Oseltamivir- and Amantadine-Resistant Influenza Viruses A (H1N1)

Surveillance of amantadine and oseltamivir resistance among influenza viruses was begun in Hong Kong in 2006. In 2008, while both A/Brisbane/59/2007-like and A/Hong Kong/2652/2006-like viruses (H1N1) were cocirculating, we detected amantadine and oseltamivir resistance among A/Hong Kong/2652/2006-like viruses (H1N1), caused by genetic reassortment or spontaneous mutation.

A high rate of oseltamivir resistance in seasonal influenza virus A (H1N1) infection was reported in Europe in the winter of 2007-08 (1). Of subtype H1N1 isolates tested in 18 European countries, 59 (13.5%) of 437 were resistant to oseltamivir and carried the substitution of histidine by tyrosine at residue 274 (H274Y) of the neuraminidase (NA) gene. Genetic analysis showed that all oseltamivirresistant strains of subtype H1N1 virus remained susceptible to amantadine.
In Hong Kong Special Administrative Region, People's Republic of China, amantadine and oseltamivir are not commonly used to treat patients with influenza. Surveillance of amantadine and oseltamivir resistance among influenza viruses was begun in 2006 after a high rate of amantadine resistance was reported among subtype H3N2 viruses (2) and a stockpile of oseltamivir was purchased for pandemic preparedness. Oseltamivir resistance was first detected in January 2008. We report detection of subtype H1N1 virus isolates that became resistant to amantadine and oseltamivir because of genetic reassortment and spontaneous mutation.

The Study
As part of ongoing surveillance, respiratory samples are routinely collected from patients seeking treatment with respiratory illness in clinics and hospitals of Hong Kong. Virus isolation is performed according to a standard protocol (3). From January 2006 through June 2008, a total of 827 influenza viruses (H1N1) were tested for oseltamivir resistance by an enzyme inhibition assay (NA-STAR; Applied Biosystems, Foster City, CA, USA) or by nucleotide sequencing of the NA gene to detect the H274Y mutation. The isolates were also tested for resistance to amantadine by an in-house-designed PCR (protocol available on request) and nucleotide sequencing of the matrix (M2) gene segment. A 575-nt fragment of the NA gene and a 199-nt fragment of the M2 gene were amplified by the designed primers N1-1H and N1-2H, and MA 692 and MA 891 (available on request), respectively, and sequenced by using a Genetic Sequencer 3100 or 3130XL (Applied Biosystems). Sequences obtained were aligned by using Simmonics (4) and analyzed by MEGA version 2.1 (5 A subtype H1N1 virus resistant to oseltamivir, but sensitive to amantadine, was first detected in Hong Kong in January 2008, which coincided with the emergence of clade 2B A/Brisbane/59/2007-like viruses. From January through June 2008, 87 (12.5%) of 697 isolates tested were resistant to oseltamivir. They all carried the H274Y mutation in the NA gene and showed an ≈1,000-fold reduction in susceptibility to oseltamivir (50% inhibitory concentration values increased from 0.5 nmol/L to 500 nmol/L). Analysis of the M2 genes showed that 3 (3.4%) of 87 oseltamivirresistant isolates also carried the S31N mutation associated with amantadine resistance. These 3 viruses were isolated from 2 infants and an elderly woman in different hospitals during 2008 (March, April, and June, respectively). To eliminate the possibility of mixed strains, we designed a real-time PCR using single nucleotide polymorphisms to detect antimicrobial drug-susceptible strains (with S31 and H274) and drug resistant strains (with S31N and H274Y) in the samples (method available on request). Results showed that the 3 isolates did not possess S31 and H274.
To further study the genetic mechanism of emergence of antiviral drug resistance among subtype H1N1 viruses, we performed nucleotide sequencing on a partial segment of the hemagglutinin (HA) gene on 84 (97%) of 87 oseltamivir-resistant and 37 (5%) of 697 oseltamivir-susceptible viruses isolated in our laboratory in 2008. PCR and DNA sequencing of the HA gene were carried out by using the primers H1-1 and H1-2, which flank a fragment of 612 nt of the HA segment (7). Sequences were obtained from 121 virus isolates (Table).
Of 37 oseltamivir-susceptible viruses, analysis of the HA, NA, and M2 genes showed that 20 were susceptible to amantadine and similar to the clade 2B A/Brisbane/59/2007 virus (GenBank accession no. CY030232) (8)   only, amantadine-and oseltamivir-resistant, and amantadine-and oseltamivir-susceptible), incubated the cultures at 33°C, and examined the cytopathic effect (CPE) daily for 7 days. By observing the timing of CPE appearance and relative progressive rate of CPE, we found no substantial difference in growth efficiency and similar replication patterns for the 4 different types of subtype H1N1 strains.

Conclusions
Reassortment Monitoring of antiviral resistance among influenza isolates showed that resistance pattern changed among subtype H1N1 viruses when different lineages were introduced.
While oseltamivir resistance appears to be largely confined to clade 2B viruses and amantadine resistance to clade 2C viruses, oseltamivir-and amantadine-resistant viruses due to spontaneous mutation or reassortment were detectable when clade 2B and 2C viruses were cocirculating. If antiviral resistance markers are combined with sequence data of HA, NA, and M genes, systematic monitoring would make it possible to track the spread of influenza viruses globally and to clarify the underlying mechanism for the spread of such resistance.