Genetic Composition and Complexity of Virus Populations at Tungro-Endemic and Outbreak Rice Sites

We have recently demonstrated the geographic isolation of rice tungro bacilliform virus (RTBV) populations in the tungro-endemic provinces of Isabela and North Cotabato, Philippines. In this study, we examined the genetic structure of the virus populations at the tungro-outbreak sites of Lanao del Norte, a province adjacent to North Cotabato. We also analyzed the virus populations at the tungro-endemic sites of Subang, Indonesia, and Dien Khanh, Vietnam. Total DNA extracts from 274 isolates were digested with EcoRV restriction enzyme and hybridized with a full-length probe of RTBV. In the total population, 22 EcoRV-restricted genome profiles (genotypes) were identified. Although overlapping genotypes could be observed, the outbreak sites of Lanao del Norte had a genotype combination distinct from that of Subang or Dien Khanh but a genotype combination similar to that identified earlier from North Cotabato, the adjacent endemic province. Sequence analysis of the intergenic region and part of the ORF1 RTBV genome from randomly selected genotypes confirms the geographic clustering of RTBV genotypes and, combined with restriction analysis, the results suggest a fragmented spatial distribution of RTBV local populations in the three countries. Because RTBV depends on rice tungro spherical virus (RTSV) for transmission, the population dynamics of both tungro viruses were then examined at the endemic and outbreak sites within the Philippines. The RTBV genotypes and the coat protein RTSV genotypes were used as indicators for virus diversity. A shift in population structure of both viruses was observed at the outbreak sites with a reduced RTBV but increased RTSV gene diversity

Low genetic diversities of rabies virus populations within different hosts in Brazil

The low rates of nonsynonymous evolution observed in natural rabies virus (RABV) isolates are suggested to have arisen in association with the structural and functional constraints operating on the virus protein and the infection strategies employed by RABV within infected hosts to avoid strong selection by the immune response. In order to investigate the relationship between the genetic characteristics of RABV populations within hosts and the virus evolution, the present study examined the genetic heterogeneities of RABV populations within naturally infected dogs and foxes in Brazil, as well as those of bat RABV populations that were passaged once in suckling mice. Sequence analyses of complete RABV glycoprotein (G) genes showed that RABV populations within infected hosts were genetically highly homogeneous whether they were infected naturally or experimentally (nucleotide diversities of 0-0.95 x 10(-3)). In addition, amino acid mutations were randomly distributed over the entire region of the G protein, and the nonsynonymous/synonymous rate ratios (d(N)/d(S)) for the G protein gene were less than 1. These findings suggest that the low genetic diversities of RABV populations within hosts reflect the stabilizing selection operating on the virus, the infection strategies of the virus, and eventually, the evolutionary patterns of the virus. (C) 2009 Elsevier B.V. All rights reserved.

Inter-and Intra-Site Genetic Diversity of Natural Field Populations of Rice Tungro Bacilliform Virus in the Philippines

The genetic structure of rice tungro bacilliform virus (RTBV) populations within and between growing sites was analyzed in a collection of natural field isolates from different rice varieties grown in eight tungro-endemic sites of the Philippines. Total DNA extracts from 345 isolates were digested with EcoRV restriction enzyme and hybridized with a full-length probe of RTBV, a procedure shown in preliminary experiments capable of revealing high levels of polymorphism in RTBV field isolates. In the total population, 17 distinct EcoRV-based genome profiles (genotypes) were identified and used as indicators for virus diversity. Distinct sets of genotypes occurred in Isabela and North Cotabato provinces suggesting a geographic isolation of virus populations. However, among the sites in each province, there were few significant differences in the genotype compositions of virus populations. The number of genotypes detected at a site varied from two to nine with a few genotypes dominating. In general the isolates at a site persisted from season to season indicating a genetic stability for the local virus population. Over the sampling time, IRRI rice varieties, which have green leafhopper resistance genes, supported similar virus populations to those supported by other varieties, indicating that the variety of the host exerted no apparent selection pressures. Insect transmission experiments on selected RTBV field isolates showed that dramatic shifts in genotype and phenotype distributions can occur in response to host /environmental shifts.

Genetic Diversity of Rice Tungro Spherical Virus in Tungro-Endemic Provinces of the Philippines and Indonesia

The two adjacent genes of coat protein 1 and 2 of rice tungro spherical virus (RTSV) were amplified from total RNA extracts of serologically indistinguishable field isolates from the Philippines and Indonesia, using reverse transcriptase polymerase chain reaction (RT-PCR). Digestion with HindIII and BstYI restriction endonucleases differentiated the amplified DNA products into eight distinct coat protein genotypes. These genotypes were then used as indicators of virus diversity in the field. Inter- and intra-site diversities were determined over three cropping seasons. At each of the sites surveyed, one or two main genotypes prevailed together with other related minor or mixed genotypes that did not replace the main genotype over the sampling time. The cluster of genotypes found at the Philippines sites was significantly different from the one at the Indonesia sites, suggesting geographic isolation for virus populations. Phylogenetic studies based on the nucleotide sequences of 38 selected isolates confirm the spatial distribution of RTSV virus populations but show that gene flow may occur between populations. Under the present conditions, rice varieties do not seem to exert selective pressure on the virus populations. Based on the selective constraints in the coat protein amino acid sequences and the virus genetic composition per site, a negative selection model followed by random-sampling events due to vector transmissions is proposed to explain the inter-site diversity observed

Experimental observations of rapid maize streak virus evolution reveal a strand-specific nucleotide substitution bias

Background. Recent reports have indicated that single-stranded DNA (ssDNA) viruses in the taxonomic families Geminiviridae, Parvoviridae and Anellovirus may be evolving at rates of ∼10-4 substitutions per site per year (subs/site/year). These evolution rates are similar to those of RNA viruses and are surprisingly high given that ssDNA virus replication involves host DNA polymerases with fidelities approximately 10 000 times greater than those of error-prone viral RNA polymerases. Although high ssDNA virus evolution rates were first suggested in evolution experiments involving the geminivirus maize streak virus (MSV), the evolution rate of this virus has never been accurately measured. Also, questions regarding both the mechanistic basis and adaptive value of high geminivirus mutation rates remain unanswered. Results. We determined the short-term evolution rate of MSV using full genome analysis of virus populations initiated from cloned genomes. Three wild type viruses and three defective artificial chimaeric viruses were maintained in planta for up to five years and displayed evolution rates of between 7.4 × 10-4 and 7.9 × 10-4 subs/site/year. Conclusion. These MSV evolution rates are within the ranges observed for other ssDNA viruses and RNA viruses. Although no obvious evidence of positive selection was detected, the uneven distribution of mutations within the defective virus genomes suggests that some of the changes may have been adaptive. We also observed inter-strand nucleotide substitution imbalances that are consistent with a recent proposal that high mutation rates in geminiviruses (and possibly ssDNA viruses in general) may be due to mutagenic processes acting specifically on ssDNA molecules. © 2008 Walt et al; licensee BioMed Central Ltd.

Ross River virus evolution : implications for vaccine development

Ross River virus is a mosquito-borne alphavirus that causes approximately 5000 cases of epidemic polyarthritis in Australia each year and has direct medical-associated costs of approximately US$15 million annually. While mosquito control programs are able, at best, to contain rather than prevent this disease, natural infection with Ross River virus confers lifelong protection against subsequent clinical infection. A killed-virus vaccine has been developed, which is in Phase III clinical trials. Analyses of intra-host genetic diversity and of long-term evolutionary changes in Ross River virus populations suggest that antigenic variation is unlikely to pose a threat to the efficacy of this vaccine.

An unbiased genetic screen reveals the polygenic nature of the influenza virus anti-interferon response

UNLABELLED: Influenza A viruses counteract the cellular innate immune response at several steps, including blocking RIG I-dependent activation of interferon (IFN) transcription, interferon (IFN)-dependent upregulation of IFN-stimulated genes (ISGs), and the activity of various ISG products; the multifunctional NS1 protein is responsible for most of these activities. To determine the importance of other viral genes in the interplay between the virus and the host IFN response, we characterized populations and selected mutants of wild-type viruses selected by passage through non-IFN-responsive cells. We reasoned that, by allowing replication to occur in the absence of the selection pressure exerted by IFN, the virus could mutate at positions that would normally be restricted and could thus find new optimal sequence solutions. Deep sequencing of selected virus populations and individual virus mutants indicated that nonsynonymous mutations occurred at many phylogenetically conserved positions in nearly all virus genes. Most individual mutants selected for further characterization induced IFN and ISGs and were unable to counteract the effects of exogenous IFN, yet only one contained a mutation in NS1. The relevance of these mutations for the virus phenotype was verified by reverse genetics. Of note, several virus mutants expressing intact NS1 proteins exhibited alterations in the M1/M2 proteins and accumulated large amounts of deleted genomic RNAs but nonetheless replicated to high titers. This suggests that the overproduction of IFN inducers by these viruses can override NS1-mediated IFN modulation. Altogether, the results suggest that influenza viruses replicating in IFN-competent cells have tuned their complete genomes to evade the cellular innate immune system and that serial replication in non-IFN-responsive cells allows the virus to relax from these constraints and find a new genome consensus within its sequence space.

IMPORTANCE: In natural virus infections, the production of interferons leads to an antiviral state in cells that effectively limits virus replication. The interferon response places considerable selection pressure on viruses, and they have evolved a variety of ways to evade it. Although the influenza virus NS1 protein is a powerful interferon antagonist, the contributions of other viral genes to interferon evasion have not been well characterized. Here, we examined the effects of alleviating the selection pressure exerted by interferon by serially passaging influenza viruses in cells unable to respond to interferon. Viruses that grew to high titers had mutations at many normally conserved positions in nearly all genes and were not restricted to the NS1 gene. Our results demonstrate that influenza viruses have fine-tuned their entire genomes to evade the interferon response, and by removing interferon-mediated constraints, viruses can mutate at genome positions normally restricted by the interferon response.

Coevolución virus-planta: impacto de las infecciones virales en poblaciones silvestres de Chiltepín (capsicum annuum var. Aviculare (dierbach) d’arcy & eshbaugh) en México.

El impacto negativo que tienen los virus en las plantas hace que estos puedan ejercer un papel ecológico como moduladores de la dinámica espacio-temporal de las poblaciones de sus huéspedes. Entender cuáles son los mecanismos genéticos y los factores ambientales que determinan tanto la epidemiología como la estructura genética de las poblaciones de virus puede resultar de gran ayuda para la comprensión del papel ecológico de las infecciones virales. Sin embargo, existen pocos trabajos experimentales que hayan abordado esta cuestión. En esta tesis, se analiza el efecto de la heterogeneidad del paisaje sobre la incidencia de los virus y la estructura genética de sus poblaciones. Asimismo, se explora como dichos factores ambientales influyen en la importancia relativa que los principales mecanismos de generación de variabilidad genética (mutación, recombinación y migración) tienen en la evolución de los virus. Para ello se ha usado como sistema los begomovirus que infectan poblaciones de chiltepín (Capsicum annuum var. aviculare (Dierbach) D´Arcy & Eshbaugh) en México. Se analizó la incidencia de diferentes virus en poblaciones de chiltepín distribuidas a lo largo de seis provincias biogeográficas, representando el área de distribución de la especie en México, y localizadas en hábitats con diferente grado de intervención humana: poblaciones sin intervención humana (silvestres); poblaciones toleradas (lindes y pastizales), y poblaciones manejadas por el hombre (monocultivos y huertos familiares). Entre los virus analizados, los begomovirus mostraron la mayor incidencia, detectándose en todas las poblaciones y años de muestreo. Las únicas dos especies de begomovirus que se encontraron infectando al chiltepín fueron: el virus del mosaico dorado del chile (Pepper golden mosaic virus, PepGMV) y el virus huasteco del amarilleo de venas del chile (Pepper huasteco yellow vein virus, PHYVV). Por ello, todos los análisis realizados en esta tesis se centran en estas dos especies de virus. La incidencia de PepGMV y PHYVV, tanto en infecciones simples como mixtas, aumento cuanto mayor fue el nivel de intervención humana en las poblaciones de chiltepín, lo que a su vez se asoció con una menor biodiversidad y una mayor densidad de plantas. Además, la incidencia de infecciones mixtas, altamente relacionada con la presencia de síntomas, fue también mayor en las poblaciones cultivadas. La incidencia de estos dos virus también varió en función de la población de chiltepín y de la provincia biogeográfica. Por tanto, estos resultados apoyan una de las hipótesis XVI clásicas de la Patología Vegetal según la cual la simplificación de los ecosistemas naturales debida a la intervención humana conduce a un mayor riesgo de enfermedad de las plantas, e ilustran sobre la importancia de la heterogeneidad del paisaje a diferentes escalas en la determinación de patrones epidemiológicos. La heterogeneidad del paisaje no solo afectó a la epidemiología de PepGMV y PHYVV, sino también a la estructura genética de sus poblaciones. En ambos virus, el nivel de diferenciación genética mayor fue la población, probablemente asociado a la capacidad de migración de su vector Bemisia tabaci; y en segundo lugar la provincia biogeográfica, lo que podría estar relacionado con el papel del ser humano como agente dispersor de PepGMV y PHYVV. La estima de las tasas de sustitución nucleotídica de las poblaciones de PepGMV y PHYVV mostró una rápida dinámica evolutiva. Los árboles filogenéticos de ambos virus presentaron una topología en estrella, lo que sugiere una expansión reciente en las poblaciones de chiltepín. La reconstrucción de los patrones de migración de ambos virus indicó que ésta expansión parece haberse producido desde la zona central de México siguiendo un patrón radial, y en los últimos 30 años. Es importante tener en cuenta que el patrón espacial de la diversidad genética de las poblaciones de PepGMV y PHYVV es similar al descrito previamente para el chiltepín lo que podría dar lugar a la congruencia de las genealogías del huésped y la de los virus. Dicha congruencia se encontró cuando se tuvieron en cuenta únicamente las poblaciones de hábitats silvestres y tolerados, lo que probablemente se debe a una codivergencia en el espacio pero no en el tiempo, dado que la evolución de virus y huésped han ocurrido a escalas temporales muy diferentes. Finalmente, el análisis de la frecuencia de recombinación en PepGMV y PHYVV indicó que esta juega un papel importante en la evolución de ambos virus, dependiendo su importancia del nivel de intervención humana de la población de chiltepín. Este factor afectó también a la intensidad de la selección a la que se ven sometidos los genomas de PepGMV y PHYVV. Los resultados de esta tesis ponen de manifiesto la importancia que la reducción de la biodiversidad asociada al nivel de intervención humana de las poblaciones de plantas y la heterogeneidad del paisaje tiene en la emergencia de nuevas enfermedades virales. Por tanto, es necesario considerar estos factores ambientales a la hora de comprender la epidemiologia y la evolución de los virus de plantas.XVII SUMMARY Plant viruses play a key role as modulators of the spatio-temporal dynamics of their host populations, due to their negative impact in plant fitness. Knowledge on the genetic and environmental factors that determine the epidemiology and the genetic structure of virus populations may help to understand the ecological role of viral infections. However, few experimental works have addressed this issue. This thesis analyses the effect of landscape heterogeneity in the prevalence of viruses and the genetic structure of their populations. Also, how these environmental factors influence the relative importance of the main mechanisms for generating genetic variability (mutation, recombination and migration) during virus evolution is explored. To do so, the begomoviruses infecting chiltepin (Capsicum annuum var. aviculare (Dierbach) D'Arcy & Eshbaugh) populations in Mexico were used. Incidence of different viruses in chiltepin populations of six biogeographical provinces representing the species distribution in Mexico was determined. Populations belonged to different habitats according to the level of human management: populations with no human intervention (Wild); populations naturally dispersed and tolerated in managed habitats (let-standing), and human managed populations (cultivated). Among the analyzed viruses, the begomoviruses showed the highest prevalence, being detected in all populations and sampling years. Only two begomovirus species infected chiltepin: Pepper golden mosaic virus, PepGMV and Pepper huasteco yellow vein virus, PHYVV. Therefore, all the analyses presented in this thesis are focused in these two viruses. The prevalence of PepGMV and PHYVV, in single and mixed infections, increased with higher levels of human management of the host population, which was associated with decreased biodiversity and increased plant density. Furthermore, cultivated populations showed higher prevalence of mixed infections and symptomatic plants. The prevalence of the two viruses also varied depending on the chiltepin population and on the biogeographical province. Therefore, these results support a classical hypothesis of Plant Pathology stating that simplification of natural ecosystems due to human management leads to an increased disease risk, and illustrate on the importance of landscape heterogeneity in determining epidemiological patterns. Landscape heterogeneity not only affected the epidemiology of PepGMV and PHYVV, but also the genetic structure of their populations. Both viruses had the highest level of genetic differentiation at the population scale, probably associated with the XVIII migration patterns of its vector Bemisia tabaci, and a second level at the biogeographical province scale, which could be related to the role of humans as dispersal agents of PepGMV and PHYVV. The estimates of nucleotide substitution rates of the virus populations indicated rapid evolutionary dynamics. Accordingly, phylogenetic trees of both viruses showed a star topology, suggesting a recent diversification in the chiltepin populations. Reconstruction of PepGMV and PHYVV migration patterns indicated that they expanded from central Mexico following a radial pattern during the last 30 years. Importantly, the spatial genetic structures of the virus populations were similar to that described previously for the chiltepin, which may result in the congruence of the host and virus genealogies. Such congruence was found only in wild and let-standing populations. This is probably due to a co-divergence in space but not in time, given the different evolutionary time scales of the host and virus populations. Finally, the frequency of recombination detected in the PepGMV and PHYVV populations indicated that this mechanism plays an important role in the evolution of both viruses at the intra-specific scale. The level of human management had a minor effect on the frequency of recombination, but influenced the strength of negative selective pressures in the viral genomes. The results of this thesis highlight the importance of decreased biodiversity in plant populations associated with the level of human management and of landscape heterogeneity on the emergence of new viral diseases. Therefore it is necessary to consider these environmental factors in order to fully understand the epidemiology and evolution of plant viruses.

RNA virus error catastrophe: Direct molecular test by using ribavirin

RNA viruses evolve rapidly. One source of this ability to rapidly change is the apparently high mutation frequency in RNA virus populations. A high mutation frequency is a central tenet of the quasispecies theory. A corollary of the quasispecies theory postulates that, given their high mutation frequency, animal RNA viruses may be susceptible to error catastrophe, where they undergo a sharp drop in viability after a modest increase in mutation frequency. We recently showed that the important broad-spectrum antiviral drug ribavirin (currently used to treat hepatitis C virus infections, among others) is an RNA virus mutagen, and we proposed that ribavirin's antiviral effect is by forcing RNA viruses into error catastrophe. However, a direct demonstration of error catastrophe has not been made for ribavirin or any RNA virus mutagen. Here we describe a direct demonstration of error catastrophe by using ribavirin as the mutagen and poliovirus as a model RNA virus. We demonstrate that ribavirin's antiviral activity is exerted directly through lethal mutagenesis of the viral genetic material. A 99.3% loss in viral genome infectivity is observed after a single round of virus infection in ribavirin concentrations sufficient to cause a 9.7-fold increase in mutagenesis. Compiling data on both the mutation levels and the specific infectivities of poliovirus genomes produced in the presence of ribavirin, we have constructed a graph of error catastrophe showing that normal poliovirus indeed exists at the edge of viability. These data suggest that RNA virus mutagens may represent a promising new class of antiviral drugs.

Exponential increases of RNA virus fitness during large population transmissions.

The great adaptability shown by RNA viruses is a consequence of their high mutation rates. Here we investigate the kinetics of virus fitness gains during repeated transfers of large virus populations in cell culture. Results always show that fitness increases exponentially. Low fitness clones exhibit regular increases observed as biphasic periods of exponential evolutionary improvement, while neutral clones show monophasic kinetics. These results are significant for RNA virus epidemiology, optimal handling of attenuated live virus vaccines, and routine laboratory procedures.

Phylodynamic evidence of the migration of turnip mosaic potyvirus from Europe to Australia and New Zealand

Turnip mosaic virus (TuMV) is a potyvirus that is transmitted by aphids and infects a wide range of plant species. We investigated the evolution of this pathogen by collecting 32 isolates of TuMV, mostly from Brassicaceae plants, in Australia and New Zealand. We performed a variety of sequence-based phylogenetic and population genetic analyses of the complete genomic sequences and of three non-recombinogenic regions of those sequences. The substitution rates, divergence times and phylogeographical patterns of the virus populations were estimated. Six inter- and seven intralineage recombination-type patterns were found in the genomes of the Australian and New Zealand isolates, and all were novel. Only one recombination-type pattern has been found in both countries. The Australian and New Zealand populations were genetically different, and were different from the European and Asian populations. Our Bayesian coalescent analyses, based on a combination of novel and published sequence data from three nonrecombinogenic protein-encoding regions, showed that TuMV probably started to migrate from Europe to Australia and New Zealand more than 80 years ago, and that distinct populations arose as a result of evolutionary drivers such as recombination. The basal-B2 subpopulation in Australia and New Zealand seems to be older than those of the world-B2 and -B3 populations. To our knowledge, our study presents the first population genetic analysis of TuMV in Australia and New Zealand. We have shown that the time of migration of TuMV correlates well with the establishment of agriculture and migration of Europeans to these countries.

How many Coccolithovirus genotypes does it take to terminate an Emiliania huxleyi bloom?

Giant viruses are known to be significant mortality agents of phytoplankton, often being implicated in the terminations of large Emiliania huxleyi blooms. We have previously shown the high temporal variability of E. huxleyi-infecting coccolithoviruses (EhVs) within a Norwegian fjord mesocosm. In the current study we investigated EhV dynamics within a naturally-occurring E. huxleyi bloom in the Western English Channel. Using denaturing gradient gel electrophoresis and marker gene sequencing, we uncovered a spatially highly dynamic Coccolithovirus population that was associated with a genetically stable E. huxleyi population as revealed by the major capsid protein gene (mcp) and coccolith morphology motif (CMM), respectively. Coccolithoviruses within the bloom were found to be variable with depth and unique virus populations were detected at different stations sampled indicating a complex network of EhV-host infections. This ultimately will have significant implications to the internal structure and longevity of ecologically important E. huxleyi blooms.

Development and validation of a qPCR-based method for quantifying Shiga toxin-encoding and other lambdoid bacteriophages

P>To address whether seasonal variability exists among Shiga toxin-encoding bacteriophage (Stx phage) numbers on a cattle farm, conventional plaque assay was performed on water samples collected over a 17 month period. Distinct seasonal variation in bacteriophage numbers was evident, peaking between June and August. Removal of cattle from the pasture precipitated a reduction in bacteriophage numbers, and during the winter months, no bacteriophage infecting Escherichia coli were detected, a surprising occurrence considering that 1031 tailed-bacteriophages are estimated to populate the globe. To address this discrepancy a culture-independent method based on quantitative PCR was developed. Primers targeting the Q gene and stx genes were designed that accurately and discriminately quantified artificial mixed lambdoid bacteriophage populations. Application of these primer sets to water samples possessing no detectable phages by plaque assay, demonstrated that the number of lambdoid bacteriophage ranged from 4.7 x 104 to 6.5 x 106 ml-1, with one in 103 free lambdoid bacteriophages carrying a Shiga toxin operon (stx). Specific molecular biological tools and discriminatory gene targets have enabled virus populations in the natural environment to be enumerated and similar strategies could replace existing propagation-dependent techniques, which grossly underestimate the abundance of viral entities.

Population dynamics of DENV-1 genotype V in Brazil is characterized by co-circulation and strain/lineage replacement

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Replicative phenotyping adds value to genotypic resistance testing in heavily pre-treated HIV-infected individuals--the Swiss HIV Cohort Study

Background Replicative phenotypic HIV resistance testing (rPRT) uses recombinant infectious virus to measure viral replication in the presence of antiretroviral drugs. Due to its high sensitivity of detection of viral minorities and its dissecting power for complex viral resistance patterns and mixed virus populations rPRT might help to improve HIV resistance diagnostics, particularly for patients with multiple drug failures. The aim was to investigate whether the addition of rPRT to genotypic resistance testing (GRT) compared to GRT alone is beneficial for obtaining a virological response in heavily pre-treated HIV-infected patients. Methods Patients with resistance tests between 2002 and 2006 were followed within the Swiss HIV Cohort Study (SHCS). We assessed patients' virological success after their antiretroviral therapy was switched following resistance testing. Multilevel logistic regression models with SHCS centre as a random effect were used to investigate the association between the type of resistance test and virological response (HIV-1 RNA <50 copies/mL or ≥1.5log reduction). Results Of 1158 individuals with resistance tests 221 with GRT+rPRT and 937 with GRT were eligible for analysis. Overall virological response rates were 85.1% for GRT+rPRT and 81.4% for GRT. In the subgroup of patients with >2 previous failures, the odds ratio (OR) for virological response of GRT+rPRT compared to GRT was 1.45 (95% CI 1.00-2.09). Multivariate analyses indicate a significant improvement with GRT+rPRT compared to GRT alone (OR 1.68, 95% CI 1.31-2.15). Conclusions In heavily pre-treated patients rPRT-based resistance information adds benefit, contributing to a higher rate of treatment success.