Resistance to the antiviral drug amantadine is spreading morerapidly among avian influenza viruses of H5N1 subtype in Southeast Asiathan in North America, according to the study done by investigators atSt. Jude Children’s Research Hospital.
The St. Jude team reached this conclusion by analyzing sequencedata of the so-called M2 protein of avian influenza viruses ofdifferent subtypes isolated in North America and Southeast Asia during19912004; and by evaluating the frequency of drug-resistant strains.Sequence data refers to the makeup of a gene coding for a particularprotein, in this case, the M2 protein. A properly functioning M2protein is key to the virus’ ability to replicate. The St. Juderesearchers demonstrated that the largest proportion of Asiandrug-resistant H5 and H9 avian influenza viruses occurred in China. Areport on these findings appears in the current online edition of Virology.
H5 influenza viruses concern world health officials because H5N1subtype has been spreading throughout chicken flocks and wild birds inSoutheast Asia since it emerged in 1997. Between late 2003 and early2004, outbreaks of highly pathogenic avian H5N1 influenza occurredamong poultry in eight Asian countries, causing the death ordestruction of tens of millions of birds. As of August 5, 2005, 112cases of human H5N1 infection have been confirmed in Indonesia,Vietnam, Thailand and Cambodia, of which 57 were fatal, according tothe World Health Organization. Humans contract H5N1 only from closecontact with infected birds and only one probable human-to-humantransmission was reported in Vietnam. This has so far prevented H5N1from becoming a major threat to humans.
“However, if H5N1 variants acquire the capacity for sustainedhuman-to-human transmission, the world will face the threat of aserious pandemic,” said Robert G. Webster, Ph.D., a member of theInfectious Diseases department and holder of the Rose Marie ThomasChair at St. Jude. “Humans don’t have resistance to H5N1, and currentlyvaccines to H5N1 are still being developed. And, the available evidenceshows that the most recent strains isolated from humans in Asia are nolonger sensitive to inhibition by the amantadine family of drugs.”
Resistance to the antiviral drug amantadine is caused bysubstitutions of one of five amino acids in the part of the M2 proteincalled the transmembrane domain—the part of M2 located within the coatof the influenza virus. The M2 protein is an ion channel located in theenvelope of the virus that permits hydrogen ions (protons) to enter theflu virion. This influx of protons allows the virus to shed its coatafter it enters a cell—an essential step in the replication of thevirus. Amantadine inhibits the function of the M2 protein and thusstops viral replication.
“By analyzing the sequence of the transmembrane part of the M2 genewe were able to determine how frequently amantadine resistance occursin avian influenza A subtypes isolated in various parts of theworld—especially among those subtypes that had the potential to cause apandemic,” said Natalia A. Ilyushina, Ph.D., a postdoctoral fellow inthe 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 60influenza viruses isolated in Southeast Asia and 74 viruses from NorthAmerica that represented the H5, H6, H7 and H9 subtypes. The scientistsalso examined information from the National Library of Medicine’sGenBank database on 408 viruses isolated from avian hosts worldwide.
Based on the study, the St. Jude team reported that there were noavian amantadine-resistant strains isolated from 1979-83 in thenortheastern United States and Southeast Asia. However, 31 percent ofH5 and 11 percent of H9 influenza viruses from Southeast Asia isolatedin 200004 carried M2 mutations. Isolates of H5 and H9 subtypes fromNorth America during that time remained sensitive to amantadine, while16 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 ofthe paper. “The specific amantadine-resistance mutations in M2 that weidentified can occur randomly throughout the world. But we now havesolid proof that in Southeast Asia, and especially in China, thesemutations are undergoing strong selective pressure.”
Selective pressure refers to the extent to which an organism hasacquired either a beneficial genetic trait that gives it a survivaladvantage in a particular environment and therefore makes the organismmore likely to survive and multiply; or a trait that makes itvulnerable to something in the environment, and therefore more likelyto become extinct.
The increasing incidence of amantadine-resistant H5N1 viruses inChina indicates that these variants appear to have survival advantagesover the wild, drug-sensitive strains. In addition, the infected birdsdie so rapidly there is no time for the virus to acquire a large numberof mutations, among which could be changes in the M2 protein.Therefore, the high rate of M2 mutations in China probably arises fromsome human activity that encourages selection of such changes, theresearchers said. For example, treating chickens with amantadine toprevent infection with H5N1 would put selective pressure on the M2 geneto 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 onthis virus ensures plenty of opportunity for H5N1 to acquire amantadineresistance, which would bring additional difficulties in controllingthe pandemic.”
This concern over activities in China that might increase selectivepressure for amantadine resistance in H5N1 has been voiced on thepostings of ProMED over the past several months. ProMED (Program forMonitoring Emerging Diseases) is an electronic outbreak reportingsystem established by the International Society for Infectious Diseasesto permit rapid and accurate monitoring of infectious disease outbreaksglobally; and to serve as a central location for news, updates, anddiscussions of infectious disease outbreaks that threaten humans. Forexample, a ProMED transmission on July 6, 2005 noted that JosephDomenech, the chief veterinary officer for the United Nations Food andAgriculture Organization commented that the use of human flu drugs byChinese farmers is “a big concern.”
This work was supported in part by the National Institute of Allergyand Infectious Disease of the National Institutes of Health and ALSAC.
St. Jude Children's Research Hospital
St. Jude Children's Research Hospital is internationallyrecognized for its pioneering work in finding cures and saving childrenwith cancer and other catastrophic diseases. Founded by lateentertainer Danny Thomas and based in Memphis, Tenn., St. Jude freelyshares its discoveries with scientific and medical communities aroundthe world. No family ever pays for treatments not covered by insurance,and families without insurance are never asked to pay. St. Jude isfinancially supported by ALSAC, its fund-raising organization.
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