Signs of drug resistance found in African H5N1 viruses

Mar 19, 2009 (CIDRAP News) – Scientists who analyzed 67 H5N1 avian influenza viruses from across Africa report that the viruses fall into three distinct sublineages, or families, and that some have mutations that make them resistant to antiviral drugs.

The scientists also found that some of the African viruses have genetic markers that are characteristic of human flu viruses rather than avian strains, according to their report, published yesterday in the online journal PLoS One.

"These findings raise concern for the possible human health risk presented by viruses with these genetic properties and highlight the need for increased efforts to monitor the evolution of A/H5N1 viruses across the African continent," says the report by a large international team of scientists. The group includes several from African countries and the UN Food and Agriculture Organization.

Lethal H5N1 viruses made their African debut on Nigerian poultry farms in January 2006, the report notes. Soon afterward the virus cropped up in Egypt, Niger, and Cameroon, and in April 2006 it was found in Sudan, Burkina Faso, Djibouti, and Ivory Coast. The virus surfaced in Ghana and Togo in mid-2007 and in Benin in December 2007.

All but two human cases of H5N1 disease in Africa have occurred in Egypt, whose official case count is 58, with 23 deaths. Nigeria and Djibouti have each had one human case.

Rapid spread of 3 sublineages
The scientists looked at 494 H5N1 gene sequences from 67 African isolates, including the complete hemagglutinin and neuraminidase gene segments, all collected between February 2006 and early 2008 and representing all 11 affected countries. The analysis also included hundreds of gene sequences from European and Middle Eastern H5N1 viruses.

The researchers determined that all the African viruses belong to clade 2.2 and are related to the H5N1 viruses that have been circulating throughout Europe, Russia, and the Middle East since late 2005. Clade 2.2 traces back to the outbreak of avian flu in thousands of migratory birds at China's Qinghai Lake in the spring of 2005, the article notes.

Detailed analysis of the hemagglutinin genes showed that the viruses fall into three sublineages (labeled I, II, and IV). All three groups "had been co-circulating since the beginning of the epidemic in Africa," suggesting that all three had been introduced into Africa separately, as reported in previous studies, the report says.

Just how the three groups entered Africa and spread so rapidly is still unclear. But the viruses emerged in Africa when related strains were present in European migratory birds, "and such birds may have played a significant role in the introduction of the virus," the scientists write.

The three sublineages had geographic dimensions, but the patterns were complex. All the Egyptian isolates were in sublineage IV, which they shared with isolates from Gaza and Israel. Strains from Burkina Faso, Ivory Coast, Ghana, and Cameroon formed a single cluster in sublineage I. However, the authors found all three groups in Nigeria, a finding that agreed with an earlier study.

Viruses collected in Sudan were in sublineage I and closely related to those from Nigeria, Burkina Faso, and Ivory Coast, rather than to those from nearby Egypt and Djibouti. Overall, the findings "may suggest that a certain degree of geographical segregation has occurred in Africa" since the initial viral introductions, the report states.

Antiviral resistance, markers of human flu
In searching signs of antiviral resistance, the team found four bird isolates from Egypt carrying a mutation linked with resistance to the older class of flu drugs, the adamantanes (amantadine and rimantadine). They also found viruses from two human cases in Egypt that had a mutation (known as N294S) that confers resistance to oseltamivir (Tamiflu) and slightly reduced sensitivity to zanamivir (Relenza).

However, no mutations conferring resistance to oseltamivir or zanamivir were found in any of the African viruses from birds.

The authors also found a number of isolates with genetic markers usually found in human flu viruses rather than avian strains. In particular, they checked the African viruses for 13 genetic markers consistently found in the flu viruses that caused the pandemics of 1918, 1957, and 1968.

They found two, both in the PB2 gene. One of these, known as E627K and associated with increased H5N1 virulence in mice, was found in all the African isolates. Another was found in just two bird viruses from Egypt.

In other findings, the report says that two different reassortant viruses representing combinations of two of the three sublineages were found in Nigeria in 2006 and 2007. One of these became the predominant strain in Nigeria's poultry in 2007.

Evidence of international spread
"The continued circulation of A/H5N1 viruses in the African continent not only affects the local economy but also impacts on animal and human health," the report states. It concludes with a call for constant efforts to monitor and control avian flu across Africa.

David A. Halvorson, DVM, an avian flu expert at the University of Minnesota in St. Paul, said the report appears to confirm that there were three separate introductions of H5N1 into Africa and that those strains continue to circulate.

Further, he said the study shows that genetic sequencing "shows evidence for international spread within Africa as well as evidence for local spread; and that there have been no additional introductions since the first ones."

Halvorson also commented that the findings regarding antiviral resistance are not surprising: "These mutations are typical of viruses as they circulate in a host. It seems they can mutate to resistance without any antiviral compound present."

Cattoli G, Monne I, Fusaro A, et al. Highly pathogenic avian influenza subtype H5N1 in Africa: a comprehensive phylogenetic analysis and molecular characterization of isolates. PLoS One 2009 March;4(3) [Full text]

See also:

Jul 6, 2006, CIDRAP News story "Report says avian flu entered Nigeria 3 times"

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