The authors have declared that no competing interests exist.
Conceived and designed the experiments: DZ L. Liang HC. Performed the experiments: DZ L. Liang YJ YL L. Liu. Analyzed the data: DZ HC. Contributed reagents/materials/analysis tools: L. Liu. Wrote the paper: DZ HC.
Despite great efforts to control the infection of poultry with H5N1 viruses, these pathogens continue to evolve and spread in nature, threatening public health. Elucidating the characteristics of H5N1 avian influenza virus will benefit disease control and pandemic preparation. Here, we sequenced the genomes of 15 H5N1 avian influenza viruses isolated in Vietnam in 2006 and 2007 and performed phylogenetic analyses to compare these sequences with those of other viruses available in the public databases. Molecular characterization of the H5N1 viruses revealed that seven genetically distinct clades of H5N1 viruses have appeared in Vietnam. Clade 2.3.4 viruses existed in Vietnam as early as 2005. Fifteen viruses isolated during 2006 and 2007 belonged to clade 1 and clade 2.3.4, and were divided into five genotypes. Reassortants between the clade 1 and clade 2.3.4 viruses were detected in both North and South Vietnam. We also assessed the replication and pathogenicity of these viruses in mice and found that these isolates replicated efficiently and exhibited distinct virulence in mice. Our results provide important information regarding the diversity of H5N1 viruses in nature.
Highly pathogenic avian influenza (HPAI) H5N1 viruses have now spread through poultry populations in many countries. These viruses have also crossed species barriers to infect different hosts
Vietnam is one of the highest frequencies of HPAI H5N1 outbreaks. HPAI H5N1 virus was first identified in Vietnam in 2001
To better understand the molecular and biological properties of H5N1 avian influenza viruses, we selected 15 H5N1 strains isolated from poultry in Vietnam during 2006 and 2007 and sequenced their entire genomes. We performed phylogenetic analyses combining with the sequence data from the Vietnam influenza viruses and other representative viruses available in public databases, and then genotyped the viruses on the basis of their whole genomes. We also assessed the replication and pathogenicity of these viruses in mice. Understanding the molecular and biological features of avian H5N1 viruses will help reveal the potential evolutionary and transmission features of H5N1 viruses, and benefit disease control and pandemic preparedness.
The 15 HPAI H5N1 viruses used in this study were isolated from domestic poultry, including chickens, Muscovy ducks, and ducks on farms in Vietnam. Details of these viruses are given in
Isolates |
Province | Position |
Year | Sublineage |
M2 ionchannel | ||
MDK/VN/1185/06 | Ca Mau | S | 2006 | Clade 1 | I26 | N31 | |
CK/VN/1180/06 | Ca Mau | S | 2006 | Clade 1 | I26 | N31 | |
MDK/VN/1159/06 | Ca Mau | S | 2006 | Clade 1 | I26 | N31 | |
MDK/VN/1181/06 | Ca Mau | S | 2006 | Clade 1 | I26 | N31 | |
DK/VN/1213/07 | Bac Lieu | S | 2007 | Clade 1 | I26 | N31 | |
CK/VN/1214/07 | Bac Lieu | S | 2007 | Clade 1 | I26 | N31 | |
CK/VN/20/07 | Cao Bang | N | 2007 | Clade 2.3.4 | |||
MDK/VN/22/07 | Ca Mau | S | 2007 | Clade 2.3.4 | N31 | ||
DK/VN/31/07 | Soc Trang | S | 2007 | Clade 2.3.4 | |||
DK/VN/34/07 | Son La | N | 2007 | Clade 2.3.4 | N31 | ||
CK/VN/41/07 | Hai Duong | N | 2007 | Clade 2.3.4 | |||
DK/VN/43/07 | Cao Bang | N | 2007 | Clade 2.3.4 | I26 | A27 | N31 |
CK/VN/44/07 | Ha Tay | N | 2007 | Clade 2.3.4 | |||
CK/VN/45/07 | Hanoi | N | 2007 | Clade 2.3.4 | |||
MDK/VN/46/07 | Hai Duong | N | 2007 | Clade 2.3.4 |
MDK, Muscovy duck; CK, chicken; DK, duck; VN, Vietnam.
The letters S and N denote southern Vietnam and northern Vietnam, respectively.
Based on the World Health Organization influenza (H5N1) nomenclature system.
Viral RNA was extracted from allantoic fluid by using TRIZOL Reagent (Invitrogen), and was then reverse-transcribed. A set of fragment-specific primers (primer sequences available on request) was used for the PCR amplification and sequence analysis. The PCR products were purified with the Watson PCR purification kit (Watson) and sequenced by using the CEQ DTCS-Quick Start Kit on a CEQ 8800 DNA sequencer (Beckman Coulter). Sequence data were compiled with the SEQMAN program (DNASTAR, Madison, WI). Phylogenetic trees were generated with MEGA 4.0 by neighbor-joining (NJ) methods and bootstrap tests (1000 replicates; seed = 64238) based on the sequences for the open reading frames (ORFs).
The nucleotide sequences obtained in this study are available from GenBank (accession nos. JX420123- JX420242).
Groups of eight six-week-old female BALB/c mice (Beijing Experimental Animal Center, Beijing) were lightly anesthetized with CO2 and inoculated intranasally with 106.0 EID50 of H5N1 influenza virus in a volume of 50 µL
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People’s Republic of China. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (approval number: BRDW-XBS–02).
To determine the molecular features of H5N1 avian influenza viruses in Vietnam, all eight gene segments of the 15 viruses were sequenced and those sequences were compared with the sequences in public databases of 25 representative influenza viruses from Vietnam and 4 representative viruses from China. The HA genes of these 44 viruses belonged to seven different clades of the WHO influenza (H5N1) nomenclature system (WHO, 2010), which could be further divided into five different groups on the basis of their evolutionary relationships (
The trees were generated by using CLUSTALx1.83 and MEGA4.0 software by the NJ method (Bootstrap test:1000 replicates, seed = 64238 ) on the basis of the following gene sequences: nucleotides 29–1,695 (1,667 bp) of HA, 21–1,358 (1,338 bp) of NA, and 25–2,163 (2,139 bp) of PA. The length of each pair of branches represents the distance between the sequence pairs, and the units at the bottom of the tree indicate the number of substitution events. The 15 H5N1 isolates from Vietnam are marked in bold italic. Abbreviations: CK, chicken; DK, duck; MDK, Muscovy duck; QL, quail; GS, goose; GD, Guangdong; GX, Guangxi; HK, Hong Kong; VN, Vietnam; SX, Shanxi.
Group 1 contained five clade 0 viruses, three of which were isolated from the eggs of Vietnamese waterfowl in 2005
Analysis of the deduced amino acid sequences of the HA genes showed that all of the 15 isolates sequenced had a series of basic amino acids at the HA cleavage site (-RRRKR/−RRKKR-), a characteristic of highly pathogenic influenza viruses in chickens
The NA genes of the 15 strains sequenced and DK/VN/208/05 mainly formed three groups (
The PA genes of the 15 sequenced strains mainly formed two groups (
The PB2 and PB1 genes of the 15 strains also formed two groups, similar to those of the PA genes; however, the PB2 and PB1 genes of MDK/VN/22/07 were included in group 1. Similarly, the same trends were seen in the phylogenetic tree for the NS genes of the viruses. The NP and M genes of all of these viruses shared over 97% homology and were considered to belong to one lineage. Mutations at certain positions in the M2 protein, including I26, A27, and N31, were associated with the amantadine resistance of influenza viruses
On the basis of genomic diversity, the 15 strains sequenced in this study were divided into five genotypes (
The eight gene segments are indicated at the top of each bar. The number in each bar shows the group of genes indicated in
To determine the ability of H5N1 avian influenza viruses to replicate and be pathogenic in mammals, we tested 12 viruses in mice. Groups of 8 mice were inoculated intranasally with 106EID50 of virus; three mice were euthanized on day 3 p.i. and their organs, including lung, spleen, kidney, and brain, were collected for virus titration in eggs, while the remaining five mice were observed for two weeks for changes in body weight or signs of death. As shown in
(A) Weight changes of mice inoculated with different H5N1 viruses. Groups of five mice were intranasally inoculated with 106 EID50 (50 µL) or with PBS as a control and weighed daily for 14 days. (B) Survival percentage of mice inoculated with H5N1 viruses.
Virus | Genotype | Virus replication in organs (log10EID50/mL ± SD |
No. of deadmice |
MLD50 | |||
Lung | Spleen | Kidney | Brain | ||||
MDK/VN/1185/06 | A | 4.6±0.3 |
+ |
− | − | 3 | − |
CK/VN/1180/06 | A | 5.6±0.1 | 2.8±0.8 | 2.8±0 | 2.7±0.0 | 5 | 2.5 |
MDK/VN/1181/06 | A | 4.8±0.7 | + | + | + | 3 | − |
CK/VN/1214/07 | A | 5.0±0.2 | − | − | − | 0 | >6.5 |
MDK/VN/22/07 | D | 4.8±0.7 | + | − | − | 2 | 6.2 |
DK/VN/31/07 | B | 6.6±0.1 | 4.2±0.7 | 3.8±0.2 | 2.3±0.5 | 5 | 2.6 |
DK/VN/34/07 | C | 5.8±0.3 | 3.8±0.5 | 1.4±0.0 | − | 5 | − |
CK/VN/41/07 | C | 6.2±0.6 | 2.3±0.9 | − | − | 4 | − |
DK/VN/43/07 | E | 5.7±0.9 | + | + | + | 5 | 3.5 |
CK/VN/44/07 | B | 5.6±0.1 | 1.8±0.4 | − | − | 0 | >6.5 |
CK/VN/45/07 | B | 6.4±0.1 | 2.7±0.0 | 2.2±0.7 | 1.7±0.1 | 5 | 2.6 |
MDK/VN/46/07 | C | 6.9±0.5 | 3.4±0.1 | 2.8±0.6 | 2.6±0.8 | 5 | 3.2 |
Six-week-old BALB/c mice were used for this study.
Standard deviation.
The data were acquired when mice were inoculated intranasally with 106 EID50 of H5N1 virus in a volume of 50 µL.
The titer shown are the means ± standard deviations of the mice inoculated.
+, Viruses were only detected from undiluted samples; -, the viruses were not detected in the organs.
The data were not acquired.
HPAI H5N1 viruses were detected in Vietnam as early as 2001
Analysis of the 15 viruses sequenced in this study further revealed that the dominant viruses circulating in Vietnam in 2006 and 2007 belonged to clade 1 and clade 2.3.4. A previous study reported that HPAI H5N1 viruses were concentrated in specific geographical regions, with clade 1 viruses mainly in Southern Vietnam and clade 2.3.4 viruses mainly in Northern Vietnam
Most Eurasian HPAI viruses isolated since 1997 can replicate in mammals
The virulence of influenza virus is determined by multiple gene products and amino acid sites. Several determinant sites in the PB2, PA, HA, NS1, and M1 genes are associated with the virulence of avian influenza viruses in mammals
In summary, we characterized the genetic and biological diversity of HPAI H5N1 viruses isolated in Vietnam and uncovered important information that contributes to our comprehensive understanding of these viruses. Influenza viruses spread in nature all year around in tropical regions
We thank Dr. Thanh Long To from the National Center for Veterinary Diagnosis in Vietnam for providing the H5N1 viruses.