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The genetic match between vaccine strains and circulating seasonal influenza A viruses in Vietnam, 2001–2009

Please cite this paper as: Vuong et al. (2013). The genetic match between vaccine strains and circulating seasonal influenza A viruses in Vietnam, 2001–2009. Influenza and Other Respiratory Viruses 7(6), 1151–1157. Background Vietnam is currently developing domestic capability to manufacture influenza vaccines but information on the genetic and antigenic characteristics of locally circulating seasonal influenza viruses is limited. To assess the relevance of WHO recommended vaccine strains to the situation in Vietnam, we analyzed the genetic relatedness of the hemagglutinin (HA) gene of seasonal influenza A viruses circulating in Vietnam from 2001 to 2009 to WHO recommended vaccine strains over the same period. Methods and Principal findings We sequenced the HA gene of 32 H1N1 and 44 H3N2 seasonal influenza A isolates from laboratory‐based sentinel surveillance sites in Hanoi from 2001 to 2005 and from a national influenza surveillance system from 2005 to 2009. H1 and H3 HA phylogenetic trees rooted to vaccine strains A/Beijing/295/1995 (H1N1) and A/Moscow/10/1999 (H3N2), respectively, were constructed with contemporary HA sequences of isolates from neighboring countries. We found some genetic differences between seasonal influenza H3N2 viruses and three WHO influenza vaccine strains recommended for use in the Northern and Southern Hemispheres for the 2001–2004 and 2007–2008 seasons and close genetic identity of circulating H3N2 strains with the recommended WHO Southern Hemisphere vaccine strains for 2004 and 2009 seasons. The genetic similarity of circulating H1N1 strains with the WHO recommended vaccine strains are described for the study period 2001–2009. Conclusions The HA gene of seasonal influenza virus strains in Vietnam (especially influenza A/H3N2) showed varying degrees of genetic identity compared with those of the Northern or Southern Hemisphere vaccine strains recommended by WHO. The close relatedness of the HA of Vietnamese strains and contemporary strains from nearby countries indicate a good genetic match of circulating strains during study period. Greater representation of virus isolates from South East Asia in the vaccine strain selection process is desirable of influenza vaccine development in Vietnam.

Background Vietnam is currently developing domestic capability to manufacture influenza vaccines but information on the genetic and antigenic characteristics of locally circulating seasonal influenza viruses is limited. To assess the relevance of WHO recommended vaccine strains to the situation in Vietnam, we analyzed the genetic relatedness of the hemagglutinin (HA) gene of seasonal influenza A viruses circulating in Vietnam from 2001 to 2009 to WHO recommended vaccine strains over the same period.
Methods and Principal findings We sequenced the HA gene of 32 H1N1 and 44 H3N2 seasonal influenza A isolates from laboratory-based sentinel surveillance sites in Hanoi from 2001 to 2005 and from a national influenza surveillance system from 2005 to 2009. H1 and H3 HA phylogenetic trees rooted to vaccine strains A ⁄ Beijing ⁄ 295 ⁄ 1995 (H1N1) and A ⁄ Moscow ⁄ 10 ⁄ 1999 (H3N2), respectively, were constructed with contemporary HA sequences of isolates from neighboring countries. We found some genetic differences between seasonal influenza H3N2 viruses and three WHO influenza vaccine strains recommended for use in the

Introduction
Influenza surveillance with genetic and antigenic characterization of virus isolates has become routine in many countries. Data and viruses submitted to the WHO's Global Influenza Surveillance and Response System (GISRS) from National Influenza Centers (NIC) provides an understanding of the global circulation of influenza viruses and is essential for vaccine strain selection. However, virus strains submitted to GISRS and strains selected for inclusion in Northern and Southern Hemisphere vaccines may not be globally representative. Although GISRS encourages representative and timely sharing of viruses, specimens from tropical countries are under-represented. There is, therefore, a need to increase the number of viruses submitted for analysis from tropical areas to better assess the relevance of the WHO recommended vaccine strains in tropical countries that are considering the introduction of influenza vaccination. 2 The  1,[6][7][8][9] The National Influenza Surveillance System (NISS) was established in 2005 and comprises 15 sentinel sites (seven sites located in the Northern Region and eight surveillance sites located in the Southern, Highland, and Central Regions of Vietnam), and four regional laboratories. In global temperate regions, there are two flu season annually, corresponding to the occurrence of winter in the Northern and Southern Hemispheres, and influenza vaccination is delivered annually to protect against the influenza strains emergent during each winter influenza season. 2,7,8 Unlike those regions, the Hanoi-based surveillance from 2001 to 2006 and the first 5 years of national surveillance in Vietnam from 2006 to 2009 revealed two peaks of seasonal influenza transmission each year, as has been reported in neighboring tropical countries. 1,3,4,6,7,10,11 Vietnam is developing seasonal influenza vaccine manufacturing capacity, and the implementation of a rational influenza vaccination strategy requires information on the optimal timing and antigenic composition of the vaccine. Because data on the genetic and antigenic characteristics of locally circulating seasonal influenza viruses and the relevance of WHO recommended vaccine strains to the epidemiology of influenza in Vietnam are limited, we have analyzed the hemagglutinin gene of seasonal influenza A ⁄ H1N1 and A ⁄ H3N2 isolated in Vietnam from 2001 through 2009. The overall objective is to better understand the HA gene match between Vietnamese strains and WHO recommended vaccine strains and to inform the development of influenza vaccination policy in Vietnam. 6

Ethical approval
The study was conducted as a part of the Vietnam Ministry of Health approved influenza surveillance system using samples taken from patients presenting to health care facilities with an ILI. The National Institute of Hygiene and Epidemiology and CDC, Atlanta provided ethical committee approval for the study. All participants provided written informed consent.

Sample collection
Patients of any age presenting to one of the sentinel sites (central hospitals, district hospitals, and polyclinics) with an ILI (measured fever ‡38°C and cough and ⁄ or sore throat) within 3 days of onset were eligible for inclusion. 1 Nasopharyngeal swabs (NPS) or throat swabs (TS) were collected by trained nurses using cotton swabs (Hanacomedical. CO.LTD -Japan). Swabs were stored in in-house viral transport media (VTM). 1,7 In the sentinel sites in Hanoi, nasopharyngeal swabs (NPS) were collected from outpatients and specimens were transferred to the respiratory virus laboratory of NIHE on the day of collection 1,2 .
At the 15 sentinel sites of the NISS, throat swabs were collected from the first two ILI patients each weekday (10 swabs per week ⁄ per sentinel site). 1 The TS were refrigerated in in-house VTM and transferred on Friday or on Monday of the following week on wet ice to a participating reference laboratory. Participating laboratories included NIHE, the Pasteur Institute, Ho Chi Minh city (IP HoChi Minh); the Pasteur Institute Nha Trang (IPNha Trang); and Tay Nguyen Institute of Hygiene and Epidemiology (TIHE). 1

Viral isolation
Swabs that were positive for influenza A by RT-PCR were selected for viral isolation, with 100 ll of sample inoculated onto MDCK cells (Madin-Darby canine kidney cells -American Type Culture Collection-ATCC) according to NISS protocols. 1,5,7 Viruses were harvested and stored in )80°C, and all isolates from regional laboratories were transferred to the NIC-NIHE for further analysis. 1,6,12 All isolates were subtyped using the hemagglutination inhibition assay (HAI) with reference antigens and antisera from the World Health Organization (WHO) reagent kit. 1,6

Molecular characterization
Subtyped influenza A isolates with a minimum of 8 HA unit by HA assay were selected for genetic analysis by sequencing at NIC-NIHE, Hanoi. 6 (Table 2) RNA extraction and Polymerase Chain Reaction (PCR) RNA extraction was conducted on 140 ll aliquot of each isolate using the Viral RNA extraction kit (Qiagen-US) according to the manufacturer's instructions. The RNA was transcribed to cDNA using the influenza A virus universal primer (Uni 12) AGC AAA AGC AGG as described. 13 The hemagglutinin (HA) gene was amplified with segmentspecific primers. 6,7,13 Nucleotide sequencing and phylogenetic analysis The PCR products were purified with PCR purification kit (Qiagen-US), and sequencing reactions performed with a Big Dye Terminator v 3.1. Cycle sequencing kit (Applied Biosystem, Foster city, CA, USA) according to manufacturer's instructions. Reaction products were resolved on an ABI 3100 automatic DNA sequencer and sequences assembled using DNA star v 8.0. Sequences were aligned with CLUSTAL X for the major coding regions of HA segments. The sequences of influenza A vaccine viruses were included along with HA sequences from neighboring countries: China, Thailand, Hong Kong SAR, Cambodia, and Japan obtained from the influenza virus resource (National Center for Biotechnology information; http:ncbi.nlm.nih. gov ⁄ genomes ⁄ FLU ⁄ FLU.html.). Because the HA1 domain of HA, the major antigenic protein of influenza A viruses, contains all the antigenic sites of HA and is continually under selection pressure driven by the host's immune response, 1130 nucleotides corresponding to the HA1 ⁄ H1 domain and 1115 nucleotides for the HA1 ⁄ H3 domain were used to construct phylogenetic trees by maximum likelihood (ML) method with bootstrap supported values. To quantify amino acid sequence diversity of each lineage, we used MEGA 5 software package and blasted against the NCBI database to determine the closest sequence available for representative strains of each lineage. The clade numbering for nucleotide-based and amino acid-based clustering was somewhat different due to synonymous substitutions at the nucleotide level.

Nucleotide sequence accession numbers
All sequences newly reported in this study have been deposited in the GenBank database under accession numbers JX506656 to JX506727.  (Table 1). During the study period, only one influenza A subtype, A ⁄ H1N1 or A ⁄ H3N2, predominated each year with the exception of 2009, when influenza A ⁄ H3N2 circulated prior to the appearance and subsequent dominance of A ⁄ H1N1 ⁄ 09 pdm (Figure 1). Because of this dominance pattern, we did not isolate influenza A ⁄  Table 2). Influenza A ⁄ H3N2 virus was identified as the predominant circulating influenza A subtype in 2007 by RT-PCR testing; however, viruses recovered from positive H3N2 samples did not reach the required HA titer for confirmation of subtype and sequence analysis. A total of thirty-four (34) A ⁄ H1N1 and forty-four (44) of A ⁄ H3N2 isolates met the quality criteria for sequencing and were used in this study (      and Southern (A ⁄ Fujian ⁄ 411 ⁄ 2002) Hemisphere vaccine strains was 21AE3 and 2AE3, respectively (Fig 3, table 2). During the 2005 influenza season, we collected twelve H3N2 isolates and most of them (9 ⁄ 12) were grouped in clade IV, with a small number of isolates (3) in clade V which was closely related to the A ⁄ Fujian    Figure 3, Table 2).

Discussion
We Larger genetic variation of HA sequences were observed in A ⁄ H3N2 viruses, with six clades being detected during the study period. This is consistent with the known faster evolution of H3N2 viruses compared with H1N1 or influenza B viruses. 15 (Figure 3) Although analysis of the HA gene does provide some indication on the emergence of antigenically divergent strains, inferences about the protection afforded by WHO recommended influenza vaccine strains in Vietnam are limited by the lack of antigenic data, due to the unavailability of essential reagents in the time period prior to participating in the GISRS (2001)(2002)(2003)(2004)(2005). Genetic data are, however, useful because some genetic changes can generate significant antigenic changes that drive the evolution of influenza. 16 Full antigenic characterization of viruses from Vietnam and other South East Asian countries is required to ensure that vaccine strains selected by WHO will provide protection in these areas. Such characterization is particularly important given that H3N2 virus may evolve and emerge from an East and Southeast Asia network, 15 the large number of people living in this region, and the more complex epidemiology compared with temperate regions. Therefore, sharing materials, such as epidemiological data and representative isolates, from a variety of countries has important implications for influenza vaccine strain selection and the optimal implementation of influenza vaccination programs.
Vietnam is located in the Northern Hemisphere and is in close in proximity to southern China, which has been considered an epicenter of human influenza outbreaks. 17 However, the circulation of seasonal influenza A virus often peaks between May and November in Vietnam, 1 similar to Southern Hemisphere epidemics, 15 and our finding suggest that Vietnamese H3N2 isolates were more similar to WHO Southern Hemisphere vaccine strains in recent years. Thus, strain selection for Southern Hemisphere countries needs to consider strains circulating in Vietnam, and vice versa.
Currently, Vietnam lacks a national policy for influenza vaccination as there is insufficient data on the burden of disease and the local epidemiology and virology. Our results provide information on the HA gene that reveals evolution of the virus that might be expected to lead to antigenic change. Complete genome analysis and serological testing (HAI) of influenza A viruses will be necessary to obtain a more comprehensive picture of virus evolution and to guide vaccine strategy in the future in Vietnam.