Robert Roos  News Editor

Jul 31, 2006 (CIDRAP News) – In an experiment designed to mimic events that could launch an influenza pandemic, a synthetic influenza virus made by combining an H5N1 avian flu virus with a human flu virus turned out to be no more contagious in an animal model than the natural H5N1 virus, US scientists are reporting this week.

Researchers at the Centers for Disease Control and Prevention (CDC) made two hybrid viruses and infected ferrets with them, according to a report to be published in the Proceedings of the National Academy of Sciences. The viruses failed to spread from infected ferrets to healthy ones in neighboring cages.

"We found that they [the viruses] were not able to transmit efficiently," said CDC researcher Dr. Jackie Katz, speaking at a Jul 28 teleconference. "In fact, they were also not as able to cause severe disease as the original H5N1 virus."

The deadly H5N1 virus has infected 232 people and killed 134 since late 2003, but it has not yet found a way to spread easily from person to person. But scientists fear the virus could pick up that ability if it combined, or reassorted, with a human flu virus, which could happen if someone became infected with both types simultaneously. The CDC set out to create such a hybrid and test it in ferrets. The animals are considered good models for flu virus research because their susceptibility to flu viruses is similar to that of humans.

CDC officials cautioned against taking much comfort from the experimental results. Although the synthetic hybrid didn"e;t spread among the ferrets in the experiments so far, that doesn"e;t mean the scenario couldn"e;t happen in nature, they said. (Also, experts say the H5N1 virus could become transmissible through accumulated small mutations, without reassortment.)

"These data do not mean that H5N1 cannot convert to be transmissible from person to person; they mean it"e;s probably not a simple process and more than simple genetic exchanges are necessary," CDC Director Dr. Julie Gerberdng said at the teleconference.

The H5N1 virus used in the study was a strain collected in 1997, when the pathogen first infected humans in Hong Kong. The human flu virus used in the study was an H3N2 strain, which has been common in recent decades. The research involved four steps, according to Katz.

First, investigators assessed whether the H3N2 and H5N1 viruses would spread in ferrets, whose cages were arranged so that viruses could spread via respiratory droplets. The human virus did spread efficiently, whereas the avian virus didn"e;t, which signaled that the ferrets were serving as good models for human infection, Katz said.

The next step was to generate reassortant viruses. "We made two viruses that contained surface protein genes from the H5N1 virus and internal genes from the human H3N2 virus," Katz said. "We found we could make these viruses and that some of them were viable."

Third, the scientists infected some ferrets with the hybrid viruses and waited to see if they would spread to healthy ferrets. The hybrid viruses caused less severe illness than the original H5N1 strain, and they failed to spread.

Finally, the investigators wanted to know if the hybrid viruses would naturally mutate to become more transmissible if they were passed through several ferrets in succession. So the researchers infected ferrets and, after the ferrets showed symptoms, took nasal secretions and used them to infect other ferrets, repeating this step five times. Further, the researchers assessed whether the virus could spread more easily after all these generations or "passages."

"We found that the virus did not acquire any additional capacity to transmit efficiently from infected ferrets to healthy ferrets," Katz said.

Katz didn"e;t explain why the CDC used a 1997 strain of H5N1 instead of a more recent strain, but said more recent isolates will be used in further experiments. Later versions of both H5N1 and H3N2 will be used to make further hybrids for testing in ferrets, she said. Scientists have identified a number of mutations in the H5N1 virus since 1997.

"We did test the more recent strains [of H5N1] for their ability to transmit, and like the 1997 strains, they could not transmit efficiently from one animal to the next," she said. "We need to continue to study this."

The CDC officials were asked whether reassortment "dumbs down" or weakens the virus. Katz replied that the hybrids were less virulent than H5N1, but cautioned that the results apply only to the 1997 strain.

Gerberding commented, "The pandemics of 1957 and 1968 were caused by reassortant viruses. Those were not dumb viruses."

In answering other questions, Katz said some scientists believe the 1918 pandemic virus, unlike the 1957 and 1968 viruses, arose through slowly accumulating mutations in an avian virus rather than through a reassortment event. "We"e;re looking at the approach of the 1957 and 1968 pandemics where there was a more sudden change," she said.

The most important lesson of the research so far, according to Katz, is "the knowledge that this process isn"e;t simple, the procedure for the virus to acquire the properties of transmissibility."

She said the CDC also created a hybrid that involved H3N2 virus surface proteins and H5N1 internal genes—the reverse of the hybrid she first described—and "that was not sufficient for transmissibility either. . . . That points to the fact that it"e;s a complex interaction of the surface genes and the internal genes."

Gerberding warned that the findings shouldn"e;t lead to complacency.

"I"e;m not reassured from the public health perspective," she said. "This virus is still out there, it"e;s still evolving, and influenza is always unpredictable. . . . So let"e;s not use the word "e;reassuring"e; with respect to what might happen with H5N1."

Because of the risk that the reassortant viruses could spread, the research was done under stringent containment, involving Biosafety Level 3 with extra precautions, Katz said.

Maines TR, Chen LM, Matsuoka Y, et al. Lack of transmission of H5N1 avian-human reassortant influenza viruses in a ferret model. Proc Natl Acad Sci 2006 (published online Jul 31) [Abstract]