The researchers linked many powerful computer systems together to analyze enormous amounts of genetic data collected from all publicly available isolated strains of the H5N1 virus – the cause of avian flu. They then developed a new Web-based application that will allow health officials and the public visualize how the virus moved across the globe using Google Earth.

The green lines on this interactive map represent how pandemic influenza (H1N1) has moved from points in the United States to geographic locations across the globe. Screenshot taken using Google Earth.

The resulting visualizations, based on results of the data analysis, represent the most comprehensive map to date of how avian flu has been transmitted among sites in Asia, Africa and Europe.

But underlying those findings is a new way of analyzing genetic data that generates more complete information about the flu’s spread. The method, combined with the increasing availability of sequenced genomes of isolated flu strains, is expected to help public health officials make more knowledgeable predictions about how the H1N1 flu pandemic will evolve.

“We are taking into account more data but at the same time, we’re making simpler visualizations, allowing users to choose what they want to see,” said Daniel Janies, associate professor of biomedical informatics at Ohio State and senior author of the study.

“We’ve created an environment where people can avail themselves of flu information specific to their region of the world or their area of interest. We waded through all of the complexities so people in the public health realm who want to determine how a flu virus got from point A to point B can find that out, and we’ll have better public health outcomes as a result.”

The visualizations and application are available online at http://routemap.osu.edu.

The research appears online in the journal Cladistics.

The research environment has changed dramatically since 1997, when an avian flu outbreak in Hong Kong alerted health officials to its dangers to humans, Janies noted. The technology behind the Human Genome Project has improved to enable the rapid sequencing of numerous genomes, and avian flu’s broad transmission has encouraged scientists to place viral sequence data into the public domain. At the same time, computational power has continued to expand.

Janies and colleagues obtained high-quality avian flu sequences contained in the repositories at the National Institutes of Health’s GenBank and the Global Initiative on Sharing Avian Influenza Data (GISAID). They then focused on studying two genes within the virus whose mutations are believed to have the most impact on H5N1 behavior: hemagglutinin, which produces the protein that recognizes the host cell receptor, and neuraminidase, an enzyme that helps the virus escape one cell so it can enter other cells.

The researchers used 1,646 sequences of hemagglutinin and 1,335 of neuraminidase in this study.

Biologists construct what are called phylogenetic trees to trace evolutionary relationships among species or strains believed to share a common ancestor. These trees’ branching diagrams can be designed to track similarities in physical characteristics, for example, in the study of dinosaurs, for which genetic data cannot be easily recovered. Or, in the study of influenza, the trees can show how viral strains are related based on shared mutations.

In the past, scientists – including Janies – have selected a single phylogenetic tree to represent related viruses that share mutations. But in this paper, the researchers used the power of supercomputers to generate millions of trees representing relationships among these thousands of viruses. They then picked a pool of thousands of high-quality trees based on a scoring system in the bioinformatics field to use in their analysis of disease transmission.

The scientists then asked of these trees – what are the geographic connections between the isolated viral strains?

These resulting diagrams were then used as the basis for an interactive map that traces the genetic, geographic and evolutionary history of avian influenza over 12 years. The highly pathogenic lineage of avian flu that crossed Asia and Africa can be traced to an isolate taken from a goose in 1996. Little genetic data is available for H5N1 viruses isolated before that.

To avoid creating a complex map that looks like “spaghetti thrown on the screen,” Janies and colleagues also simplified the map’s design. Green lines represent transmission pathways most strongly supported by the research findings. Yellow lines indicate less certainty. Lines also are colored differently depending on whether they indicate an incoming or outgoing virus from a specific location. And users can search for specific transmission routes rather than seeing all transmission events on the map at once.

The maps represent scientists’ best approximation of avian flu transmission based on the information available, Janies explained. Without access to every complete genome of every flu virus that ever infected a bird or human, researchers can never fully track evolutionary relationships, genetic histories and specific locations of each outgoing and incoming viral transmission.

Daniel Janies

“Collect and share as much data as possible and let the data tell the story,” he said. “We’re honest about the uncertainty our results may have – but even with partial data, we can infer much about a virus in an area based on its sources.”

The method has already been applied to studies of the H1N1 flu currently infecting millions of people in the United States. International cooperation spearheaded by the NIH, GISAID and the Centers for Disease Control and Prevention has resulted in ready availability of H1N1 sequences for study.

“With what we have so far, we can see the spread of H1N1 out of the United States and all over the world. There is a different dynamic, in that this is a virus carried by humans, who are cosmopolitan and moving both ways,” Janies said. “It’s also a virus that has been transmitted all over the world in a matter of months, and it’s still similar to its ancestors.”

H5N1, on the other hand, has been creeping across Asia and into Europe and Africa for more than a decade and picked up mutations along the way, he noted. While H1N1 has spread more quickly, it is far less deadly to humans than H5N1 – meaning it is still useful for the world to keep an eye on avian flu, Janies said.

His group’s visualizations will help make that possible.

The computing power used in this study was supplied by the Ohio Supercomputer Center and the Ohio State University Medical Center. The research is funded by the U.S. Army Research Laboratory and Office, Ohio State’s Department of Biomedical Informatics and the Mathematical Biosciences Institute (MBI) at Ohio State.

Janies conducted the work with Rasmus Hovmöller, Boyan Alexandrov and Jori Hardman of Ohio State’s Department of Biomedical Informatics. Hovmöller is also an investigator in the MBI.

Written by Emily Caldwell – Ohio State University

In a joint effort by national laboratory-, university- and private-sector institutions, researchers are developing new tools for rapidly characterizing biological pathogens that could give rise to potentially deadly pandemics such as Influenza A (H1N1).

Credit: Los Alamos National Laboratory

The first tool, an automated genotyping system, is a joint effort between Los Alamos National Laboratory, the University of California at Los Angeles (UCLA) School of Public Health, and Agilent Technologies. This system will be utilized in the Global Bio Lab at UCLA and will use high-throughput technology for automated global-public-health surveillance.

The automated genotyping system, built to specification by Agilent Technologies, was delivered to Los Alamos in late May for verification of design and capability testing. The $1.7 million BioCel Automation System was designed in collaboration by Los Alamos and UCLA researchers, and professionals at Agilent’s automation solutions division, previously known as Velocity11. The system will be able to automatically determine the genetic sequence of viruses such as influenza hundreds of times faster than any other method available today.

By using this system and future high-throughput tools in pandemic response mode, public-health officials will be able to rapidly and reliably determine the strain of a virus, allowing more time for mitigation or containment strategies to be employed if necessary. Moreover, these BioCel systems will also be useful in research mode for monitoring animal populations for the emergence of new and potentially deadly pathogens before the pathogens are able to infect humans. The UCLA Global Bio Lab will become part of the High Throughput Laboratory Network (HTLN), which, when built out, will provide an international and interconnected capacity that provides uniformity in testing methods—reducing the potential for errors or confusion arising from variable testing methodologies currently used.

“As the recent outbreak of the swine flu shows, we need to do a much more extensive and thorough job of surveillance,” said Dr. Tony Beugelsdijk, leader of the HTLN project at Los Alamos National Laboratory. “This program will provide the world with the tools for this task.”

Current genetic identification methods require lots of time and manpower. The new genotyping system features two robots and the ability to fully sequence 10,000 or more influenza viruses per year. This makes it much faster and more reliable than current methods, and reduces the amount of manpower necessary to process a large number of samples.

These artists' representations show how a High-Throughput Laboratory Network (HTLN) might look when fully developed at a site. The aboratory provides a way to rapidly identify and verify strains of pathogens hundreds of times fater than methods currently available, giving public-health officials options for potentially mitigating or containing an emerging pandemic.

“This system is the next-generation tool to rapidly and accurately test and identify biological pathogens in mass quantities of samples,” said Nick Roelofs, vice president and general manager of Agilent Life Sciences Solutions Unit. “Capable of performing tests 100 times faster than any current method, it will provide reliable, real-time data to the global health community. Given current health concerns about the swine flu, the system addressees an immediate and vital need in the public health arena.”

Later this summer the system will be delivered to UCLA, where researchers will operate the system for public health research and surveillance, and train others to use the new tool. If necessary, the system has surge capacity and the ability to test samples in response to a pandemic should the need arise.

“The automated genotyping system will vastly increase the speed and volume by which influenza samples are analyzed,” added Dr. Scott Layne, professor of epidemiology at the UCLA School of Public Health. “The pace of emerging infectious disease outbreaks in the world is increasing and demands new kinds of technologies be created and applied. These technologies will help us to safeguard public health and save lives.”

LANL and UCLA researchers are currently determining protocols for culturing and screening processes that can be used with the high-throughput laboratory. Establishing such protocols is the next step toward making the Global Bio Lab at UCLA fully operational.

The Global Bio Lab at UCLA is funded by the U.S. Department of Defense and the California Office of Homeland Security and is being developed in partnership between Los Alamos and UCLA.

About Agilent Technologies (www.agilent.com)

Agilent Technologies Inc. is the world’s premier measurement company and a technology leader in communications, electronics, life sciences, and chemical analysis. The company’s 19,000 employees serve customers in more than 110 countries. Agilent had net revenues of $5.8 billion in fiscal 2008. Information about Agilent is available on the Web at www.agilent.com.About UCLA School of Public Health

The UCLA School of Public Health is dedicated to enhancing the public’s health by conducting innovative research, training future leaders and health professionals, translating research into policy and practice, and serving local, national, and international communities. For more information see www.ph.ucla.edu

Los Alamos National Laboratory, Los Alamos National Security, LLC

Approach targets both the H and N portions of the virus

What happens if the next big influenza mutation proves resistant to the available anti-viral drugs? This question is presenting itself right now to scientists and health officials this week at the World Health Assembly in Geneva, Switzerland, as they continue to do battle with H1N1, the so-called swine flu, and prepare for the next iteration of the ever-changing flu virus.

Promising new research announced by Rensselaer Polytechnic Institute could provide an entirely new tool to combat the flu. The discovery is a one-two punch against the illness that targets the illness on two fronts, going one critical step further than any currently available flu drug.

“We have been fortunate with H1N1 because it has been responding well to available drugs. But if the virus mutates substantially, the currently available drugs might be ineffective because they only target one portion of the virus,” said Robert Linhardt, the Ann and John H. Broadbent Jr. ’59 Senior Constellation Professor of Biocatalysis and Metabolic Engineering at Rensselaer. “By targeting both portions of the virus, the H and the N, we can interfere with both the initial attachment to the cell that is being infected and the release of the budding virus from the cell that has been affected.”

The findings of the team, which have broad implications for future flu drugs, will be featured on the cover of the June edition of European Journal of Organic Chemistry.

The influenza A virus is classified based on the form of two of its outer proteins, hemagglutinin (H) and neuraminidase (N). Each classification — for example H5NI “bird flu” or H1N1 “swine flu” — represents a different mutation of hemagglutinin and neuraminidase or H and N.

Flu drugs currently on the market target only the neuraminidase proteins, and disrupt the ability of the virus to escape an infected cell and move elsewhere to infect other healthy cells. The new process developed by Linhardt is already showing strong binding potential to hemagglutinin, which binds to sialic acid on the surface of a healthy cell, allowing the virus to entire the cell.

“We are seeing promising preliminary results that the chemistry of this approach will be effective in blocking the hemagglutinin portion of the disease that is currently not targeted by any drug on the market,” he said.

In addition, Linhardt and his team have shown their compound to be just as effective at targeting neuraminidase as the most popular drugs on the market, according to Linhardt.

The approach can also be modified to specifically target the neuraminidase or the hemagglutinin, or both, depending on the type of mutation that is present in the current version of the flu, according to Linhardt.

In the next steps of his research, Linhardt will look at how their compounds bind to hemagglutinin, and he will test the ability to block the virus first in cell cultures and then in infected animal models.

“It is still early in the process,” he said. “We are several steps away from a new drug, but this technique is allowing us to move very quickly in creating and testing these compounds.”

The technique that Linhardt used is the increasingly popular technique of “click chemistry.” Linhardt is among the first researchers in the world to utilize the technique to create new anti-viral agents. The process allows chemists to join small units of a substance together quickly to create a new, full substance.

In this case, Linhardt used the technique to quickly build a new derivative of sialic acid. Because it is chemically very similar to the sialic acid found on the surface of a cell, the virus could mistake the compound as the real sialic acid and bind to it instead of the cell, eliminating the connections to hemagglutinin and neuraminidase that are required for initial infection and spread of the infection in the body. The currently available drugs are translation-state inhibitors whose chemical structure allows them to only effectively target the neuraminidase.

The research was funded by the National Institutes of Health. Linhardt was joined in the research by Michel Weïwer, Chi-Chang Chen, and Melissa Kemp of Rensselaer.