Severe acute respiratory syndrome (SARS) is a novel human illness caused by a previously unrecognized coronavirus (CoV) termed SARS-CoV. There are conflicting reports on the animal reservoir of SARS-CoV. Many of the groups that argue carnivores are the original reservoir of SARS-CoV use a phylogeny to support their argument. However, the phylogenies in these studies often lack outgroup and rooting criteria necessary to determine the origins of SARS-CoV. Recently, SARS-CoV has been isolated from various species of Chiroptera from China (e.g., Rhinolophus sinicus) thus leading to reconsideration of the original reservoir of SARS-CoV. We evaluated the hypothesis that SARS-CoV isolated from Chiroptera are the original zoonotic source for SARS-CoV by sampling SARS-CoV and non-SARS-CoV from diverse hosts including Chiroptera, as well as carnivores, artiodactyls, rodents, birds and humans. Regardless of alignment parameters, optimality criteria, or isolate sampling, the resulting phylogenies clearly show that the SARS-CoV was transmitted to small carnivores well after the epidemic of SARS in humans that began in late 2002. The SARS-CoV isolates from small carnivores in Shenzhen markets form a terminal clade that emerged recently from within the radiation of human SARS-CoV. There is evidence of subsequent exchange of SARS-CoV between humans and carnivores. In addition SARS-CoV was transmitted independently from humans to farmed pigs (Sus scrofa). The position of SARS-CoV isolates from Chiroptera are basal to the SARS-CoV clade isolated from humans and carnivores. Although sequence data indicate that Chiroptera are a good candidate for the original reservoir of SARS-CoV, the structural biology of the spike protein of SARS-CoV isolated from Chiroptera suggests that these viruses are not able to interact with the human variant of the receptor of SARS-CoV, angiotensin-converting enzyme 2 (ACE2). In SARS-CoV we study, both visually and statistically, labile genomic fragments and, putative key mutations of the spike protein that may be associated with host shifts. We display host shifts and candidate mutations on trees projected in virtual globes depicting the spread of SARS-CoV. These results suggest that more sampling of coronaviruses from diverse hosts, especially Chiroptera, carnivores and primates, will be required to understand the genomic and biochemical evolution of coronaviruses, including SARS-CoV.

BackgroundIn 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.ResultsUsing 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.ConclusionsAs 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.

Avian influenza (H5N1) has been of great social and economic importance since it first infected humans in Hong Kong in 1997. A highly pathogenic strain has spread from China and has killed humans in east Asia, west Africa, south Asia, and the Middle East. Recently, several molecular phylogenetic studies have focused on the relationships of various clades of H5N1 and their spread over time, space, and various hosts. These studies examining the geographical spread of H5N1 have based their conclusions on a single tree. This tree often results from the analysis of the genomic segment coding for the haemagglutinin (HA) or neuraminidase (NA) proteins and a limited sample of viral isolates. Here we present the first study using multiple candidate trees to estimate geographical transmission routes of H5N1. In addition, we use all high-quality HA and NA sequences available to the public as of June 2008. We estimated geographical transmission routes of H5N1 by optimizing multistate characters with states representing different geographical regions over a pool of presumed minimum-length trees. We also developed means to visualize our results in Keyhole Markup Language (KML) for virtual globes. We provide these methods as a web application entitled ''Routemap'' (http:// routemap.osu.edu). The resulting visualizations are akin to airline route maps but they depict the routes of spread of viral lineages. We compare our results with the results of previous studies. We focus on the sensitivity of results to sampling of tree space, character coding schemes, optimization methods, and taxon sampling. In conclusion, we find that using one tree and a single character optimization method will ignore many of the transmission routes indicated by genetic sequence and geographical data.

Novel pathogens have the potential to become critical issues of national security, public health and economic welfare. As demonstrated by the response to Severe Acute Respiratory Syndrome (SARS) and influenza, genomic sequencing has become an important method for diagnosing agents of infectious disease. Despite the value of genomic sequences in characterizing novel pathogens, raw data on their own do not provide the information needed by public health officials and researchers. One must integrate knowledge of the genomes of pathogens with host biology and geography to understand the etiology of epidemics. To these ends, we have created an application called Supramap (http://supramap.osu.edu) to put information on the spread of pathogens and key mutations across time, space and various hosts into a geographic information system (GIS). To build this application, we created a web service for integrated sequence alignment and phylogenetic analysis as well as methods to describe the tree, mutations, and host shifts in Keyhole Markup Language (KML). We apply the application to 239 sequences of the polymerase basic 2 (PB2) gene of recent isolates of avian influenza (H5N1). We map a mutation, glutamic acid to lysine at position 627 in the PB2 protein (E627K), in H5N1 influenza that allows for increased replication of the virus in mammals. We use a statistical test to support the hypothesis of a correlation of E627K mutations with avian-mammalian host shifts but reject the hypothesis that lineages with E627K are moving westward. Data, instructions for use, and visualizations are included as supplemental materials at: http://supramap.osu.edu/sm/supramap/publications. Ó The Willi Hennig Society 2010.We have created a web-based workflow application, Supramap (http://supramap.osu.edu). Using a web browser, a user inputs text files containing sequence and or phenotypic data, latitude and longitude coordinates, and (optionally) a date of isolation for each strain. Our application then executes a workflow that entails integrated sequence alignment and phylogenetic analysis, computation of character changes (e.g., mutations and host shifts), and geographical projection of the tree on a computing cluster. Once the analyses are complete, the user can download a phylogenetic layer expressed in KML file and view the file with a Geographic Information System (GIS). The user can use the phylogenetic layer to visualize several aspects of pathogen evolution including: spread of lineages, mutations, shifts among hosts, and phenotypic changes over geography and time. We illustrate the use of the system with a case study on H5N1 and discuss use of visualization in conjunction with statistical validation. Other tree projection effortsSupramap is superficially similar to other efforts for projecting phylogenetic trees in GIS, such as

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