms behind virus recombination is complicated by our ability to isolate and analyse recombinants from a mixed virus population including the parental viruses. The objectives of this study were to firstly devise a method for generating populations of natural recombinant viruses, and secondly, to study the molecular processes that determine where and when recombination occurred in the enteroviral genome. During this project, an in vitro system was developed to allow the recovery of recombinant enteroviruses in the absence of their parental viruses. Two virus RNA molecules containing “lesions” rendering them unable to generate viable virus on their own were co-transfected into mouse L929 cells. The method required two parental virus RNA molecules to be present in a single cell to produce a viable recombinant virus. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and sequencing analysis confirmed the recombinant nature of progeny virus genomes. Experimental data confirmed the effectiveness of the method and provided evidence that recombination occurs in at least two phases. Initial template switching, referring to the transfer of RdRp from one RNA template to another mid-replication, occurred apparently indiscriminately and with the addition of extra virus and non-virus sequence at the junction sites. In the second phase, any additional sequence was lost during subsequent rounds of replication and selection. The approach was expanded to incorporate viruses from different enterovirus species to investigate intra- and interspecies recombination events.EThOS - Electronic Theses Online ServiceUniversity of WarwickGBUnited Kingdo

Multiple recombinant dengue type 1 viruses in an isolate from a dengue patient

Between 2000 and 2004, dengue virus type 1 (DENV-1) genotypes I and II from Asia were introduced into the Pacific region and co-circulated in some localities. Envelope protein gene sequences of DENV-1 from 12 patients infected on the island of New Caledonia were obtained, five of which carried genotype I viruses and six, genotype II viruses. One patient harboured a mixed infection, containing viruses assigned to both genotypes I and II, as well as a number of inter-genotypic recombinants. This is the first report of a population of dengue viruses isolated from a patient containing both parental and recombinant viruses

Effect of Serotype and Strain Diversity on Dengue Virus Replication in Australian Mosquito Vectors

Dengue virus (DENV) is the most important mosquito-borne viral pathogen of humans, comprising four serotypes (DENV-1 to -4) with a myriad of genotypes and strains. The kinetics of DENV replication within the mosquito following ingestion of a blood meal influence the pathogen’s ability to reach the salivary glands and thus the transmission potential. The influence of DENV serotype and strain diversity on virus kinetics in the two main vector species, Aedes aegypti and Ae. albopictus, has been poorly characterized. We tested whether DENV replication kinetics vary systematically among serotypes and strains, using Australian strains of the two vectors. Mosquitoes were blood fed with two strains per serotype, and sampled at 3, 6, 10 and 14-days post-exposure. Virus infection in mosquito bodies, and dissemination of virus to legs and wings, was detected using qRT-PCR. For both vectors, we found significant differences among serotypes in proportions of mosquitoes infected, with higher numbers for DENV-1 and -2 versus other serotypes. Consistent with this, we observed that DENV-1 and -2 generally replicated to higher RNA levels than other serotypes, particularly at earlier time points. There were no significant differences in either speed of infection or dissemination between the mosquito species. Our results suggest that DENV diversity may have important epidemiological consequences by influencing virus kinetics in mosquito vectors

Myanmar Dengue Outbreak Associated with Displacement of Serotypes 2, 3, and 4 by Dengue 1

In 2001, Myanmar (Burma) had its largest outbreak of dengue—15,361 reported cases of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), including 192 deaths. That year, 95% of dengue viruses isolated from patients were serotype 1 viruses belonging to two lineages that had diverged from an earlier, now extinct, lineage sometime before 1998. The ratio of DHF to DSS cases in 2001 was not significantly different from that in 2000, when 1,816 cases of DHF/DSS were reported and dengue 1 also was the most frequently isolated serotype. However, the 2001 ratio was significantly higher than that in 1998 (also an outbreak year) and in 1999, when all four serotypes were detected and serotypes 1, 2, and 3 were recovered in similar numbers. The large number of clinical cases in 2001 may have been due, in part, to a preponderance of infections with dengue 1 viruses

Defective Interfering Viral Particles in Acute Dengue Infections

While much of the genetic variation in RNA viruses arises because of the error-prone nature of their RNA-dependent RNA polymerases, much larger changes may occur as a result of recombination. An extreme example of genetic change is found in defective interfering (DI) viral particles, where large sections of the genome of a parental virus have been deleted and the residual sub-genome fragment is replicated by complementation by co-infecting functional viruses. While most reports of DI particles have referred to studies in vitro, there is some evidence for the presence of DI particles in chronic viral infections in vivo. In this study, short fragments of dengue virus (DENV) RNA containing only key regulatory elements at the 3′ and 5′ ends of the genome were recovered from the sera of patients infected with any of the four DENV serotypes. Identical RNA fragments were detected in the supernatant from cultures of Aedes mosquito cells that were infected by the addition of sera from dengue patients, suggesting that the sub-genomic RNA might be transmitted between human and mosquito hosts in defective interfering (DI) viral particles. In vitro transcribed sub-genomic RNA corresponding to that detected in vivo could be packaged in virus like particles in the presence of wild type virus and transmitted for at least three passages in cell culture. DENV preparations enriched for these putative DI particles reduced the yield of wild type dengue virus following co-infections of C6–36 cells. This is the first report of DI particles in an acute arboviral infection in nature. The internal genomic deletions described here are the most extensive defects observed in DENV and may be part of a much broader disease attenuating process that is mediated by defective viruses

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

The molecular determinants and consequences of recombination in the evolution of human enteroviruses

Recombination is an important biological process in a diverse range of viruses, particularly those with single-stranded ribonucleic acid (RNA) genomes including the enteroviruses. Mutations caused by the error-prone RNA-dependent RNA polymerase (RdRp) and the vast population size of these virus populations are evolutionary mechanisms that generate genetic diversity – this allows viruses to survive under changing environmental pressures (e.g. adaptive host immunity). Ribonucleic acid recombination has been identified as another contributing mechanism involved in diversification, by removing interfering or lethal mutations from a virus genome, and by establishing new viruses. Virus RNA recombination is well documented and is identified in several virus families including picornaviruses. However, recombination is a rare event and the study of the molecular mechanisms behind virus recombination is complicated by our ability to isolate and analyse recombinants from a mixed virus population including the parental viruses. The objectives of this study were to firstly devise a method for generating populations of natural recombinant viruses, and secondly, to study the molecular processes that determine where and when recombination occurred in the enteroviral genome. During this project, an in vitro system was developed to allow the recovery of recombinant enteroviruses in the absence of their parental viruses. Two virus RNA molecules containing “lesions” rendering them unable to generate viable virus on their own were co-transfected into mouse L929 cells. The method required two parental virus RNA molecules to be present in a single cell to produce a viable recombinant virus. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and sequencing analysis confirmed the recombinant nature of progeny virus genomes. Experimental data confirmed the effectiveness of the method and provided evidence that recombination occurs in at least two phases. Initial template switching, referring to the transfer of RdRp from one RNA template to another mid-replication, occurred apparently indiscriminately and with the addition of extra virus and non-virus sequence at the junction sites. In the second phase, any additional sequence was lost during subsequent rounds of replication and selection. The approach was expanded to incorporate viruses from different enterovirus species to investigate intra- and interspecies recombination events

Recombination in enteroviruses is a biphasic replicative process involving the generation of greater-than genome length 'imprecise' intermediates

Recombination in enteroviruses provides an evolutionary mechanism for acquiring extensive regions of novel sequence, is suggested to have a role in genotype diversity and is known to have been key to the emergence of novel neuropathogenic variants of poliovirus. Despite the importance of this evolutionary mechanism, the recombination process remains relatively poorly understood. We investigated heterologous recombination using a novel reverse genetic approach that resulted in the isolation of intermediate chimeric intertypic polioviruses bearing genomes with extensive duplicated sequences at the recombination junction. Serial passage of viruses exhibiting such imprecise junctions yielded progeny with increased fitness which had lost the duplicated sequences. Mutations or inhibitors that changed polymerase fidelity or the coalescence of replication complexes markedly altered the yield of recombinants (but did not influence non-replicative recombination) indicating both that the process is replicative and that it may be possible to enhance or reduce recombination-mediated viral evolution if required. We propose that extant recombinants result from a biphasic process in which an initial recombination event is followed by a process of resolution, deleting extraneous sequences and optimizing viral fitness. This process has implications for our wider understanding of 'evolution by duplication' in the positive-strand RNA viruses