Cladograms with Path to Event (ClaPTE): a novel algorithm to detect associations between genotypes or phenotypes using phylogenies.

BACKGROUND: Associations between genotype and phenotype provide insight into the evolution of pathogenesis, drug resistance, and the spread of pathogens between hosts. However, common ancestry can lead to apparent associations between biologically unrelated features. The novel method Cladograms with Path to Event (ClaPTE) detects associations between character-pairs (either a pair of mutations or a mutation paired with a phenotype) while adjusting for common ancestry, using phylogenetic trees. METHODS: ClaPTE tests for character-pairs changing close together on the phylogenetic tree, consistent with an associated character-pair. ClaPTE is compared to three existing methods (independent contrasts, mixed model, and likelihood ratio) to detect character-pair associations adjusted for common ancestry. Comparisons utilize simulations on gene trees for: HIV Env, HIV promoter, and bacterial DnaJ and GuaB; and case studies for Oseltamavir resistance in Influenza, and for DnaJ and GuaB. Simulated data include both true-positive/associated character-pairs, and true-negative/not-associated character-pairs, used to assess type I (frequency of p-values in true-negatives) and type II (sensitivity to true-positives) error control. RESULTS AND CONCLUSIONS: ClaPTE has competitive sensitivity and better type I error control than existing methods. In the Influenza/Oseltamavir case study, ClaPTE reports no new permissive mutations but detects associations between adjacent (in primary sequence) amino acid positions which other methods miss. In the DnaJ and GuaB case study, ClaPTE reports more frequent associations between positions both from the same protein family than between positions from different families, in contrast to other methods. In both case studies, the results from ClaPTE are biologically plausible.

Genotypic and phenotypic heterogeneity in the U3R region of HIV type 1 subtype C.

Approximately 20% of all HIV-1 mother-to-child transmission (MTCT) occurs in utero (IU). In a chronic HIV infection, HIV-1 exists as a complex swarm of genetic variants, and following IU MTCT, viral genomic diversity is restricted through a mechanism that remains to be described. The 5' U3R region of the HIV-1 long terminal repeat (LTR) contains multiple transcription factor (TF) binding sites and regulates viral transcription. In this study, we tested the hypothesis that sequence polymorphisms in the U3R region of LTR are associated with IU MTCT. To this end, we used single template amplification to isolate 517 U3R sequences from maternal, placental, and infant plasma derived from 17 HIV-infected Malawian women: eight whose infants remained HIV uninfected (NT) and nine whose infants became HIV infected IU. U3R sequences show pairwise diversities ranging from 0.2% to 2.3%. U3R sequences from one participant contained two, three, or four putative NF-κB binding sites. Phylogenetic reconstructions indicated that U3R sequences from eight of nine IU participants were consistent with placental compartmentalization of HIV-1 while only one of eight NT cases was consistent with such compartmentalization. Specific TF sequence polymorphisms were not significantly associated with IU MTCT. To determine if replication efficiency of the U3R sequences was associated with IU MTCT, we cloned 90 U3R sequences and assayed promoter activity in multiple cell lines. Although we observed significant, yet highly variable promoter activity and TAT induction of promoter activity in the cell lines tested, there was no association between measured promoter activity and MTCT status. Thus, we were unable to detect a promoter genotype or phenotype associated with IU MTCT.

Eco-virological approach for assessing the role of wild birds in the spread of avian influenza H5N1 along the Central Asian Flyway.

A unique pattern of highly pathogenic avian influenza (HPAI) H5N1 outbreaks has emerged along the Central Asia Flyway, where infection of wild birds has been reported with steady frequency since 2005. We assessed the potential for two hosts of HPAI H5N1, the bar-headed goose (Anser indicus) and ruddy shelduck (Tadorna tadorna), to act as agents for virus dispersal along this 'thoroughfare'. We used an eco-virological approach to compare the migration of 141 birds marked with GPS satellite transmitters during 2005-2010 with: 1) the spatio-temporal patterns of poultry and wild bird outbreaks of HPAI H5N1, and 2) the trajectory of the virus in the outbreak region based on phylogeographic mapping. We found that biweekly utilization distributions (UDs) for 19.2% of bar-headed geese and 46.2% of ruddy shelduck were significantly associated with outbreaks. Ruddy shelduck showed highest correlation with poultry outbreaks owing to their wintering distribution in South Asia, where there is considerable opportunity for HPAI H5N1 spillover from poultry. Both species showed correlation with wild bird outbreaks during the spring migration, suggesting they may be involved in the northward movement of the virus. However, phylogeographic mapping of HPAI H5N1 clades 2.2 and 2.3 did not support dissemination of the virus in a northern direction along the migration corridor. In particular, two subclades (2.2.1 and 2.3.2) moved in a strictly southern direction in contrast to our spatio-temporal analysis of bird migration. Our attempt to reconcile the disciplines of wild bird ecology and HPAI H5N1 virology highlights prospects offered by both approaches as well as their limitations.

Different regions of HIV-1 subtype C env are associated with placental localization and in utero mother-to-child transmission.

HIV infections are initiated by a limited number of variants that diverge into a diverse quasispecies swarm. During in utero mother-to-child transmission (IU MTCT), transmitted viral variants must pass through multiple unique environments, and our previously published data suggest a nonstochastic model of transmission. As an alternative to a stochastic model of viral transmission, we hypothesize that viral selection in the placental environment influences the character of the viral quasispecies when HIV-1 is transmitted in utero. To test this hypothesis, we used single-template amplification to isolate HIV-1 envelope gene (env) sequences from both peripheral plasma and the placentas of eight nontransmitting (NT) and nine IU-transmitting participants. Statistically significant compartmentalization between peripheral and placental HIV-1 env was detected in one of the eight NT cases and six of the nine IU MTCT cases. In addition, viral sequences isolated from IU MTCT placental tissue showed variation in env V1 loop lengths compared to matched maternal sequences, while NT placental env sequences did not. Finally, comparison of env sequences from NT and IU MTCT participants indicated statistically significant differences in Kyte-Doolittle hydropathy in the signal peptide, C2, V3, and C3 regions. Our working hypothesis is that the hydropathy differences in Env associated with IU MTCT alter viral cellular tropism or affinity, allowing HIV-1 to efficiently infect placentally localized cells.

Genome informatics of influenza A: from data sharing to shared analytical capabilities.

Emerging infectious diseases are critical issues of public health and the economic and social stability of nations. As demonstrated by the international response to the severe acute respiratory syndrome (SARS) and influenza A, rapid genomic sequencing is a crucial tool to understand diseases that occur at the interface of human and animal populations. However, our ability to make sense of sequence data lags behind our ability to acquire the data. The potential of sequence data on pathogens is not fully realized until raw data are translated into public health intelligence. Sequencing technologies have become highly mechanized. If the political will for data sharing remains strong, the frontier for progress in emerging infectious diseases will be in analysis of sequence data and translation of results into better public health science and policy. For example, applying analytical tools such as Supramap (http://supramap.osu.edu) to genomic data for pathogens, public health scientists can track specific mutations in pathogens that confer the ability to infect humans or resist drugs. The results produced by the Supramap application are compelling visualizations of pathogen lineages and features mapped into geographic information systems that can be used to test hypotheses and to follow the spread of diseases across geography and hosts and communicate the results to a wide audience.

Selection for resistance to oseltamivir in seasonal and pandemic H1N1 influenza and widespread co-circulation of the lineages.

BACKGROUND: In Spring 2009, a novel reassortant strain of H1N1 influenza A emerged as a lineage distinct from seasonal H1N1. On June 11, the World Heath Organization declared a pandemic - the first since 1968. There are currently two main branches of H1N1 circulating in humans, a seasonal branch and a pandemic branch. The primary treatment method for pandemic and seasonal H1N1 is the antiviral drug Tamiflu (oseltamivir). Although many seasonal H1N1 strains around the world are resistant to oseltamivir, initially, pandemic H1N1 strains have been susceptible to oseltamivir. As of February 3, 2010, there have been reports of resistance to oseltamivir in 225 cases of H1N1 pandemic influenza. The evolution of resistance to oseltamivir in pandemic H1N1 could be due to point mutations in the neuraminidase or a reassortment event between seasonal H1N1 and pandemic H1N1 viruses that provide a neuraminidase carrying an oseltamivir-resistant genotype to pandemic H1N1. RESULTS: Using phylogenetic analysis of neuraminidase sequences, we show that both seasonal and pandemic lineages of H1N1 are evolving to direct selective pressure for resistance to oseltamivir. Moreover, seasonal lineages of H1N1 that are resistant to oseltamivir co-circulate with pandemic H1N1 throughout the globe. By combining phylogenetic and geographic data we have thus far identified 53 areas of co-circulation where reassortment can occur. At our website POINTMAP, http://pointmap.osu.edu we make available a visualization and an application for updating these results as more data are released. CONCLUSIONS: As oseltamivir is a keystone of preparedness and treatment for pandemic H1N1, the potential for resistance to oseltamivir is an ongoing concern. Reassortment and, more likely, point mutation have the potential to create a strain of pandemic H1N1 against which we have a reduced number of treatment options.