Amantadine resistant avian influenza viruses more common in Southeast Asia than North America

Resistance to the antiviral drug amantadine is spreading more rapidly among avian influenza viruses of H5N1 subtype in Southeast Asia than in North America, according to the study done by investigators at St. Jude Children's Research Hospital.

The St. Jude team reached this conclusion by analyzing sequence data of the so-called M2 protein of avian influenza viruses of different subtypes isolated in North America and Southeast Asia during 1991-2004; and by evaluating the frequency of drug-resistant strains. Sequence data refers to the makeup of a gene coding for a particular protein, in this case, the M2 protein. A properly functioning M2 protein is key to the virus' ability to replicate. The St. Jude researchers demonstrated that the largest proportion of Asian drug-resistant H5 and H9 avian influenza viruses occurred in China. A report on these findings appears in the current online edition of Virology.

H5 influenza viruses concern world health officials because H5N1 subtype has been spreading throughout chicken flocks and wild birds in Southeast Asia since it emerged in 1997. Between late 2003 and early 2004, outbreaks of highly pathogenic avian H5N1 influenza occurred among poultry in eight Asian countries, causing the death or destruction of tens of millions of birds. As of August 5, 2005, 112 cases of human H5N1 infection have been confirmed in Indonesia, Vietnam, Thailand and Cambodia, of which 57 were fatal, according to the World Health Organization. Humans contract H5N1 only from close contact with infected birds and only one probable human-to-human transmission was reported in Vietnam. This has so far prevented H5N1 from becoming a major threat to humans.

"However, if H5N1 variants acquire the capacity for sustained human-to-human transmission, the world will face the threat of a serious pandemic," said Robert G. Webster, Ph.D., a member of the Infectious Diseases department and holder of the Rose Marie Thomas Chair at St. Jude. "Humans don¡¦t have resistance to H5N1, and currently vaccines to H5N1 are still being developed. And, the available evidence shows that the most recent strains isolated from humans in Asia are no longer sensitive to inhibition by the amantadine family of drugs."

Resistance to the antiviral drug amantadine is caused by substitutions of one of five amino acids in the part of the M2 protein called the transmembrane domain-the part of M2 located within the coat of the influenza virus. The M2 protein is an ion channel located in the envelope of the virus that permits hydrogen ions (protons) to enter the flu virion. This influx of protons allows the virus to shed its coat after it enters a cell-an essential step in the replication of the virus. Amantadine inhibits the function of the M2 protein and thus stops viral replication.

"By analyzing the sequence of the transmembrane part of the M2 gene we were able to determine how frequently amantadine resistance occurs in avian influenza A subtypes isolated in various parts of the world-especially among those subtypes that had the potential to cause a pandemic," said Natalia A. Ilyushina, Ph.D., a postdoctoral fellow in the Infectious Diseases Department at St. Jude. First author of the Virology paper, Ilyushina did much of the work on this project.

The St. Jude researchers analyzed the M2 gene sequences from 60 influenza viruses isolated in Southeast Asia and 74 viruses from North America that represented the H5, H6, H7 and H9 subtypes. The scientists also examined information from the National Library of Medicine's GenBank database on 408 viruses isolated from avian hosts worldwide.

Based on the study, the St. Jude team reported that there were no avian amantadine-resistant strains isolated from 1979-83 in the northeastern United States and Southeast Asia. However, 31 percent of H5 and 11 percent of H9 influenza viruses from Southeast Asia isolated in 2000ƒ{04 carried M2 mutations. Isolates of H5 and H9 subtypes from North America during that time remained sensitive to amantadine, while 16 percent of H7 isolates were resistant to this drug.

"These data are clear and convincing," said Elena A. Govorkova, Ph.D., of the St. Jude Infectious Diseases department, a co-author of the paper. "The specific amantadine-resistance mutations in M2 that we identified can occur randomly throughout the world. But we now have solid proof that in Southeast Asia, and especially in China, these mutations are undergoing strong selective pressure."

Selective pressure refers to the extent to which an organism has acquired either a beneficial genetic trait that gives it a survival advantage in a particular environment and therefore makes the organism more likely to survive and multiply; or a trait that makes it vulnerable to something in the environment, and therefore more likely to become extinct.

The increasing incidence of amantadine-resistant H5N1 viruses in China indicates that these variants appear to have survival advantages over the wild, drug-sensitive strains. In addition, the infected birds die so rapidly there is no time for the virus to acquire a large number of mutations, among which could be changes in the M2 protein. Therefore, the high rate of M2 mutations in China probably arises from some human activity that encourages selection of such changes, the researchers said. For example, treating chickens with amantadine to prevent infection with H5N1 would put selective pressure on the M2 gene to acquire mutations that made it resistant to this antiviral drug.

"H5N1 is now endemic-a permanent- resident- in Southeast Asia, including China," Webster said. "Therefore, any selective pressure on this virus ensures plenty of opportunity for H5N1 to acquire amantadine resistance, which would bring additional difficulties in controlling the pandemic."

This concern over activities in China that might increase selective pressure for amantadine resistance in H5N1 has been voiced on the postings of ProMED over the past several months. ProMED (Program for Monitoring Emerging Diseases) is an electronic outbreak reporting system established by the International Society for Infectious Diseases to permit rapid and accurate monitoring of infectious disease outbreaks globally; and to serve as a central location for news, updates, and discussions of infectious disease outbreaks that threaten humans. For example, a ProMED transmission on July 6, 2005 noted that Joseph Domenech, the chief veterinary officer for the United Nations Food and Agriculture Organization commented that the use of human flu drugs by Chinese farmers is "a big concern."