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.

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.

Molecular characterization of Drosophila C virus isolates

Reverse transcription coupled with polymerase chain reaction and restriction enzyme analysis was used to characterize 12 Drosophila C virus isolates from geographically different regions. A 1.2-kb fragment was amplified from cDNA and profiles from digestion with 20 restriction enzymes were generated. Analysis of the restriction fragment data gave estimates of nucleotide divergence of 0-10% between isolates. The isolates were grouped on the basis of genetic distance estimates derived from the restriction data. For the isolates from which a single genotype could be purified, a geographical pattern in the distribution of viral genotypes was identified. The 4 Moroccan isolates were very closely related to each other, differing in only 1 restriction profile. The 2 Australian isolates were each other's closest relatives, as were the 2 isolates first recovered in France. The PCR-RFLP technique used in this study has provided us with a simple procedure which can be used to characterize DCV isolates. A single enzyme, Tag I, generated 5 distinct and diagnostic restriction fragment patterns, which allowed easy assignment of isolates to one of the five viral genotypes identified in this study. (C) 1999 Academic Press.

The yellow fever 17D vaccine virus as a vector for the expression of foreign proteins: development of new live flavivirus vaccines

The Flaviviridae is a family of about 70 mostly arthropod-borne viruses many of which are major public health problems with members being present in most continents. Among the most important are yellow fever (YF), dengue with its four serotypes and Japanese encephalitis virus. A live attenuated virus is used as a cost effective, safe and efficacious vaccine against YF but no other live flavivirus vaccines have been licensed. The rise of recombinant DNA technology and its application to study flavivirus genome structure and expression has opened new possibilities for flavivirus vaccine development. One new approach is the use of cDNAs encopassing the whole viral genome to generate infectious RNA after in vitro transcription. This methodology allows the genetic mapping of specific viral functions and the design of viral mutants with considerable potential as new live attenuated viruses. The use of infectious cDNA as a carrier for heterologous antigens is gaining importance as chimeric viruses are shown to be viable, immunogenic and less virulent as compared to the parental viruses. The use of DNA to overcome mutation rates intrinsic of RNA virus populations in conjunction with vaccine production in cell culture should improve the reliability and lower the cost for production of live attenuated vaccines. The YF virus despite a long period ignored by researchers probably due to the effectiveness of the vaccine has made a come back, both in nature as human populations grow and reach endemic areas as well as in the laboratory being a suitable model to understand the biology of flaviviruses in general and providing new alternatives for vaccine development through the use of the 17D vaccine strain.

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.

Effects of concentrated viral communities on photosynthesis and community composition of co-occurring benthic microalgae and phytoplankton

Marine viruses have been shown to affect phytoplankton productivity; however, there are no reports on the effect of viruses on benthic microalgae (microphytobenthos). Hence, this study investigated the effects of elevated concentrations of virus-like particles on the photosynthetic physiology and community composition of benthic microalgae and phytoplankton. Virus populations were collected near the sediment surface and concentrated by tangential flow ultrafiltration, and the concentrate was added to benthic and water column samples that were obtained along a eutrophication gradient in the Brisbane River/Moreton Bay estuary, Australia. Photosynthetic and community responses of benthic microalgae, phytoplankton and bacteria were monitored over 7 d in aquaria and in situ. Benthic microalgal communities responded to viral enrichment in both eutrophic and oligotrophic sediments. In eutrophic sediments, Euglenophytes (Euglena sp.) and bacteria decreased in abundance by 20 to 60 and 26 to 66%, respectively, from seawater controls. In oligotrophic sediments, bacteria decreased in abundance by 30 to 42% from seawater controls but the dinoflagellate Gymnodinium sp. increased in abundance by 270 to 3600% from seawater controls, The increased abundance of Gymnodinium sp. may be related to increased availability of dissolved organic matter released from lysed bacteria. Increased (140 to 190% from seawater controls) initial chlorophyll a fluorescence measured with a pulse-amplitude modulated fluorometer was observed in eutrophic benthic microalgal incubations following virus enrichment, consistent with photosystem II damage. Virus enrichment in oligotrophic water significantly stimulated carbon fixation rates, perhaps due to increased nutrient availability by bacterial lysis. The interpretation of data from virus amendment experiments is difficult due to potential interaction with unidentified bioactive compounds within seawater concentrates. However, these results show that viruses are capable of influencing microbial dynamics in sediments.

Rhinovirus Genome Variation during Chronic Upper and Lower Respiratory Tract Infections.

Routine screening of lung transplant recipients and hospital patients for respiratory virus infections allowed to identify human rhinovirus (HRV) in the upper and lower respiratory tracts, including immunocompromised hosts chronically infected with the same strain over weeks or months. Phylogenetic analysis of 144 HRV-positive samples showed no apparent correlation between a given viral genotype or species and their ability to invade the lower respiratory tract or lead to protracted infection. By contrast, protracted infections were found almost exclusively in immunocompromised patients, thus suggesting that host factors rather than the virus genotype modulate disease outcome, in particular the immune response. Complete genome sequencing of five chronic cases to study rhinovirus genome adaptation showed that the calculated mutation frequency was in the range observed during acute human infections. Analysis of mutation hot spot regions between specimens collected at different times or in different body sites revealed that non-synonymous changes were mostly concentrated in the viral capsid genes VP1, VP2 and VP3, independent of the HRV type. In an immunosuppressed lung transplant recipient infected with the same HRV strain for more than two years, both classical and ultra-deep sequencing of samples collected at different time points in the upper and lower respiratory tracts showed that these virus populations were phylogenetically indistinguishable over the course of infection, except for the last month. Specific signatures were found in the last two lower respiratory tract populations, including changes in the 5'UTR polypyrimidine tract and the VP2 immunogenic site 2. These results highlight for the first time the ability of a given rhinovirus to evolve in the course of a natural infection in immunocompromised patients and complement data obtained from previous experimental inoculation studies in immunocompetent volunteers.

Estimating the basic reproductive number from viral sequence data.

Epidemiological processes leave a fingerprint in the pattern of genetic structure of virus populations. Here, we provide a new method to infer epidemiological parameters directly from viral sequence data. The method is based on phylogenetic analysis using a birth-death model (BDM) rather than the commonly used coalescent as the model for the epidemiological transmission of the pathogen. Using the BDM has the advantage that transmission and death rates are estimated independently and therefore enables for the first time the estimation of the basic reproductive number of the pathogen using only sequence data, without further assumptions like the average duration of infection. We apply the method to genetic data of the HIV-1 epidemic in Switzerland.

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 ≥1.5 log 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.

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.

Next-generation sequencing of HIV-1 RNA genomes: determination of error rates and minimizing artificial recombination.

Next-generation sequencing (NGS) is a valuable tool for the detection and quantification of HIV-1 variants in vivo. However, these technologies require detailed characterization and control of artificially induced errors to be applicable for accurate haplotype reconstruction. To investigate the occurrence of substitutions, insertions, and deletions at the individual steps of RT-PCR and NGS, 454 pyrosequencing was performed on amplified and non-amplified HIV-1 genomes. Artificial recombination was explored by mixing five different HIV-1 clonal strains (5-virus-mix) and applying different RT-PCR conditions followed by 454 pyrosequencing. Error rates ranged from 0.04-0.66% and were similar in amplified and non-amplified samples. Discrepancies were observed between forward and reverse reads, indicating that most errors were introduced during the pyrosequencing step. Using the 5-virus-mix, non-optimized, standard RT-PCR conditions introduced artificial recombinants in a fraction of at least 30% of the reads that subsequently led to an underestimation of true haplotype frequencies. We minimized the fraction of recombinants down to 0.9-2.6% by optimized, artifact-reducing RT-PCR conditions. This approach enabled correct haplotype reconstruction and frequency estimations consistent with reference data obtained by single genome amplification. RT-PCR conditions are crucial for correct frequency estimation and analysis of haplotypes in heterogeneous virus populations. We developed an RT-PCR procedure to generate NGS data useful for reliable haplotype reconstruction and quantification.