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Vaccine
journal homepage: www.elsevier.com/locate/vaccine

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Increase in viral yield in eggs and MDCK cells of reassortant H5N1 vaccine candidate viruses caused by insertion of 38 amino acids into the NA stalk
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Wenjun Zhang a , Tao Xue a , Xiaowei Wu a , Pinghu Zhang a,b , Guo Zhao a , Daxing Peng a , Shunlin Hu a , Xiaoquan Wang a , Xiaowen Liu a , Wenbo Liu a , Xiufan Liu a,
a b

Animal Infectious Disease Laboratory, School of Veterinary Medicine, Yangzhou University, 12 East Wenhui Road, Yangzhou, Jiangsu 225009, China National Drug Screening Laboratory, New Drug Screening Center, China Pharmaceutical University, Nanjing 210009,China

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a r t i c l e

i n f o

a b s t r a c t
Background: The H5N1 subtype of highly pathogenic avian inuenza viruses has spread to over 63 countries in Asia, Europe, and Africa and has become endemic in poultry. Since 2004, vaccination against H5N1 inuenza has become common in domestic poultry operations in China. Most inuenza vaccines have been produced in embryonated chicken eggs. High yield is the essential feature of a good vaccine candidate virus. Objective: Therefore, the large-scale manufacture of such a vaccine requires that the viral yield of H5N1 reassortant vaccine viruses in eggs and MDCK cells be increased. Methods: We generated two sets of reassortant H5N1 viruses based on backbone viruses A/Chicken/F/98 (H9N2) and A/Puerto Rico/8/34 (H1N1) using reverse genetics. The HAs and NAs of the reassortants were derived from the three epidemic H5N1 strains found in China. We compared the replication properties of these recombinant H5N1 viruses in embryonated chicken eggs and MDCK cells after inserting either 20 or 38 amino acids into their NA stalks. Results: In this study, we demonstrated that inserting 38 amino acids into the NA stalks can signicantly increase the viral yield of H5N1 reassortant viruses in both embryonated chicken eggs and MDCK cells, while inserting only 20 amino acids into the same NA stalks does not. Hemagglutinin inhibition testing and protection assays indicated that recombinant H5N1 viruses with 38 aa inserted into their NA stalks had the same antigenicity as the viruses with wt-NA. Conclusion: These results suggest that the generation of an H5N1 recombinant vaccine seed by the insertion of 38 aa into the NA stalk may be a suitable and more economical strategy for the increase in viral yield in both eggs and MDCK cells for the purposes of vaccine production. 2011 Published by Elsevier Ltd.

Article history: Received 31 March 2011 Received in revised form 10 August 2011 Accepted 10 August 2011 Available online xxx Keywords: Avian inuenza virus Vaccines NA stalk Viral growth

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1. Introduction The H5N1 subtype of highly pathogenic avian inuenza viruses has caused outbreaks in poultry and evolved into 10 distinct phylogenetic clades, as can be seen by HA gene analysis [1]. Moreover, the H5N1 avian inuenza virus has caused at least 316 human deaths as of March 25, 2011 [2]. Currently, vaccination is still the principal means of protection against avian inuenza in poultry in China [3]. In most laboratories, a reassortant vaccine virus selected for rapid growth in eggs is generated by the 6 + 2 approach, in which it is based on a backbone virus called A/PR8/34 (H1N1, PR8). However, most H5N1 reassortant viruses generated using this technique do not posses the same growth properties as the PR8 backbone virus and so are not suitable for the production of inactivated vac-

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Corresponding author. Tel.: +86 514 87991416; fax: +86 514 87972591. E-mail address: xiu@yzu.edu.cn (X. Liu). 0264-410X/$ see front matter 2011 Published by Elsevier Ltd. doi:10.1016/j.vaccine.2011.08.054

cines [4]. Recently, various continuous cell lines, such as MDCK, VERO, and PER.C6, have been developed as alternative platforms for inuenza vaccine production [5,6]. However, the acquisition of reassortant viruses using these platforms can be time-consuming and unpredictable. For this reason, improving the growth properties of reassortant vaccine viruses in both eggs and cells may be a critical step toward the production of sufcient inuenza vaccine [7]. So far, researchers have not focused signicant effort on the contribution of the neuraminidase (NA) gene or NA stalk to the overall yield of H5N1 vaccine viruses. It is known that the NA stalks coordination with HA plays a signicant role in viral growth and that inuenza viruses need a careful balance of HA and NA activity in order to adapt to their hosts [7,8]. Neuraminidase function is critical to viral release and to preventing the self-aggregation of the progeny viruses [9]. In 2000, a 20-amino-acid deletion was observed between positions 49 and 68 in the NA stalk of the H5N1 avian inuenza virus. Moreover, since 2000, H5N1 viruses with

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Please cite this article in press as: Zhang W, et al. Increase in viral yield in eggs and MDCK cells of reassortant H5N1 vaccine candidate viruses caused by insertion of 38 amino acids into the NA stalk. Vaccine (2011), doi:10.1016/j.vaccine.2011.08.054

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this 20-amino-acid deletion have become more common [1,1012]. Castrucci et al. and Luo et al. have investigated the biological importance of NA stalk engineering in the A/WSN/33 (HlNl) virus. Their results suggest that the length of the NA stalk may be variable and associated with the growth and pathogenesis of the WSN virus; the longer the stalk, the better the replication in eggs [13,14]. Consequently, the NA stalk may also act independently to determine the overall robustness of the H5N1 virus and directly inuence viral yield. However, the biological characteristics of the NA stalk and whether NAs of different stalk lengths have different qualities with regard to replication, virulence, or immunogenicity in H5N1 inuenza vaccine viruses has not yet been studied. Most of these approaches focus on the two highly variable inuenza virus envelope glycoproteins, HA and NA. This is because the more conserved inner virion proteins are not sufciently antigenic to induce solid protective immunity [15]. Qiao et al. reported that a FPV-based vectored vaccine co-expressing H5 and N1 proteins was shown to protect chickens not only against H5N1 but also against H7N1 HPAIV, indicating that NA can contribute to the immune response [16]. Consistent with this observation, vaccination of mice with NA expression plasmids or baculovirus-expressed NA has also been shown to induce pronounced immune response and to protect against lethal infection with homologous inuenza viruses [17,18]. The vaccination of chickens with different DNA and subunit vaccines providing N2 subtype NA was also found to confer partial protection against H5N2 HPAIV challenge [19]. Because NA exhibits lower mutation rates than HA, any additional use of NA as a component in a recombinant vaccine could expand its efcacy to a broader range of inuenza virus strains of the matching NA subtype [20]. This was conrmed by evaluation of an FPV recombinant co-expressing H5 and N1 proteins, which conferred protection not only against experimental challenge with homologous HPAIV but also against an H7N1 isolate [16]. PR8 is a laboratory strain of inuenza, known to be neurovirulent and lethal in mice. It is commonly used as a background vaccine in poultry and humans. Any large poultry operation must require large amounts of inactivated preparations, which presents a risk to vaccine operators in factories [21]. However, the F (H9N2) strain is avirulent in mammals and is more effective than PR8. In order to improve the viral yield of reassortant H5N1 vaccine viruses in eggs and cell lines, we generated two sets of recombinant H5N1 viruses based on A/Chicken/F/98 (H9N2) and A/Puerto Rico/8/34 (H1N1) backbone viruses. We then engineered reassortant H5N1 viruses via the insertion of either 20 or 38 amino acids into the NA stalk and evaluated their replication properties in embryonated chicken eggs and MDCK cells.

(HA) and hemagglutination inhibition tests (HI) were performed with 1% chicken erythrocytes as described by the OIE [24]. All experiments using H5N1 avian inuenza viruses and animals were conducted in an animal biosafety level (ABSL)-3-enhanced laboratory. 2.2. Sequence analysis, mutagenesis, and cloning of HA and NA genes The nucleotide sequences of S and L viruses in this study were made available by GenBank under accession numbers EU195389EU195396 and EU195397EU195404 [22]. The HA and NA sequences of the D strain were sequenced and identied as H5N1 subtypes (accession numbers HQ677023 and HQ677024). The cleavage motifs of three HA genes with high pathogenicity to chickens were deleted by site-directed mutagenesis [25]. Meanwhile, the HA proteins of the S and L strains were moved from position 341 R to position G (Table 1). For this reason, three HA genes were modied to induce attenuated pathogenicity in avian inuenza viruses. Compared to the NA of an earlier H5N1 strain (A/goose/Guangdong/3/1997, GD97, accession number AF364335), the NA stalks of the three isolates were missing 20 amino acids (Fig. 1A). In this study, 20 amino acids from the NA GD97 and 18 amino acids from the NA of A/Tern/Australia/G70C/75 (H11N9, AU75, accession number M17813) were added to the parental NA stalks of these three isolates. In other words, three types of NA with stalks containing 0, 20, or 38 extra amino acids were constructed (Fig. 1B). The modied HA and NA genes were subcloned into pHW2000 vectors [26,27]. Then all plasmids were sequenced to verify that the correct modications had been introduced. 2.3. Plasmids of internal genes In this study, internal genes were derived from two donor strains, A/Chicken/Shanghai/F/98 (H9N2, F, accession numbers AY253750AY253756 and AY743216) and PR8 strain. The internal genes of the F-strain had been cloned into pHW2000 vectors by our laboratory previously and were named as follows: pHW201-PB2, pHW202-PB1, pHW203-PA, pHW205-NP, pHW207M, and pHW208-NS [21,28,29]. The PR8 plasmids (pHW191PB2, pHW192-PB1, pHW193-PA, pHW195-NP, pHW197-M, and pHW198-NS) were provided by Dr. Robert Webster of the St. Jude Childrens Research Hospital (Memphis, TN, USA) [27]. 2.4. Viral rescue by DNA transfection The reassortant viruses were generated by plasmid-based reverse genetics as described previously [27]. Plasmids containing modied HA and NA genes together with plasmids carrying the six internal genes (PB2, PB1, PA, NP, M, and NS) from the PR8 or F-strains were used to transfect COS-1 cells using Lipofectamine 2000 (Invitrogen Corp., Carlsbad, CA, USA) as previously described [21]. At 48 h post-transfection, the culture mixtures were inoculated into 10-day-old SPF eggs to amplify the rescued viruses at 35 C. At 72 h, the allantoic uid was analyzed by HA and HI assay. 2.5. Replication of viruses in eggs and MDCK cells After one additional egg passage, the infectivity and virion content of the rescued vaccine viruses was determined by HA assays and titration in 10-day-old SPF eggs and was expressed as HA titers and EID50 . Conuent MDCK cell monolayers in six-well tissue culture plates were inoculated with virus inoculum at an m.o.i. of 0.02 TCID50 /cell and were cultured with 2 mL DMEM containing 1 g/mL of TPCK-trypsin and incubated at 35 C. Subsequently, the

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2. Materials and methods 2.1. Virus strains, cells, and medium Wild-type inuenza A virus H5N1 strains, A/mallard/Huadong/S/2005 (S), A/mallard/Huadong/lk/2005 (L), and A/chicken/Huadong/4/2008 (D) were isolated in eastern China by our laboratory and identied as highly pathogenic H5N1 avian inuenza viruses [22]. Viral titers were determined by calculating the 50% egg infectious dose (EID50 ) per mL of virus stock [23]. The S and L isolates were highly pathogenic in ducks [22]. COS-1 and MadinDarby canine kidney (MDCK) cells (obtained from the American Type Culture Collection, Manassas, VA, USA) were maintained in Dulbeccos modied Eagles medium (DMEM, Invitrogen, Carlsbad, CA, USA) containing 10% (v/v) fetal bovine serum (FBS, HyClone, Logan, UT, USA). All cells were maintained at 37 C and 5% (v/v) CO2 atmosphere. Polyclonal antisera against S, L, and D isolates were produced in SPF chickens. Hemagglutinin tests

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Please cite this article in press as: Zhang W, et al. Increase in viral yield in eggs and MDCK cells of reassortant H5N1 vaccine candidate viruses caused by insertion of 38 amino acids into the NA stalk. Vaccine (2011), doi:10.1016/j.vaccine.2011.08.054

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W. Zhang et al. / Vaccine xxx (2011) xxxxxx 3 Amino acid sequence at cleavage site Wild-type Modied P L R E G R G L F P L R E G R G L F P Q I E G R G L F

Table 1 Modications in HA cleavage motifs of H5 isolates. Virus

A/mallard/Huadong/S/2005 A/mallard/Huadong/lk/2005 A/chicken/Huadong/4/2008

PLRERRRKRGLF PLRERRRKRGLF PQIEGRRRKRGLF

Note: () Indicates proteolytic cleavage site. () indicates deleted amino acids. Under bar indicates amino acid sequences with mutations.

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cell supernatants were collected at 12, 24, 36, 48, and 72 h postinfection and subjected to analysis to determine the HA titers and tissue culture infective doses (TCID50 ) in MDCK cells. The EID50 s and TCID50 s were calculated with the ReedMuench method [23].

at 37 C. The decrease in HA titer, which was taken to reect NAmediated virus elution from CRBCs, was monitored for 48 h.

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2.6. NA activity assays NA activity was determined as follows. For enzymatic assays, virus dilutions in U-bottomed microtiter plates were incubated with increasing concentrations (5100 M) of the substrate 4-methylumbelliferyl N-acetylneuraminic acid (4-MUNANA; Sigma). The mixtures were incubated for 30 min at 37 C in a water bath, and the reaction was stopped by the addition of 0.1 ml of 0.1 M glycine buffer (pH 10.7) containing 25% ethanol [30]. The uorescence of the released 4-methylumbelliferone was monitored using a Sare2 microplate reader (Tecan). The kinetic parameters (MichaelisMenten constant) (Km ) and maximum velocity of the reaction (Vmax ) were calculated by tting the data to the appropriate MichaelisMenten equation using Kaleida-Graph software (Synergy Software). To evaluate the time of virus elution from chicken red blood cells (CRBCs), HA assays were performed on 25 l of twofold serial dilutions of the viral stocks in phosphate-buffered saline (PBS) [30]. Microtiter plates were stored at 4 C for 1 hour to allow viral adsorption to the CRBCs. The plates were then transferred to a water bath Ten-day-old embryonated chicken eggs were inoculated with 0.1 mL of log10 dilutions of each viral allantoic uids, diluted with infectious titers. The viral dose sufcient to cause death in 50% of embryos was calculated by the Reed and Muench method and recorded as the median chicken embryo lethal dose (ELD50 ) [31]. The mean http://www.iciba.com/death/time of death (MDT) of each chicken embryo was recorded in independent assays and observed for 120 h post-inoculation. To evaluate the pathogenicity of the viruses in chickens, the IVPI was tested according to the recommendation of the OIE [24]. Briey, 10 groups of eight six-week-old SPF white leghorn chickens were inoculated intravenously (i.v.) with either 0.1 mL wild-type and reassortant virus stocks or with 0.1 mL phosphate-buffered saline (PBS). Birds were monitored daily for clinical signs of disease. Oropharyngeal and cloacal swabs were collected for virus isolation on the third and fth days post-challenge and were inoculated into embryonated eggs for virus recovery. The sera of all surviving chickens were HI-tested for evidence of seroconversion on day 14 post-challenge [21].
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Fig. 1. Alignment and insertion of the NA stalk regions. (A) The alignment of the NA stalk regions from A/goose/Guangdong/3/1997 (GD97, top line), A/mallard/Huadong/S/2005 (S, second line), A/mallard/Huadong/lk/2005 (L, third line) and A/chicken/Huadong/4/2008 (D, bottom line) is displayed. The ClustalW method was used for this alignment and the NA stalk regions (amino acids 3690) of the four viruses were closed by a rectangle. The dashes indicate amino acid deletion. (B) The top construct containing the transmembrane, stalk, and head regions indicates partial NA segments of three isolates. Twenty amino acids from the GD97 NA stalk were inserted into the NA stalks of three isolates (middle construct). Another 18 amino acids from the A/Tern/Australia/G70C/75 (H11N9) NA stalk were added and have been underlined behind the previously inserted amino acids.

Please cite this article in press as: Zhang W, et al. Increase in viral yield in eggs and MDCK cells of reassortant H5N1 vaccine candidate viruses caused by insertion of 38 amino acids into the NA stalk. Vaccine (2011), doi:10.1016/j.vaccine.2011.08.054

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W. Zhang et al. / Vaccine xxx (2011) xxxxxx Table 2 Antigenic properties of parental and reassortant viruses. Antigena Antisera (log2 ) Anti-RE-5 RE-5 RE-4 wt-S SF0 SF38 wt-L LR0 LR38 wt-D DF0 DF38 8 2 7 7 7 8 8 8 1 1 1 Anti-RE-4 1 8 2 2 2 2 2 2 2 2 2 Anti-S 8 1 8 8 8 8 8 8 1 <1 1 Anti-L 7 1 7 7 7 8 8 8 1 <1 1 Anti-D 2 2 1 1 1 1 1 1 7 7 7

2.8. Antigenicity of recombinant viruses The antigenicity of the modied reassortant viruses was analyzed by HI testing using chicken antisera immunized with inactivated S, L, and D strains as previous described [24]. Inactivated antigens of A/Duck/Anhui/1/2006 (RE-5) and A/chicken/Shanxi/2/2006 (RE-4), which are currently used as vaccine strains in mainland China, were also used to estimate the antigenicity of the reassortant viruses [1]. 2.9. Vaccination studies of reassortant viruses 2.9.1. Preparation of formalin-inactivated vaccines Viruses were inoculated into the allantoic cavities of 10-dayold embryonated eggs and were harvested after 72 h of incubation at 35 C. The HA protein content of the freshly harvested allantoic uid was determined by SDS-PAGE. The oil-adjuvant-inactivated vaccines were prepared as described previously [32]. The HA protein content of the nal vaccine preparations of each reassortant virus was 9 g/mL. A placebo vaccine contained virus-free allantoic uid from 13-day-old embryonated chicken eggs. 2.9.2. Experiment in SPF chickens Ten groups of 10 seven-day-old white Leghorn SPF chickens received intramuscular injections of either 0.3 mL PBS or 0.3 mL inactivated vaccine preparations (oil-SF0, oil-SF38, oil-LR0, oilLR38, oil-DF0, and oil-DF38) containing 2.7 g HA protein. Blood samples were taken from 10 chickens of each group on day 21 post-vaccination (pv) and sera were isolated for HI assays. Ten chickens from each group were challenged with 106 EID50 homologous viruses (S, L or D strain) at 3 weeks pv via intranasal (IN; 50 L) and eye drop (ED; 50 L) routes. Oropharyngeal and cloacal swabs from the chickens were collected on the third and fth days postchallenge for viral isolation and titration. Chickens were observed for signs of disease and death for 2 weeks after the challenge. 2.10. Statistical analysis Comparisons of experimental groups were estimated by Students t-test with two-tailed analysis to determine signicant differences. P values of less than 0.05 were considered statistically signicant [33]. 3. Results 3.1. Phylogenetic characteristics of multiple clades of H5N1 viruses in Asia HPAI H5N1 viruses mutate rapidly, and this has led to multiple clades of viruses that can affect birds. To identify viruses representative of clades 2.3.4 and 7 for the purposes of vaccine production, a group of related clade 2 and clade 7 viruses were subjected to phylogenetic analysis and some of these were subjected to antigenic analysis. The S and L strains showed close phylogenetic relationships to other viruses within clade 2.3.4 and the D strain was close to clade 7, suggesting that these would be suitable representatives of HA genetic diversity within these two clades. Chicken antisera were generated by vaccination with inactivated wild-type viruses and used in HI assays to determine the antigenic relatedness of the three viruses. Antisera to the S and L viruses showed strong inhibition of hemagglutination by clade 2.3.4 virus antigens (RE-5, wt-S, and wt-L). They also showed low HI titers with clade 7 virus antigens (RE-4 and wt-D) (Table 2), indicating that the S and L strains could be considered representative antigens for the generation of clade 2.3.4 H5N1 candidate reassortant

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Note: The rectangles indicate HI results of the antisera reacting with homologous HA antigens. a RE-4 and RE-5 are inactivated antigens of reference vaccines A/chicken/Shanxi/2/2006 (H5N1, clade 7) and A/Duck/Anhui/1/2006 (H5N1, clade 2.3.4). The other antigens are allantoic uids containing live viruses.

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viruses. Although belonging to clade 7, the D virus showed a distinct phylogenetic relationship to A/chicken/Shanxi/2/2006 (RE-4) and a close phylogenetic relationship to the more recently identied 08-09 clade 7 viruses found in China [12]. However, the antiserum of the D virus showed low HI titers with wt-S, wt-L, RE-5, and RE-4 (Table 2), indicating that it may be necessary to replace vaccine virus RE-4 with a reassortant virus derived from the D virus. 3.2. Generation of inuenza viruses containing different NA stalk mutants The stalk lengths of NAs were altered by mutagenesis. The reassortant viruses carried three subtypes of NA stalks, with either 0, 20 (from GD97), or 38 (from GD97 and AU75 stain) extra amino acids generated by reverse genetics. Nine 6:2 reassortant H5N1 viruses, SF0, SF20, SF38, LF0, LF20, LF38, DF0, DF20, and DF38, were generated using F-strain backbones. In addition, another nine reassortant viruses, SR0, SR20, SR38, LR0, LR20, LR38, DR0, DR20, and DR38, were constructed using the internal genes of the PR8 strain (Table 3). 3.3. Growth of NA stalk mutant viruses in eggs and MDCK cells

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As shown in Table 3, in embryonated chicken eggs, the viruses with 20aa-insertions in their NA stalks (SF20, SR20, LF20, LR20, DF20, and DR20) showed similar scores on their HA titers to those possessing wt-NA stalks (SF0, SR0, LF0, LR0, DF0, and DR0) except for LF20. The HA titer of LF20 was higher than LF0, which had a wt-NA. The HA titers of all the viruses with 38aa-insertions in their NA stalks (SF38, SR38, LF38, LR38, DF38, and DR38) gave results between 2 and 16 times higher than those of the viruses with homologous wt-NA stalks. Similarly, the EID50 of the reassortant viruses with 38aa-insertion-NA stalks showed signicantly higher results than those of the viruses with wt-NA stalks. In contrast, except for SF20, the viruses with 20aa-insertions had EID50 s similar to those with wt-NA. The titer of SF20 was about 0.65 log10 EID50 /mL higher than SF0. These results suggest that enhanced growth of reassortant viruses is achieved in eggs by adding 38 amino acids to the NA stalks. HA and TCID50 assays were performed to quantitate viral infectivity and compare the replication kinetics of the reassortant viruses in MDCK cells. Compared to the infectivity titers of the wt-NA viruses, each 38aa virus showed signicantly enhanced

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Please cite this article in press as: Zhang W, et al. Increase in viral yield in eggs and MDCK cells of reassortant H5N1 vaccine candidate viruses caused by insertion of 38 amino acids into the NA stalk. Vaccine (2011), doi:10.1016/j.vaccine.2011.08.054

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W. Zhang et al. / Vaccine xxx (2011) xxxxxx 5 HA (mean log2 SD)b Internal gene F PR8 9.67 0.33 9.56 0.19 SF0 SF20 SF38 SR0 SR20 SR38 7.11 7.56 10.78 7.56 8.22 9.56 7.11 8.78 10.78 7.67 8.11 9.67 7.33 8.11 10.56 7.44 7.67 9.22 0.38 0.19 0.19** 0.19 0.19 0.19** 0.51 0.19 0.19** 0.33 0.38 0.33** 0.33 0.19 0.19** 0.19 0.33 0.19** 8.63 0.44 8.81 0.07 7.20 7.95 9.87 7.36 8.12 8.37 7.77 7.93 9.19 7.62 8.14 8.97 7.13 8.04 9.40 7.22 7.45 8.61 0.06 0.10* 0.03** 0.06 0.20 0.12* 0.18 0.17 0.16** 0.11 0.44 0.14* 0.10 0.41 0.24** 0.07 0.24 0.17** Mean log10 EID50 /mL SDc

Table 3 Hemagglutination and infectivity titers of reassortant viruses in embryonated chicken eggs. Vaccine straina HA/NA

F/H9N2 PR8-RG/H1N1 S

LF0 LF20 LF38 LR0 LR20 LR38

DF0 DF20 DF38 DR0 DR20 DR38

a b c * **

Recombinant vaccine strains were rescued from COS-1 cells and were passaged through the eggs only once. HA titers were determined using 1% chicken red blood cells. Viral titers were expressed as the mean log10 EID50 /mL SD obtained from three or more independent experiments. Signicantly (P < 0.05) increased titers relative to those of only viruses containing wt-NA were observed. Signicantly (P < 0.05) increased titers relative to those of both viruses possessing wt-NA and viruses containing 20-aa-insertion-NA.

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growth at 24, 36, 48, and 72 h post-infection, respectively (P < 0.05) (Fig. 2AC). Similarly, the HA peak titers of 38aa viruses were signicantly higher than those of wt-NA viruses at 48 h postinfection (P < 0.05), however, the HA peak titers of viruses with 20aa-insertions were similar to those of viruses with wt-NAs (Fig. 2DF). Similarly, the TCID50 of viruses with 20aa-insertions, except for LF20, were similar to those of viruses with wt-NAs (P > 0.05). However, LF20 reached a signicantly higher titer than LF0. These results suggest that, in MDCK cells, sufciently enhanced viral growth can be mediated by 38aa-insertion but not by 20aainsertion.

3.5. Enzymatic properties of the NA activity of H5 viruses To evaluate possible effects of these insertions on NA activity, we prepared suspensions of different H5 viruses with identical hemagglutination titers and tested each suspension for (i) ability to adsorb to and elute from CRBCs, and (ii) NA activity against the low-molecular-weight substrate 4-MU-NANA. Previous studies have shown that NA stalk length affects the elution of viruses bound to CRBCs [13,34]. To determine whether the 38-aa-insertion in the NA stalks of reassortant viruses affected the release of progeny virions from CRBCs, we observed their ability to elute from CRBCs. Reassortant viruses with 38aa-insertion stalks (SF38, LR38, and DF38) were found to elute more readily than any of the homologous reassortant viruses, with elution complete between 1.5 and 11 h at 37 C. All viruses with 20aa-insertion stalks (SF20, LR20, and DF20) eluted at a slightly slower rate. The wild-type and reassortant viruses with parental NAs were able to elute after long periods of time, whereas the wt-DT and DF0 strains were completely eluted from the CRBCs after 22 h of incubation (Table 4). In this way, the rate of elution from CRBCs of H5 reassortant viruses was found to correspond to the length of the NA stalk: the longer the stalks, the faster the elution. The Km values that reected afnity for the substrate were very similar among homologous viruses with NA stalks of different lengths (P > 0.05). They remained within the same range as Km values obtained with other neuraminidases of the N1 subtype [3538]. The Vmax , which is determined by both the specic activity and the amount of enzyme in the reaction, was equivalent to that of wild-type viruses and reassortant viruses with parental NAs. The Vmax of viruses with 20-aa-insertions (SF20, LR20, and DF20) was lower than hat of viruses with parental NAs (P < 0.05). However, the Vmax of viruses with 38-aa-insertions (SF38, LR38, and DF38) was markedly higher than that of SF20, LR20, or DF20 (P < 0.05). It was even higher than that of viruses with wt-NAs. Although there was no apparent relationship between NA stalk length and enzyme

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3.4. Growth of reassortants between the F and PR8 donor strains In eggs, the infectivity titers of viruses bearing 38aa-insertionNAs and F-strain backbones, SF38, LF38, and DF38, reached 9.87 0.03, 9.19 0.16, and 9.40 0.24 log10 EID50 /mL, respectively, all of which were signicantly higher than those of viruses possessing 38aa-insertion-NAs and PR8 backbones (SR38, LR38, and DR38) (P < 0.05). Similarly, reassortant viruses SF38, LF38, and DF38 had higher HA titer results than SR38, LR38, or DR38 (P < 0.05) (Table 3). In contrast, in MDCK cells, viruses SR38, LR38, and DR38 grew to the peak titers of 9.14 0.17, 9.23 0.10, and 8.34 0.16 log10 TCID50 /mL, respectively, reaching signicantly higher titers than viruses SF38, LF38 and DF38 did at 72 h post-infection (P < 0.05) (Fig. 2AC). In addition, the peak HA titers of SR38, LR38, and DR38 were also higher than those of SF38, LF38, and DF38 in MDCK cells at 48 h post-infection, respectively (P < 0.05) (Fig. 2DF). These results demonstrate the suitable contribution of the combination of the 38aa-insertion-NA and the internal genes of the F-strain to the better growth of H5N1 vaccine seed viruses in eggs and the combination of the 38aa-insertion-NA and the internal genes of the PR8 strain to the replication of different H5N1 vaccine viruses in MDCK cells.

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Please cite this article in press as: Zhang W, et al. Increase in viral yield in eggs and MDCK cells of reassortant H5N1 vaccine candidate viruses caused by insertion of 38 amino acids into the NA stalk. Vaccine (2011), doi:10.1016/j.vaccine.2011.08.054

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Fig. 2. Growth curves of reassortant viruses in MDCK cells. MDCK cells were infected at an MOI of 0.02 TCID50 /cell with reassortants (( ) SF0, LF0, and DF0; ( ) SF20, LF20, and DF20; ( ) SF38, LF38, and DF38; () SR0, LR0, and DR0; () SR20, LR20, and DR20; and ( ) SR38, LR38, and DR38). At the indicated times (12, 24, 36, 48, and 72 hpi), infectious particles in culture medium were titrated by TCID50 assay ((A) HA genes derived from S strain, (B) HA genes derived from L strain, and (C) HA genes derived from D strain) and hemagglutinin assay ((D) HA genes derived from S strain, (E) HA genes derived from L strain, and (F) HA genes derived from D strain). Each data point represents the mean SD from three independent experiments. In (A), (B), and (C), signicantly (P < 0.05) increased titers compared with that of only viruses containing wt-NA (*) and compared to those of viruses that are either wt-NA or 20aa-insertion-NA (**). In (D), (E), and (F), the same comparisons were only made at 48 h post-inoculation when each virus grow to peak titer.

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activity toward the small MUNANA substrates, 38-aa-insertions were still found to be superior to 20-aa-insertions in NA stalks. 3.6. Pathogenicity in chickens and embryonated chicken eggs Highly pathogenic avian inuenza viruses are typically lethal to embryonated chicken eggs and death can be observed as early as 24 h post-inoculation. Ten-day-old chicken embryos were

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inoculated with reassortants (SF0, SF38, LR0, LR38, DF0, and DF38) or wild-type viruses. High proportions of embryos from each group were viable at 24 h post-inoculation. The ELD50 s of reassortants (SF0, SF38, LR0, LR38, DF0, and DF38) were lower than 3.47 log10 ELD50 /mL 0.40 (Table 5). In contrast, wild-type viruses (wt-S, wt-L, and wt-D) were highly lethal to embryos even, their ELD50 s being higher than 7.93 log10 ELD50 /mL 0.31 (Table 5). Similarly, the MDTs of the reassortant viruses tended to be more than

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W. Zhang et al. / Vaccine xxx (2011) xxxxxx 7 Km ( M)a 61.4 62.37 64.43 63.40 68.30 69.50 65.97 69.43 46.13 47.83 43.63 53.60 4.53 6.64 2.17 6.16 5.33 3.76 2.91 3.30 1.16 1.94 1.70 6.02 Vmax (uorescence U/s)a 9.6 8.31 7.93 13.47 13.73 12.37 6.70 9.97 6.50 7.97 5.07 10.13 0.79 0.74 0.65 0.85 0.57 0.67 0.46 0.76 0.72 0.78 0.67 1.36 Vmax ratiob 1.00 0.81 0.83 1.40 1.00 0.90 0.49 0.73 1.00 1.42 0.82 1.63 Elution time (h) 8 6.5 3 1.5 15 11 3 2 22 26 15.5 11

Table 4 Neuraminidase activities of reassortant viruses with different NA stalks. Virus wt-S SF0 SF20 SF38 wt-L LR0 LR20 LR38 wt-D DF0 DF20 DF38
a b

Results are given as the mean standard deviation from three independent determinations on duplicate samples using H5 virus dilutions. Vmax ratio of reassortant viruses to homologous wild-type viruses.

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83 h post-inoculation, much later than those of wild-type viruses. More importantly, the ELD50 s and MDTs of SF38, LR38, and DF38 were lower and longer than those of SF0, LR0, and DF0, respectively (Table 5). These results suggest that 38aa-insertion into NA stalks reduces the pathogenicity of reassortant viruses in chicken embryos. To determine the pathogenic potential of the reassortant vaccine viruses in poultry, groups of chickens were inoculated intravenously with 106 EID50 of either reassortant vaccine virus or parental virus. The chickens inoculated with the parental viruses died within two days (wt-S or L strain) or four days (wt-D strain) after inoculation and gross lesions were observed in all cases. In contrast, all chickens inoculated with reassortant vaccine viruses remained healthy throughout the 14-day observation period. Consequently, according to the OIE criteria, the IVPIs of the wild-type viruses were not less than 2.85, indicating highly pathogenic phenotypes, and those of the reassortant viruses were 0, indicating avirulent phenotypes (Table 5). All chickens seroconverted. On day 14, Oropharyngeal and cloacal swabs were taken from chickens inoculated with the reassortants, and no virus was isolated from either type of swab (Table 5). More importantly, in the oropharyngeal and cloacal swabs collected on the third and fth days post-inoculation, the positive percentages of virus isolation in groups SF38, LR38, and DF38 were lower than those of groups SF0, LR0, and DF0 (Table 5). Our results demonstrate that the removal of the multibasic amino acid cleavage site from the HAs results in diminished pathogenicity in H5N1 reassortant vaccine viruses in chickens. Moreover, the pathogenicity of reassortant viruses possessing 38aa-insertions was further reduced in chick-

ens relative to that of reassortant viruses containing homologous wt-NAs. 3.7. Antigenicity of reassortant viruses Vaccine manufacture requires that the modied virus preserve the antigenicity of the parental virus. To estimate whether the modications to HA and NA stalks inuence viral antigenicity, HI tests were performed with chicken antisera. For reassortant vaccine viruses, homologous antisera resulted in high HI titers (Table 2). More importantly, modied vaccine viruses with 38aa-insertion NAs showed titer results similar to those with homologous wt-NA stalks. These results indicate that, within the limits of the sensitivity of the HI assay, reassortant viruses with 38aa-insertions maintain the antigenic characteristics of parental viruses and reassortant viruses with wt-NAs. 3.8. Vaccine efcacy in SPF chickens Single intramuscular (i.m.) doses (0.3 mL formalin-inactivated vaccine preparations containing 2.7 g HA protein) of formalininactivated vaccines were prepared from several reassortant viruses with wt-NAs and 38aa-insertion NAs. To evaluate protective efcacy, we vaccinated seven-day-old chickens with one dose each. Three weeks after immunization, all reassortant vaccines (oil-SF0, oil-SF38, oil-LR0, oil-LR38, oil-DF0, and oil-DF38) had induced high HI titers to homologous wild-type viruses (Table 6). The formalin-inactivated vaccines with 38aa-insertion NAs (oilSF38, oil-LR38, and oil-DF38) completely protected chickens from

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Table 5 Pathogenicity of parental and reassortant viruses in embryonated chicken eggs and SPF chickens. Virus ELD50 a MDT (h)b IVPI Isolation from swabs (shedding/total) Oropharyngeal 3 dpi PBS wt-S SF0 SF38 wt-L LR0 LR38 wt-D DF0 DF38
a b c

Seroconversion (HI titers)c

Cloacal 3 dpi 0 6/8 2/8 2/8 0/8 1/1 4/8 0/8 5 dpi 0 2/8 0/8 0/8 8/8 (7.50 0.53) 1/8 8/8 (7.13 0.64) 0/8 8/8 (6.75 0.46) 8/8 (7.00 0.76) 8/8 (7.38 0.52)

5 dpi 0 5/8 0/8 3/8 0/8 2/8 0/8

9.27 0.51 3.47 0.40 2.70 0.26 8.57 0.25 3.10 0.40 2 >120 7.93 0.31 2.60 0.26 2 >120

36 83 106 36 94 0 51 106 0

0 3 0 0 2.86 0 2/8 2.85 0 2/8

0 8/8 3/8 4/8 0/8 1/1 5/8 0/8

8/8 (7.00 0.76)

Viral stocks containing different infection titers were diluted with 101 108 and 0.1 mL dilutions and then inoculated. ELD50 titers are shown as log10 ELD50 /mL SD. Recorded every 12 h for ve days after the inoculation. HI titers are shown as mean log2 SD.

Please cite this article in press as: Zhang W, et al. Increase in viral yield in eggs and MDCK cells of reassortant H5N1 vaccine candidate viruses caused by insertion of 38 amino acids into the NA stalk. Vaccine (2011), doi:10.1016/j.vaccine.2011.08.054

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W. Zhang et al. / Vaccine xxx (2011) xxxxxx HI titer (log2 SD)b Shedding/total (log10 EID50 SD)c Day 3 O C 1/10 (<1) 2/10 (<1) ND(dead) 1/10 (<1) 0/10 ND (dead) 0/10 0/10 4/4 (2.38 0.41) Day 5 O 0/10 0/10 ND (dead) 0/10 0/10 ND (dead) 0/10 0/10 ND (dead) C 0/10 0/10 ND (dead) 0/10 0/10 ND (dead) 0/10 0/10 ND (dead) 10/10 10/10 0/10 (12) 10/10 10/10 0/10 (12) 10/10 10/10 0/10 (23) Survival/total (dpc)d

Table 6 Protective efcacy of H5N1 formalin-inactivated vaccines in SPF chickens. Groupa

Oil-SF0-C1 Oil-SF38-C1 Placebo-C1 Oil-LR0-C2 Oil-LR38-C2 Placebo -C2 Oil-DF0-C3 Oil-DF38-C3 Placebo -C3

7.4 0.66 7.6 0.49 0 7 0.67 7.1 0.74 0 6.2 0.63 6.1 0.57 0

3/10(1.67 0.58) 2/10 (1.5 0.71) ND (dead) 1/10 (<1) 0/10 ND (dead) 2/10 (<1) 0/10 4/4 (2.76 0.53)

ND: Dead and not done. a C1, C2, and C3: Challenged with 106 EID50 A/mallard/Huadong/S/2005 (H5N1, S, clade 2.3.4), A/mallard/Huadong/lk/2005 (H5N1, L, clade2.3.4), A/Chicken/Huadong/4/2008 (H5N1, D, clade7), respectively. b Sera were collected from 10 chickens at three weeks p.v. for HI antibody detection against homologous wild-type viruses. c O: Oropharyngeal swabs. C: cloacal swabs. d dpc: Days post-challenge.

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subsequent challenge with 106 EID50 of the homologous wild-type virus, as did the formalin-inactivated vaccines with wt-NAs (oilSF0, oil-LR0, and oil-DF0) (Table 6). Under these conditions, none of the vaccinated birds developed disease signs or died, though a minority of those vaccinated with oil-SF0, oil-SF38, oil-LR0, or oilDF0 shed viruses in their oropharynxes and cloacae on the third day post-challenge (Table 6). In the control groups, all chickens mockvaccinated with placebo shed high titers of virus and died within three days of the challenge (Table 6). These results indicate that 38aa-insertions into NA stalks do not reduce the immunogenicity of reassortant viruses. 4. Discussion One of the most challenging tasks involved in producing large quantities of H5N1 avian inuenza vaccine is the production of 6:2 reassortant viruses capable of efcient replication in eggs and cells the substrates for current manufactured inuenza vaccines while retaining optimal antigenicity and immunogenicity. In this study, we assessed molecular modications toward the ends of enhanced growth in H5N1 vaccine seed viruses in embryonated chicken eggs and MDCK cells approved for avian and human H5 subtype vaccine production. The internal genes of the reassortant viruses were derived from F or PR8 strains and their mHA and NA genes were derived from three prevailing H5N1 viruses. Multiple clades of H5N1 co-circulate in birds in Asia, Africa, and Europe. In China, H5N1 clades 2.3.4, 2.3.2, and 7 have been found to co-circulate in poultry in live bird markets, backyard ocks, and slaughtering sites [12]. Inactivated inuenza vaccines are thought to elicit strain-specic protection [39]. Consequently, co-circulation of multiple antigenically distinct H5N1 virus clades presents a signicant challenge to preventive efforts involving vaccine production. By matching the vaccine candidate viruses to major antigenic groups of circulating H5N1 viruses, we managed to update vaccine reference viruses for immediate use. The S and L viruses were found to be well-representative antigens for the generation of candidate reassortant viruses against H5N1 clade 2.3.4. The D virus showed potential as a replacement for the vaccine virus RE-4 against clade 7 [1]. Such a repository may enable more rapid development of high-yield vaccine viruses for use with poultry. It has been shown that one of the ways in which the inuenza virus adapts to new hosts is by alterations in the NA stalk [8,40]. The HA/NA functional balance of viruses with long-stalk NAs gives them an advantage over viruses with short-stalk NAs with regard to growth in MDCK cells [33]. It was also shown that HA glycosylation

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and NA stalk length are interdependent [41]. Most H5N1 inuenza viruses with a 20-amino-acid deletion between positions 49 and 68 in the stalk region contain glycosylation at aa170 (or aa169), but H5N1 inuenza viruses with whole stalk regions lack this glycosylation. HA glycosylation can lead to reduced afnity of HA to receptors [41]. NA stalk length is correlated to biological activity. Long stalks (44 amino acids in length) probably enhance the functionality of NA over viruses with stalk lengths of only 24 amino acids. Such viruses include most H5N1 viruses [13,34] Research has shown that reassortant viruses with longer or shorter NA stalks display attenuated phenotypes relative to those with wt-NAs [42]. Castrucci et al. indicated that the stalkless mutant WSN (H1N1) virus did not grow in eggs, while the reassortant viruses possessing longer NA stalks replicated well in eggs. In our study, H5N1 reassortant viruses with 38aa-insertions in their NA stalks grew more vigorously in MDCK cells and eggs than those with homologous wt-NAs, suggesting that the H5N1 viruses with long-stalk NAs are superior viruses with short-stalk NAs with regard to growth in MDCK cells and eggs. Most of the reassortant viruses with 20aa-insertions into their NA stalks did not grow signicantly better that those with wt-NAs, indicating that 20aa-insertions into NA stalks might be insufcient for improved growth in these two substrates. We not only constructed the three types of NA stalks in our previous experiment but also inserted 57 aa into the NA stalks of those viruses. We found, however, that this 57-aa-insertion could not rescue viral activity. Luo et al. investigated amino acid deletions and insertions in the NA stalk of the inuenza A/WSN/33 virus (H1N1) and found that deletions of up to 28 amino acids and insertions of up to 41 amino acids did not abolish the formation of infectious progeny [14]. In contrast, 57-aa-insertions into NA stalks are not conducive to the formation and replication of viral particles. It has been reported that the replacement of NA with PR8 NA can disrupt the functional balance of HA and NA, enhancing growth in eggs [43]. However, the optimal HA/NA functional balance differed between MDCK cells and eggs, possibly because of the difference in cell receptors [33]. Therefore, it may be difcult to develop ideal HA and NA activity levels for high yield in both eggs and MDCK. In our study, all reassortant viruses with 38aa-insertions grew better than those with wt-NAs, indicating that 38aa-insertion is sufcient to improve the growth of inuenza viruses from different H5N1 clades in these two substrates regardless of the internal backbone. Different host factors between eggs and MDCK cells may inuence the growth of vaccine viruses with different internal genes [44,45]. Thus, it is possible that the specicity of the internal genes

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may determine the preference of reassortant viruses to different host cells. We previously reported that seed viruses maintained F-strain backbones and exhibited strong growth in embryonated chicken eggs [21]. Similarly, those with 38aa-insertions and Fstrain backbones grew better in eggs than in MDCK cells. However, those with PR8 backbones reached higher titers in MDCK cells. This may indicate that the backbone is involved in host range determinants between avian and mammalian species, which are also mediated by the host factors of each animal, as previously reported for the amino acids at positions 627 and 701 of PB2 [46,47]. For example, Lys has only been found at position 627 in inuenza vaccines isolated from humans and those deliberately adapted to mammalian cells [47]. In addition, Li et al. demonstrated that acquisition of an Asp-to-Asn mutation at 701 in PB2 enables an avian inuenza virus to cross the host species barrier and replicate in mammals [46]. In the F-strain, the amino acids at positions 627 and 701 were Glu and Asp. In contrast, in the PB2 PR8 strain, Lys and Asn occupied the same positions, the result of an adaptation to mammalian hosts. However, other unknown characteristics of the internal backbone may also be involved in the process of improving growth. H5N1 vaccine viruses with PR8 backbones of different origins presented unequal growth in eggs. Because of the interactions between polymerase subunit proteins and NP, PR8(UW) showed growth superior to that of PR8 (Cambridge) [43]. The Glu at position 55 of NS1 shows partial responsibility for the ability of an H5N1 vaccine seed virus to grow in MDCK cells [33]. Seven differences in amino acid sequence and large deletions from the C-terminal part of NS1 protein may cause increased efciency of replication in Vero cells [48,49]. These traits were also enhanced by replacement of the PR8 NS gene with that of a Vero-adapted reassortant virus [50]. Therefore it can be determined that the insertion of 38 amino acids into the NA stalk combined with the use of an F-strain backbone may be a determinant of stronger viral growth in eggs. In contrast, 38aa-insertion into NA stalks combined with PR8 backbones was not found to be a determinant of superior viral growth in MDCK cells. The reassortant viruses showed increased ability to release virions from CRBCs, in keeping with the presence of long-stalked NAs. To explain these results, we attributed the faster elution rate of reassortant viruses to the insertions in the NA stalks, which appears to increase the ability of the enzyme to destroy receptors on the erythrocytes. It may be the result of the higher accessibility of the enzyme active site toward large-substrate CRBCs [30]. We also determined NA activity against a low-molecular-weight substrate, 4-MU-NANA, in the same viral preparations that were used in the elution experiments. The NA activity of viruses with longer NA stalks (20- or 38-aa-insertions) against this substrate was not consistently higher than that of viruses with parental NAs. Even 20aa-insertions in stalks signicantly reduced the activity of the NA to the substrate 4-MU-NANA. However, the NA activity of viruses with 38-aa-insertions was signicantly higher than that of viruses with 20-aa-insertions, indicating that 38-aa-insertions in NA stalks may contribute to better spatial structure at the active site than the 20-aa-insertion. Large-substrate CRBCs and small-substrate 4MU-NANA assays showed 38-aa-insertions to be more conducive to increasing the overall functionality of NA enzyme than 20-aainsertions. This is correlated with the viral yield of reassortant viruses in eggs and MDCK cells. It has been reported that a shortened NA stalk is a strong determinant of the adaptation and virulence of waterfowl inuenza viruses in chickens [35]. 27-amino-acid deletion in the NA stalk supports the H2N2 inuenza viruss ability to replicate in the respiratory systems of chickens [51]. Moreover, the short-stalk NAs of the H5N1 viruses circulating in Asia may contribute to their virulence in humans [38]. Similarly, our results indicate that long-stalk NAs with 38aa-insertions reduce the virulence of H5N1 reassortant

viruses in eggs and chickens. As the results of NA activity assays, the NA activity of viruses with 38aa-insertion NAs was different from those of viruses with wt-NAs and 20-aa-insertion NAs, which might lead to the different susceptibility of viruses in different species and further decrease virulence in eggs and chickens. In summary, by adding 38 amino acids to the NA stalks, we were able to produce H5N1 inuenza vaccine candidates that could replicate efciently in eggs and MDCK cells and showed less pathogenic in eggs and chickens. In addition, these reassortant vaccine viruses were both antigenically matched to the parental H5N1 avian inuenza viruses and properly immunogenic in chickens. Consequently, they were found to be economical for use against the challenge of creating reliable and safe H5N1 avian inuenza viruses. Acknowledgments The present work was supported by Major State Basic Research Development Program of China (973 Program) (grant number 2011CB505003), the Earmarked Fund for Modern Agro-industry Technology Research System (nycytx-41-G07) and the Jiangsu High School Natural Science Foundation (No. 10KJA230055). We thank E. Hoffmann and R.G. Webster for the pHW2000 plasmid the expression plasmids of PR8. References
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Please cite this article in press as: Zhang W, et al. Increase in viral yield in eggs and MDCK cells of reassortant H5N1 vaccine candidate viruses caused by insertion of 38 amino acids into the NA stalk. Vaccine (2011), doi:10.1016/j.vaccine.2011.08.054

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