Conceived and designed the experiments: XW TN NC CVN AK ROD. Performed the experiments: XW TN TD CBS KI HTD DTM SJ AB BS YM. Analyzed the data: XW TN TD ZMZ MC FL ME SEL. Wrote the paper: XW. Revised paper: ROD TD.
The authors have declared that no competing interests exist.
Highly pathogenic avian influenza (HPAI) H5N1 viruses have caused dramatic economic losses to the poultry industry of Vietnam and continue to pose a serious threat to public health. As of June 2008, Vietnam had reported nearly one third of worldwide laboratory confirmed human H5N1 infections. To better understand the emergence, spread and evolution of H5N1 in Vietnam we studied over 300 H5N1 avian influenza viruses isolated from Vietnam since their first detection in 2001. Our phylogenetic analyses indicated that six genetically distinct H5N1 viruses were introduced into Vietnam during the past seven years. The H5N1 lineage that evolved following the introduction in 2003 of the A/duck/Hong Kong/821/2002-like viruses, with clade 1 hemagglutinin (HA), continued to predominate in southern Vietnam as of May 2007. A virus with a clade 2.3.4 HA newly introduced into northern Vietnam in 2007, reassorted with pre-existing clade 1 viruses, resulting in the emergence of novel genotypes with neuraminidase (NA) and/or internal gene segments from clade 1 viruses. A total of nine distinct genotypes have been present in Vietnam since 2001, including five that were circulating in 2007. At least four of these genotypes appear to have originated in Vietnam and represent novel H5N1 viruses not reported elsewhere. Geographic and temporal analyses of H5N1 infection dynamics in poultry suggest that the majority of viruses containing new genes were first detected in northern Vietnam and subsequently spread to southern Vietnam after reassorting with pre-existing local viruses in northern Vietnam. Although the routes of entry and spread of H5N1 in Vietnam remain speculative, enhanced poultry import controls and virologic surveillance efforts may help curb the entry and spread of new HPAI viral genes.
The emergence of highly pathogenic avian influenza (HPAI) H5N1 virus as a human pathogen occurred in 1997 in Hong Kong Special Administrative Region (SAR)
A global effort is underway to control or eradicate H5N1 in poultry and prevent human exposure, both of which may also reduce the risk of pandemic emergence. The scientific rationale for these programs is provided by ecologic, virologic, epidemiologic, and immunologic studies. In particular, molecular and functional characterization of H5N1 viruses from poultry will help inform development and implementation of public health control measures involving diagnosis, immunization and antiviral drug therapy.
The putative ancestor of the currently circulating H5N1 viruses is A/goose/Guangdong/1/96 (Gs/GD/1/96), named after the province in southern China which is also thought to be the epicenter of H5N1
In 2005, unprecedented outbreaks of H5N1 in wild migratory birds at Qinghai Lake in western China apparently facilitated its spread to more than 30 countries in Europe, the Middle East and Africa
Although HA gene evolution merits much attention because of its central role in antigenic drift and interaction with the host, the remaining seven viral genes are also critical to inform disease control efforts as they contribute to antiviral drug susceptibility, host range and virulence, among other phenotypic properties
Vietnam (VN) is one of the countries most affected by H5N1 outbreaks in poultry and has accumulated 106 laboratory-confirmed human cases since 2003. Recurring epizootics of H5N1 were reported in poultry from 2003 until 2005
The nationwide H5N1 surveillance program launched in 2004 by the Government of Vietnam and implemented by the National Centre for Veterinary Diagnostics (NCVD), in collaboration with the Vietnam Department of Animal Health (DAH) and DAH Regional Animal Health offices, involved the collection and analysis of 20,567 samples from 38 provinces between that year and May of 2007. A total of 2,111 positive specimens were detected by RT-PCR or real-time RT-PCR specific for H5 HA RNA (
2004 |
2005 |
2006 |
2007 |
Total | |
Total samples | 2,272 | 13,889 | 64 | 4,342 | 20,567 |
Positive Samples | 515 (22.7%) |
1,317 (9.4%) | 2 (3.1%) | 277 (6.4%) | 2,111 (10.3%) |
Number of provinces |
28 | 34 | 12 | 35 | 38 |
Results from RT-PCR.
Results from real-time RT-PCR.
Percentage of samples positive out of total collected.
See
In order to investigate the origin of the HA genes of H5N1 viruses isolated in Vietnam prior to May 2007, we first constructed their phylogeny. Six different HA clades were identified and designated according to the recently described nomenclature system for the highly pathogenic H5N1 viruses as well as by potential precursors: clade 0, HK97-like (HK/483/97); clade 1, HK821-like (Dk/HK/821/02); clade 2.3.2, E319-like (Dk/China/E319-2/03); clade 2.3.4, FJ584-like (Ck/Fujian/584/05); clade 3, GX22-like (Dk/GX/22/01); clade 5, F1-like (swine/Fujian/F1/01) (
(A) Phylogenetic tree of subtype H5 HA genes from avian influenza viruses. The phylogenetic tree was constructed by Maximum Likelihood using GARLI version 0.951
Precursor virus | Abbreviation |
HK/483/97 | HK97-like |
Dk/HK/821/02 | HK821-like |
Dk/China/E319-2/03 | E319-like |
Dk/GX/4016/05 | GX4016-like |
Ck/GX/604/05 | GX406-like |
Ck/Fujian/584/06 | FJ584-like |
Dk/GX/22/01 | GX22-like |
Sw/Fujian/F1/01 | F1-like |
The timing of H5N1 clade isolation from poultry indicated that some lineages were present for one year or less, while others continued to circulate in Vietnam for more than 3 years. For instance, following their first detection in 2003, clade 5 viruses circulated in Vietnam for approximately 1 year and were replaced by clade 1 viruses in 2004. The clade 1 viruses first detected in late 2003 continued to circulate until 2007. In addition, other viral clades co-circulated in the region in 2005; e.g., clade 0 and clade 2.3.2 (
Each segment was marked in a different color corresponding to its putative precursor virus. The segment numbers are ordered 1 to 8 from top to bottom.
In 2006, there were very few outbreaks reported in Vietnam possibly due to increased use of poultry vaccines after August, 2005. Our surveillance detected two positive samples among 64 tested using real-time RT-PCR. However, no H5N1 viruses were isolated from these positive samples. A sharp decrease in poultry outbreaks throughout Vietnam during 2006 led to a transition into a passive mode of surveillance for H5N1 and accounted for the low number of samples collected for testing in comparison to other years (
Phylogenetic analyses of the NA and the so-called “internal gene” segments (PB2, PB1, PA, NP, M, NS) of all available H5N1 viruses from Vietnam (>300 viruses) were performed to establish the diversity of the influenza gene pool present in Vietnam. Phylogenetic trees for each gene segment revealed the potential precursor virus for each lineage of the NA and internal gene segments (
The phylogenetic tree was constructed by Maximum Likelihood using GARLI version 0.951
Virus | Virus segment | Year | Genotype | |||||||
HA |
NA | PB2 | PB1 | PA | NP | MP | NS | |||
Gs/VN/113/01 | clade 3 GX22-like; 98.8 |
GX22-like; 99.1 | ND |
ND | ND | ND | ND | ND | 2001 | VN1 |
Ck/VN/NCVD8/03 | clade 5 F1-like; 97.7 | F1-like; 98.7 | F1-like; 99.0 (GX4016-like; 98.7) |
F1-like; 97.8 | F1-like; 97.8 | F1-like; 98.5 | E319-like; 99.2 (GX4016-like; 99.3) | F1-like; 98.7 (GX4016-like; 97.3) | 2003 | VN2 |
Dk/VN/NCVD-7/07 | clade 1 HK821-like; 97.5 | HK821-like; 97.6 | HK821-like; 98.4 | HK821-like; 98.3 | HK821-like; 97.8 | HK821-like; 98.4 | HK821-like; 98.3 | HK821-like; 97.3 | 2003–2007 | VN3 |
Ck/VN/10/05 | clade 2.3.2 GX4016-like; 98.9 (E319-like; 98.6) (GX604-like; 98.6) | GX4016-like; 99.1 (E319-like; 98.7) | F1-like; 98.6 (GX4016-like; 99.1) | GX4016-like; 99.2 | GX4016-like; 99.3 | E319-like; 99.1 (FJ584-like; 98.0) (GX4016-like; 98.6) | E319-like; 98.9 (GX4016-like; 99.6) | F1-like;97.6 (GX4016-like;98.9) | 2005 | VN4 |
Dk/VN/8/05 | clade 0 HK97-like; 97.8 | HK97-like; 97.7 | HK97-like; 97.6 | HK97-like; 99.4 | HK97-like; 98.2 | HK97-like; 98.2 | HK97-like; 99.1 | HK97-like; 99.7 | 2005 | VN5 |
Ck/VN/NCVD-44/07 | clade 2.3.4 FJ584-like; 97.9 | FJ584-like; 98.0 | GX604-like; 98.3 (E319-like; 97.2) | FJ584-like; 98.7 (E319-like; 98.1) | FJ584-like; 98.4 | E319-like; 99.1 (FJ584-like; 97.7) (GX4016-like; 98.7) | FJ584-like; 99.0 | FJ584-like; 98.7 | 2007 | VN6 |
Ck/VN/NCVD-20/07 | clade 2.3.4 FJ584-like; 98.1 | GX4016-like; 97.6 (E319-like; 97.3) | GX604-like; 98.3 (E319-like; 97.3) | FJ584-like; 98.8 (E319-like; 98.2) | FJ584-like; 98.0 | E319-like; 99.1 (FJ584-like; 97.7) (GX4016-like; 98.6) | FJ584-like; 98.8 | FJ584-like; 98.3 | 2007 | VN7 |
M.Dk/VN/NCVD-22/07 | clade 2.3.4 FJ584-like; 98.2 | GX4016-like; 97.5 (E319-like; 97.3) | GX604-like; 97.9 (E319-like; 97.1) | FJ584-like; 98.7 (E319-like; 98.1) | HK821-like; 96.8 | HK821-like; 98.6 | FJ584-like; 98.6 | HK821-like; 98.2 | 2007 | VN8 |
Dk/VN/NCVD-43/07 | clade 2.3.4 FJ584-like; 97.3 | HK821-like; 98.1 | HK821-like; 97.2 | FJ584-like; 96.5 (E319-like; 96.3) | HK821-like; 98.3 | HK821-like; 98.6 | HK821-like; 98.9 | HK821-like; 97.3 | 2007 | VN9 |
Based on the WHO HA nomenclature for HPAI H5N1
The number indicates the nucleotide sequence identity percentile between each gene segment of a representative Vietnam H5N1 virus and the corresponding gene from the putative progenitor virus.
ND denotes the associated segment sequence is not available.
The precursor virus outside the parenthesis is the primary precursor virus, which had a closer phylogenetic relationship to a specific Vietnam virus lineage than the other potential precursor virus shown within the parenthesis. If a lineage had more than one potential precursor, only one virus was marked as the primary precursor-like virus and other closely related viruses were listed within parentheses.
The diverse gene segment lineages identified in H5N1 viruses isolated in Vietnam between 2001 and 2007 provide a large gene pool for reassortment. By combining the phylogenetic information from all the genes of each isolate, we were able to determine the number of viral genotypes present in Vietnam from 2001 to 2007 (
In order to trace the time and location of H5N1 genotype emergence and spread within Vietnam, we plotted the geographical location of each genotype onto a map of the country. While the specific collection sites of the VN1 genotype virus within Vietnam was not known, VN2 genotype virus, which contains all F1-like (clade 5) segments except M, was first detected in 2003. During 2003, both the VN2 and VN3 genotypes were detected only in northern Vietnam despite equal sampling of birds in southern Vietnam during the same time period. In 2004, however, VN3 genotype viruses were isolated in both southern and northern Vietnam
The viral genotype of each H5N1 isolate was mapped chronologically to show the time of genotype isolation in certain regions of Vietnam. VN5 was not shown in this figure since the geographic locations of these viruses were not known. The strain name, genotype, and collection location of each isolate are listed in
Despite markedly lower levels of H5N1 detection in 2006, perhaps as a result of a successful vaccination campaign coupled with a decline in sampling due to a transition to passive surveillance in poultry, five new genotypes were detected in 2007 (VN6-VN9). Each of these genotypes contained the HA gene from the clade 2.3.4 viruses, which were introduced into Vietnam in 2007. In addition, they contained gene segments from other viruses isolated earlier in Vietnam, suggesting that these genotypes emerged locally from reassortment of the newly introduced clade 2.3.4 viruses with pre-existing ones. Reassortment yielding genotypes VN6, VN7, and VN9 appear to have occurred near the Hanoi area as the majority of viruses from these genotypes have been detected in this region of Vietnam (
The VN3 genotype was predominant throughout Vietnam and neighboring countries between 2003 and 2005. The large size of Vietnam's bird population and the high rates of infection in this period were conducive to short viral generation times and rapid evolution. This genotype continued to circulate in 2007, but it was isolated with lower frequency than the VN6 genotype in northern Vietnam (
To complement the genetic analysis of HK821α and HK821β, we compared their amino acid sequences at positions with known functional roles. Interestingly, the receptor binding site of both HK821α and HK821β contained residue 123P (corresponding to residue 127 in H3 influenza A virus numbering) instead of 123S, which was found in the HK821P and FJ584-like virus sub-lineages
A panel of ferret antisera to the WHO reference subtype H5N1 viruses (clades 1, 2.1.3, 2.2 and 2.3.4) was used in hemagglutination inhibition assays with the 40 HPAI H5N1 viruses isolated during 2007. The homologous clade HI titers were at least four-fold higher than those in the heterologous reactions, indicating a strong correlation of genetic and antigenic characteristics. A/chicken/Fujian/584/2006-like (clade 2.3.4) and A/duck/Hong Kong/821/2002-like (clade 1) viruses had distinct properties, as illustrated in
There are 13 WHO reference antigens (marked in red diamonds) and each of the 40 HPAI H5N1 Vietnam viruses isolated in 2007 (marked in blue diamonds;
Virus | Residue position |
||||||||||
86 | 94 | 155 | 174 | 181 | 212 | 227 | 269 | 282 | 310 | 322 | |
clade 1 | V/A/I | D/N/V | S/N | V | P | R/K | E | L | M | R | Q |
clade 2.1 | A/T | S/N | S/N | V | P | K | E | L | M | R | Q |
clade 2.2 | A | N/D | N/D | V | P | K | E | L | I | R | Q |
clade 2.3 | A/V | N | N/D | I/V | S/P | K | D | V | I/M | K | L/Q |
Residue position denotes the amino acid position in the mature HA protein sequence.
HPAI H5N1 viruses were first identified in Vietnam in 2001 and have since caused repeated poultry outbreaks throughout the country leading to dramatic economic losses to the nation's poultry industry. Early introductions of H5N1 in Vietnam (in 2001) led to only sporadic detection of the virus in domestic birds
During the 2003 H5N1 avian influenza outbreaks in Vietnam, the predominant viruses identified belonged to the so-called Z genotype, which were first identified in China in 2002 (referred to as the VN3 genotype in this study)
The VN3 genotype, which was predominate in northern and southern Vietnam throughout 2004 and 2005, has continued to circulate at least through the early part of 2007 despite introduction of other genotypes in 2005 and 2007 (
Four distinct genotypes containing clade 2.3.4 (FJ584-like) HA genes were identified in Vietnam after May 2007: VN6-VN9. Interestingly, these viruses were primarily collected in northern Vietnam and appear to be replacing the VN3 genotype in this part of the country (
The diversity of genotypes present in Vietnam also provides a pluripotent reservoir from which an epidemic or pandemic strain may arise. The discovery of four previously unidentified genotypes (VN4, VN7, VN8 and VN9) confirms previous observations that reassortment between H5N1 viruses occurs frequently. Reassortment between clade 1 viruses (HK821-like) and clade 2.3.4 viruses (FJ584-like) and between clade 2.3.2 (GX4016-like) and clade 5 (F1-like) viruses suggests a high level of genetic compatibility between viruses with diverse parental genotypes. In addition, the emergence of three new genotypes (VN7, VN8, and VN9) following the introduction of clade 2.3.4 viruses (VN6) in 2007, and the concurrent eclipse of VN3 in northern Vietnam suggests that these genotypes may have a selective advantage. One obvious possibility is the antigenic novelty of the clade 2.3.4 HA. Another example of the possible advantage of antigenic diversity is the expansion of HK821β-like viruses that emerged in southern Vietnam in 2007 (
As noted in previous studies, some viruses may have spread bi-directionally between countries
New genotypes were first identified in northern provinces of Vietnam and appear to have gradually spread to southern provinces via unknown mechanisms. The majority of isolates were collected from specific locations in Vietnam with high human population densities, especially in areas around Hanoi and Ho Chi Minh City, which also contain most of the country's industrial and backyard poultry flocks. Although sampling outside of these large cities was less intensive, it is possible that avian influenza transmission from northern to southern Vietnam occurs via direct poultry trade routes between major population centers in each region
Inactivated H5N1 and H5N2 vaccines have been used in Vietnam since August of 2005 to control H5N1 viruses
In summary, our studies of viruses isolated in Vietnam during 2001–2007 demonstrate that multiple viral introductions and reassortment events have occurred frequently in Vietnam leading to the emergence of at least four novel genotypes. Thus, disease prevention and control must be applied inside the country and at the borders, to prevent introduction of new HPAI H5N1 strains, which expand the H5N1 viral gene pool. Strengthening vaccination and associated surveillance programs in poultry would help decrease the current avian influenza virus genetic reservoir in Vietnam, and H5N1 viruses in particular, thus reducing the threat to public health.
The National Centre for Veterinary Diagnostics (NCVD, Hanoi, Vietnam) surveillance program was initiated in 2004 in collaboration with the Vietnam Department of Animal Health (DAH) and DAH Regional Animal Health offices to detect H5N1 infections in poultry. Collection sites included poultry farms, backyard flocks, and live bird markets in 38 provinces representing regions in both northern and southern Vietnam. Cloacal swab specimens were collected in tubes with virus transport medium. Swab samples, as well as sick or dead bird specimens, were submitted to NCVD and regional laboratories. Tissue samples collected from slaughtered sick birds included the bronchus, lung, kidney, spleen, and brain. Tissue specimens were placed immediately on ice in sterile plastic bags and later stored at −80°C. From 2001–2003, samples were screened for H5N1 virus by inoculation in 10–11 day old embryonated chicken eggs (ECEs) for virus amplification. Allantoic fluid from ECEs was tested for the presence of virus by performing a hemagglutination assay using chicken red blood cells. In 2004 and 2005, an influenza subtype H5 specific RT-PCR test was used to identify samples containing H5N1 viral sequences. Since 2006, real-time RT-PCR has been used to identify samples containing H5N1 RNA, which were then selected for inoculation into ECEs for isolation.
Following transfer of swab samples and/or viral isolates to the Centers for Disease Control and Prevention, Atlanta, GA, highly pathogenic H5N1 viruses were isolated from positive samples by infection of either 10–11 day old ECEs or MDCK cells. A total of 158 H5N1 viruses were isolated from 2001 to May of 2007. RNA isolation and sequencing procedures were described previously
In addition to the 158 isolates collected by NCVD and sequenced for this study (
To identify the potential precursor viruses for each Vietnam isolate, we applied our newly developed Gene In Network (GIN) method
Phylogenetic inferences relied on Maximum Parsimony (MP) and Neighbor-Joining (NJ) methods using PAUP* 4.0 Beta
Antigenic characterization of HPAI H5N1 isolated in Vietnam during 2007 was performed using the hemagglutinin inhibition (HI) assay with ferret antisera to the WHO reference H5 subtype viruses, including A/Turkey/65-596/06, A/whooper swan/Mongolina/244/05, A/Indonesia/5/05×PR8, A/Indonesia/5/05, A/Anhui/1/05×PR8, A/chicken/Malaysia/935/06, A/Anhui/1/05, A/Japanese white-eye/Hong Kong/1038/06, A/Anhui/2/05, A/Vietnam/1203/04×PR8, A/Vietnam/1203/04, A/Vietnam/HN30408/05, and A/Thailand/16/04. The HI assay was performed with a starting serum dilution of 1∶5. The multidimensional scaling analysis was performed using SPSS 15.0 (SPSS, Inc.) to analyze HI titers in order to correlate data between 2007 Vietnam H5N1 viruses and WHO H5N1 reference antigens. The HI data for each antigen-antiserum pair was normalized by dividing the maximum HI value for that antiserum. The pairwise
Gene sequences generated during this study have been deposited into the GenBank database, and accession numbers can be found in
Summary of the HPAI H5N1 viruses analyzed in this study.
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Number of H5N1 isolates collected and percentage of each identified genotype in Vietnam per year.
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Geographic origin of HPAI H5N1 viruses from Vietnam.
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Phylogenetic analysis of the H5N1 highly pathogenic avian influenza viruses isolated in Vietnam. Figures A-H represent segments HA, NA, PB2, PB1, PA, NP, MP, and NS, respectively. Posterior probabilities and bootstrap values are given above and below branches. A red dot was marked beside each predicted precursor virus. Previously reported genotypes, such as A, B, C, D, E, G, X0, X1, X2, X3, Y, V, Z, and Z+
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Phylogenetic tree of the HA gene of HK821-like avian influenza viruses isolated from Vietnam. HK821-like viruses formed three sub-lineages: HK821P, HK821α, and HK821β. The tree was rooted by Dk/HK/821/02. The phylogenetic tree was constructed by Maximum Likelihood using GARLI version 0.951 by selecting GTR+I+G model from Modeltest 3.7. Posterior probabilities and bootstrap values were given above and below branches, respectively.
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We are grateful to the Vietnam Department of Animal Health (DAH) and DAH Regional Animal Health offices for submission of swab material and surveillance results to NCVD. We also thank Elizabeth Smith and William Stembridge for viral sequencing. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention or the Agency for Toxic Substances and Disease Registry.