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Published Date: 2005-08-27 23:50:00
Subject: PRO/AH> Avian influenza, human - East Asia (118): evolution
Archive Number: 20050827.2538

AVIAN INFLUENZA, HUMAN - EAST ASIA (118): EVOLUTION
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Date: Sat 27 Aug 2005
From: ProMED-mail <promed@promedmail.org>
Source: Emerging Infectious Diseases, Vol. 11, No. 10, October 2005 (ahead
of print) [edited]
<http://www.cdc.gov/ncidod/EID/vol11no10/05-0644.htm>

Evolution of H5N1 Avian Influenza Viruses in Asia
-----------------------------------------------
["Evolution of H5N1 Avian Influenza Viruses in Asia" is a report compiled
by The World Health Organization Global Influenza Program Surveillance
Network. The Summary and Introduction are reproduced in full, followed by
extracts from the body of the paper. Interested readers should refer to the
original text for information on methods, the text figures and tabulated
data, and references. Novel features of this report are the definition of 3
distinct clades by phylogenetic analysis, the complete absence of
reassortment of avian and human influenza virus genes, and the 1st
documentation of the occurrence of antigenic drift. - Mod.CP]
Summary
----------
An outbreak of highly pathogenic avian influenza A (H5N1) has recently
spread to poultry in 9 Asian countries. H5N1 infections have caused >52
human deaths in Viet Nam, Thailand, and Cambodia from January 2004 to April
2005. Genomic analyses of H5N1 isolates from birds and humans showed 2
distinct clades with a non-overlapping geographic distribution. All the
viral genes were of avian influenza origin, which indicates absence of
reassortment with human influenza viruses. All human H5N1 isolates tested
belonged to a single clade and were resistant to the adamantane drugs but
sensitive to neuraminidase inhibitors. Most H5N1 isolates from humans were
antigenically homogeneous and distinct from avian viruses circulating
before the end of 2003. Some 2005 isolates showed evidence of antigenic
drift. An updated nonpathogenic H5N1 reference virus, lacking the polybasic
cleavage site in the hemagglutinin gene, was produced by reverse genetics
in anticipation of the possible need to vaccinate humans.
Introduction
----------
Highly pathogenic avian influenza viruses of the H5N1 subtype are
circulating in eastern Asia with unprecedented epizootic and epidemic
effects. 9 Asian countries reported H5N1 outbreaks in poultry in 2004:
Cambodia, China, Indonesia, Japan, Laos, Malaysia, South Korea, Thailand,
and Viet Nam. Between 2004 and the 1st 3 months of 2005, a total of 89
laboratory-confirmed human infections, 52 of which were fatal, were
reported to the World Health Organization (WHO) by public health
authorities in Viet Nam, Thailand, and Cambodia. These records indicate
that this outbreak of human H5N1 infections is the largest documented since
its emergence in humans in 1997. Efficient viral transmission among poultry
caused the virus to spread regionally, leading to the loss of >100 million
birds from disease and culling. In contrast, human-to-human transmission of
the virus is exceptional but has been described, most recently in a family
cluster in Thailand.
The 3 viral envelope proteins of influenza A virus are most medically
relevant. The hemagglutinin (HA), neuraminidase (NA), and M2 are essential
viral proteins targeted by host antibodies or antiviral drugs such as
oseltamivir and rimantadine. The HA glycoprotein forms spikes at the
surface of virions, mediating attachment to host cell sialoside receptors
and subsequent entry by membrane fusion. The NA forms knoblike structures
on the surface of virus particles and catalyzes their release from infected
cells, allowing virus spread. The M2 is a transmembrane protein that forms
an ion channel required for the uncoating process that precedes viral gene
expression.
Phylogenetic, phenotypic, and antigenic analysis of H5N1 viruses from the
2004-2005 outbreak, focusing on these 3 genes, have been carried out to
address questions relevant to the public health response to the outbreak:
1) What is the genetic diversity of H5N1 viruses involved in human
infections? 2) Can the relationship between human and avian H5N1 isolates
help explain the source of infection? 3) Do genetic changes correlate with
enhanced viral transmissibility in humans? 4) How sensitive are H5N1
isolates to antiviral drugs? 5) What is the antigenic similarity between
human H5N1 viruses and current candidate vaccines? and 6) Can candidate
vaccine reference stocks be developed in time for an effective public
health response?
Phylogenetic Analysis of the HA, NA and M2 Genes
----------
Phylogenetic analyses of the H5 HA genes from the 2004 and 2005 outbreak
showed 2 different lineages of HA genes, termed clades 1 and 2. Viruses in
each of these clades are distributed in non-overlapping geographic regions
of Asia. The H5N1 viruses from the Indochina peninsula are tightly
clustered within clade 1, whereas H5N1 isolates from several surrounding
countries are distinct from clade 1 isolates and belong in the more
divergent clade 2. Clade 1 H5N1 viruses were isolated from humans and birds
in Viet Nam, Thailand, and Cambodia but only from birds in Laos and
Malaysia. The clade 2 viruses were found in viruses isolated exclusively
from birds in China, Indonesia, Japan, and South Korea. Viruses isolated
from birds and humans in Hong Kong in 2003 and 1997 made up clades 1* and
3, respectively.
The HA genes from H5N1 viruses isolated from human specimens were closely
related to HA genes from H5N1 viruses of avian origin; human HA gene
sequences differ from the nearest gene from avian isolates from the same
year in 2-14 nucleotides (<1 percent divergence). These findings are
consistent with the epidemiologic data that suggest that humans acquired
their infections by direct or indirect contact with poultry or poultry
products.
Analysis of the amino acid sequences showed that both clades of H5 HAs from
the 2004-2005 outbreak have a multiple basic amino acid motif at the
cleavage site, a defining feature of highly pathogenic avian influenza
viruses. Among all H5N1 isolates collected in east Asia since 1997, only
those in clades 1, 1*, and 3 appear to be associated with fatal human
infections.
NA genes from human and related avian H5N1 isolates from 2003-2005 as well
as clade 3 isolates were characterized by deletions in the stalk region of
the protein (positions 49-68 for clades 1-2 and 54-72 for clade 3).
Deletions in the stalk of the NA are thought to increase retention of
virions at the plasma membrane to balance weaker binding of sialic acid
receptors by the HA with newly acquired N154 glycosylation.
The phylogenetic tree of the M genes resembled that of the HA genes,
indicating coevolution of these genes. The amino acid sequence of the M2
protein of clade 1 viruses as well as of HK/213/03 indicated a
serine-to-asparagine substitution at residue 31 (S31N), known to confer
resistance to adamantanes (including amantadine and rimantadine). All the
currently circulating clade 1 isolates are resistant to adamantanes.
Origin of Internal Genes of H5N1 Viruses from Asia
----------
The PB2, PB1, and PA polymerase genes from 2003-2005 H5N1 isolates from
humans constitute a single clade and have coevolved with the respective HA
genes. No evidence of reassortment with polymerase genes from circulating
H1N1 or H3N2 human influenza virus was found. The phylogenies of the NP and
NS genes also supported the avian origin of these genes, indicating that
all the genes from the human H5N1 isolates analyzed are of avian origin,
which confirms the absence of reassortment with human influenza genes.
Taken together, the phylogenies of the 8 genomic segments show that the
H5N1 viruses from human infections and the closely related avian viruses
isolated in 2004 and 2005 belong to a single genotype, often referred to as
genotype Z.
Antigenic Analysis of H5N1 Viruses from Asia
----------
H5N1 isolates collected in 2004 and 2005 analyzed by the HI test showed
reactivity patterns that correlated with the 3 main clades of recent
isolates identified in the HA gene phylogeny. Viruses from humans and birds
in clade 1 were found to constitute a relatively homogeneous and distinct
antigenic group characterized by poor inhibition by ferret antisera to
isolates from other clades, in particular by the ferret antiserum raised to
HK/213/03 (64-fold reduction compared to the homologous titer). The latter
isolate was previously used to develop a vaccine reference strain in
response to 2 confirmed H5N1 human infections in February 2003. Antigenic
analysis of human isolates from 2005 provided evidence of antigenic drift
among the most recently circulating H5N1 strains.
Development of Candidate H5N1 Vaccine Reference Stocks
----------
Inactivated influenza vaccines are manufactured from reassortant viruses
obtained by transferring the HA and NA genes with the desired antigenic
properties into a high-growth strain such as PR8. However, reassortants
with H5-derived HA with a polybasic cleavage site are potentially hazardous
for animal health. Because the high pathogenicity of the H5N1 viruses in
poultry, mice, and ferrets depends primarily on the polybasic cleavage site
in the HA molecule, a derivative with a deletion of this motif was
engineered in cloned HA cDNAs. 3 high-growth reassortant influenza viruses
were developed: NIBRG-14 (NIBSC), VN/04xPR8-rg (SJCRH), and
VNH5N1-PR8/CDC-rg (CDC). These candidate vaccine strains, bearing mutant H5
HA, intact NA, and the internal genes from PR8, were generated by a reverse
genetics approach using Vero cells and laboratory protocols compatible with
eventual use of the vaccine in human subjects. These 3 vaccine candidates
were characterized genetically (nucleotide sequencing of HA and NA) and
antigenically in HI assays to confirm that their antigenicity remained
unchanged relative to the wildtype virus. The candidate reference stocks
had molecular and antigenic properties equivalent to parental H5N1 donor
strains and lacked virulence in chicken, mouse, and ferret models.
Discussion and Conclusions
----------
The origin of the HA genes of the 2004-2005 outbreak as well as an earlier
isolate from a fatal human infection in Hong Kong in 2003 (clade 1*) can be
traced back to viruses isolated in 1997 in Hong Kong (clade 3) and from
geese in China (goose/Guangdong/96). The phylogeny also shows that viruses
with HK/97-like HA may have circulated in avian hosts continuously after
1997 without causing any reported human infections until the 2 confirmed
cases in Hong Kong in February 2003.
Genetic and antigenic analyses have shown that, compared to previous H5N1
isolates, 2004-2005 isolates share several amino acid changes that modulate
antigenicity and perhaps other biological functions. Furthermore, molecular
analysis of the HA from isolates collected in 2005 suggests that several
amino acids located near the receptor-binding site are undergoing change,
some of which may have an affect on antigenicity or transmissibility.
Further surveillance to determine the prevalence of such variants in
poultry will be critical to determine whether these variants compromise the
efficacy of the candidate vaccine or increase the efficiency of transmission.
The phylogenies of the 8 genomic segments from the clade 1 and 2 isolates
from 2004-2005 showed that all genes are of avian origin. All H5N1 isolates
from both clades belong to one of the genotypes recently circulating in
Eastern and Southern Asia, e.g., genotypes V and Z. An effective H5N1
vaccine is a public health priority and the cornerstone for pandemic
prevention and control. Reverse genetics approaches allow the rapid
production of high-growth PR8 reassortant viruses by engineering a virus
with a homologous HA gene lacking the polybasic amino acids associated with
high virulence. These candidate H5N1 pandemic vaccine viruses have been
made available to vaccine manufacturers to produce pilot lots for clinical
trials and are available for possible large-scale manufacturing, should the
need arise.
--
ProMED-mail
<promed@promedmail.org>
[Perhaps one of the most significant aspects of this analysis is the
finding that antigenic drift is occurring and that some recent isolates are
distinguishable from virus isolates that were chosen as the candidate
vaccine antigens. This identifies a need for continued surveillance of
poultry for the appearance of antigenic variants which may compromise the
effectiveness of the current vaccine under development. A rolling program
of vaccine development may be required to take account of possible changes
in the antigenicity of the virus. - Mod.CP]

See Also