The new temperature-sensitive mutation PA-F35S for developing recombinant avian live attenuated H5N1 influenza vaccine
© Zhang et al.; licensee BioMed Central Ltd. 2012
Received: 22 October 2011
Accepted: 23 May 2012
Published: 23 May 2012
H5N1 highly pathogenic avian influenza virus (HPAIV) is continuously circulating in many Asian countries and threatening poultry industry and human population. Vaccination is the best strategy to control H5N1 HPAIV infection in poultry and transmission to human population. The aim of this study is to identify new temperature-sensitive (ts) mutations for developing recombinant avian live attenuated H5N1 influenza vaccine.
A “6 + 2” recombinant virus C4/W1 that contained NA gene and modified HA gene from virus A/chicken/Hubei/327/2004 (H5N1) (C4), and six internal genes from virus A/duck/Hubei/W1/2004 (H9N2) (W1) was generated using reverse genetics and subsequently passaged in chicken eggs at progressively lower temperatures (32°C, 28°C and 25°C). The resulting virus acquired ts phenotype and one of its amino acid mutations, PA (F35S), was identified as ts mutation. Furthermore, when used as live attenuated vaccine, the recombinant virus with this ts mutation PA (F35S) provided efficient protection for chickens against H5N1 HPAIV infection.
These findings highlight the potential of the new ts mutation PA (F35S) in developing recombinant avian live attenuated H5N1 influenza vaccine.
H5N1 HPAIV is continuously circulating in many Asian countries and threatening poultry industry and human population. An effective vaccine to protect poultry from infections of H5N1 HPAIV is needed not only to reduce economic losses in the poultry industry, but also to minimize virus transmission from infected poultry to humans .
Live attenuated influenza vaccine (LAIV) with needle-free intranasal administration and cross-protection is attractive in controlling pandemic influenza. Recombinant influenza virus with C-terminal truncated NS1, deficient NA, cleavage-site modified HA or cytoplasmatic-tail deleted M2 has been reported as LAIV [2–5]. Besides these, low-temperature passage has been commonly used to acquire LAIV with ts phenotype [6–8]. The ts mutations were identified in internal genes of the LAIVs , and some LAIVs were subsequently used as master donor virus (MDV) to generate new LAIVs by reassortment with two surface glycoprotein genes from currently epidemic strains [10–13].
LAIV has been widely used in humans, while rare LAIV was reported to be used for poultry. Lee et al. developed an avian H9N2 LAIV by successively passaged a wild-type H9N2 influenza virus A/Chicken/Korea/S1/03 in embryonated chicken eggs at progressively lower temperatures . Although the ts mutations of the avian H9N2 LAIV were not identified, some of the amino acid mutations (PB2N265S, PB1K391E/E581G/A661T and NPD34G) were similar to the ts mutations of the MDV A/Ann Arbor/6/60 (H2N2). However, transferring these ts mutations (PB2N265S, PB1K391E/E581G/A661T and NPD34G) into the corresponding internal genes of an avian H9N2 influenza virus A/Guinea Fowl/Hong Kong/WF10/99 could not sufficiently result in an avian live attenuated H9N2 influenza backbone, further introduction of an HA tag into PB1 gene was needed . This strategy of generating avian live attenuated H9N2 influenza backbone seemed inconvenient, more simple ts mutations needed to be identified.
Therefore, the aim of this study was to acquire new and simple ts mutations within an avian H9N2 influenza backbone for developing recombinant avian live attenuated H5N1 influenza vaccine. All experiments with H5N1 HPAIV were performed in ABSL-3 containment facility. We initially generated a “6 + 2” recombinant virus C4/W1 that contained NA gene and modified HA gene (cleavage site was changed from PQRERRRKKR↓G to low pathogenic characteristic PQIETR↓G)  from H5N1 influenza virus A/Chicken/Hubei/327/2004 (C4) , and six internal genes from H9N2 influenza virus A/Duck/Hubei/W1/2004 (W1) . Recombinant virus C4/W1 was then serially passaged in chicken eggs for 10 times at 32°C and 28°C respectively and 15 times at 25°C [7, 8].
Subsequently, eight gene segments of the virus ts-C4/W1 were sequenced and compared with that of virus C4/W1. Five amino acid mutations were identified in the genome of the virus ts-C4/W1. They were PB1 (K698N), PA (F35S), HA (H119Y), M1 (R174K) and NS1 (K108N) (data not shown). To determine which amino acid mutation or mutations contributed to the ts phenotype of the virus ts-C4/W1, the five amino acid mutations were individually introduced into virus C4/W1 and five single-mutation recombinant viruses were generated. The ts phenotype of these single-mutation recombinant viruses was evaluated. As shown in Figure 1, four of the five single-mutation recombinant viruses including C4/W1-PB1(K698N), C4/W1-HA(H119Y), C4/W1-M1(R174K) and C4/W1-NS1(K108N) did not display ts phenotype, they grew up to similar titers at 37°C or 41°C. The rest single-mutation recombinant virus C4/W1-PA (F35S) grew efficiently at °C (8.9 log10 EID50/ml) in chicken eggs, but was highly impaired in growth at 41°C(under limited detection). These results indicated that PA (F35S) was the ts mutation.
Pathogenicity and growth property of recombinant viruses in chickens and mice
Virus replication in chickens#(log10EID50/ml ± SE)
Virus replication in mice&(log10EID50/ml ± SE)
4/4* (3.9 ± 0.4)
4/4 (2.4 ± 0.5)
4/4 (3.5 ± 0.2)
4/4 (5.4 ± 0.2)
3/4 (3.3 ± 0.3)
4/4 (4.7 ± 0.6)
3/3* (4.6 ± 0.2)
3/3 (3.3 ± 0.3)
2/3 (1.6 ± 0.1)
4/4 (1.8 ± 0.3)
3/3 (3.4 ± 0.3)
2/3 (2.5 ± 0.1)
In addition, live attenuated vaccine must be non-pathogenic to vaccine operators before used in poultry. Here, mice were used to assess the virulence of virus C4/W1-PA (F35S). The fifty percent mouse lethal dose (MLD50) was determined as described previously . As shown in Table 1, virus C4/W1-PA (F35S) and virus ts-C4/W1 were more attenuated (MLD50 > 8.0 log10EID50/50 μl) in mice than virus C4/W1 (MLD50 = 6.5 log10EID50/50 μl). This phenotype of attenuation could also be observed from body weight loss of mice infected i.n. with 50 μl 106 EID50 of virus C4/W1-PA (F35S), ts-C4/W1 or C4/W1. Virus C4/W1 caused maximum 20% of initial body weight loss, while no apparent body weight loss was observed when mice infected with virus C4/W1-PA (F35S) or ts-C4/W1 (Data not shown). Their growth ability in mice was also determined. Groups of 6-week-old female BALB/c mice were inoculated i.n. with 50 μl 106 EID50 of virus C4/W1-PA (F35S), ts-C4/W1 or C4/W1. At days 3 and 6 p.i., three mice in each group were sacrificed, and lungs were removed and homogenized in 1 ml PBS containing penicillin and streptomycin. Viral titers were determined by EID50 at 37°C. The results showed that viruses C4/W1-PA (F35S) and ts-C4/W1 grew up to lower titers than that of virus C4/W1 at 3 or 6 days p.i. (Table 1).
The PA subunit was reported to be critical in modulating ribonucleoprotein (RNP) activity under thermal stress (Kashiwagi et al., 2010) . Effects of the ts mutation PA (F35S) on polymerase activity was analyzed at different temperatures (37°C and 41°C) using minigenome reconstitution assay as described previously (Sun et al., 2010) . The RNP components of virus C4/W1 and C4/W1-PA(F35S) showed similar Luciferase activity at 37°C. However, at 41°C, the Luciferase activity of the RNP components of virus C4/W1 was 5.3-fold that of virus C4/W1-PA(F35S) (data not shown). The position 35 was near the endonuclease active site 41 of PA (Yuan et al., 2009) , whether the amino acid mutation PA(F35S) restricted the endonuclease activity of PA to some extent under thermal stress needed to be identified.
Protective efficacy of the live attenuated H5N1 influenza vaccine in chickens
Positive/total No. (HI antibody, log2)before challenge
Shedding/total (log10EID50/ml ± SE)
ND (dead & )
6/6 * (2.3)
Different sublineages of avian H9N2 influenza virus showed different pathogenicity to chickens or mice. Virus of A/Quail/Hong Kong/G1/97 sublineage with internal genes similar to the highly pathogenic Hong Kong/97 H5N1 influenza virus killed mice and spread to mouse brain, while virus of A/Duck/Hong Kong/Y280/97 H9N2 (Y280) sublineage were nonpathogenic to chickens or mice (Guo et al., 2000) . Choosing a nonpathogenic H9N2 influenza virus as backbone might be safer and the stain W1 used in this study as avian backbone was the Y280-sublineage.
In light of rare LAIV used for poultry, we highlighted the potential of the new and simple ts mutation PA (F35S) in developing recombinant avian H5N1 LAIV with avian H9N2 influenza virus as backbone.
We thank Dr. Richard Webby of St. Jude Children Research Hospital, Memphis, TN, USA for the pHW2000 plasmid, Dr. Hualan Chen of Harbin Veterinary Research Institute, China for pPolI-NP-Luc plasmid used in polymerase activity experiment and madam Liu Yanxiu for critically reading of the manuscript. This work was supported by National Basic Research Program of China (program 973, grant 2011CB505004), the National Plan for Science and Technology Support (2010DAD04B03).
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