Excerpted from the thesis of Nguyen Thi Hong Tham

1. Summary ofIntroduction

– Name of the Ph.D candidate: Nguyen Thi Hong Tham

– Thesis title: Study to select virus strain avian influenza currently circulating in Vietnam, which can generate highly protective immune response in

– Code: veterinary microbiology 62 62 5010

– Instutution: National Institute of VeterinaryResearch

2. Contents of abstract

2.1 Introduction

Avian influenza type A/H5N1 is dangerous disease that causes tremendous economic losses in Vietnam since 2003; According to OIE update information, to Oct. 2014, Vietnam is the country that has highest number of avian influenza outbreaks, and there have been 127 infection human cases of which 64 death. Prophylaxis by vaccination is one of the active and effective methods, but up to date, most of Avian influenza vaccine used in Vietnam was from imported source, which produces in consistent efficacy for duck (Avian influenza virus reservoir). Local produced vaccines used the imported master seed (generated from the strains emerged since 2003) has effective only in limited area where the corresponding strains are still circulated. To develop a vaccine (by any technology), the first step is always selection of vaccine strain and generate a master seed that has to match to the currently circulating strains. This study focus on the selection of the Avian influenza virus A/H5N1 currently circulating in Vietnam, which can generate highly protective immune response in ducks.

2.2. Aim and objectives

The aim of this study is to select a strain of avian influenza A/H5N1 virus isolated from Vietnam that can trigger high protective immune response in duck in order to produce autonomously local inactivated vaccine for poultry and in particular for water fowls.

Objectives: Avian influenza A/H5N1 virus isolated from Vietnam; HA Gene of HPAI A/H5N1, and protective immune response of poultry vaccinated by inactivated vaccine.

2.3 Methods

Method involved the modem and classical techniques including microbiology, cell technology, and molecular biology e.g. cell culture (MDCK), viral titration, inactivation, viral concentration, emulsification, immunization, challenging experiments, RT-PCR, realtime RT- PCR, sequencing, phylogenetic analysis.

2.4 Major results and conclusion:

We have selected a avian influenza A/H5N1 virus strain (A/Dk/VN/Qb7412), representative strain of clade 2.3.2.1c newly emerging and currently circulated strain, which, after 10 passages on embryonated eggs and MDCK cell lines resulted in two masters seed 7412PG and 7412TB for inactivated vaccine production.

Avian influenza A/H5N1 virus, strain A/Dk/VN/QB7412, possess a normal genome of 8 segments without any particular insertions or deletions. The whole genome sequence of

bp can be retrieved at Genbank with Accession Numbers KF182738 – KF182745. This is a virulent strain, capable of generation the lethal rate of 83% for experimental duck at dose of 200 µl in dilution of 1/8 of the allantoic fluid containing 8log2HA virus antigen and 100% chicken at 100 LD50

Oil-adjuvanted inactivated vaccine generated from these 2 master seeds can protect

92,31% immunized ducks when challenged with the A/Dk/VN/QB7412 of origin. This laboratory outcome has been verified in the field condition. Furthermore, these inactivated vaccine could generate cross-protection against avian influenza virus type A, clade 2.3.2.1a, 2.3.2.1b and clade 1 previously circulated.

Siectific outcome:

The successes of the thesis have proven a concept that it is always possible to select a viral strains for vaccine production from the collection of local isolates in Vietnam that have property to give protect poultry from the same or cross-clade ancestor viruses, opening the possibility to select for vaccine strain when there is new strain emerged.

Practical outcome:

The results have provided 2 master seed strains, readily used for autonomous production of avian influenza inactivated vaccine (on embryonated eggs and MDCK cell line), or alternatively be subjective for further reverse genetic modification to generate a vaccine strains that are safer in performance.

New contribution:

The outputs may have 4 new contribution:

Establismentof protocol of viral selection for vaccinated strain from local
Generations of 2 master seeds for inactivated vaccine production that gave high protective immune status in
Reveal a hotspot mutation “new experimental host cell adaptation” in the HA2
Establishment of challenging dose for duck useful in efficacytest