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Hum Vaccin Immunother. 2017 January; 13(1): 111–116.
Published online 2016 September 20. doi:  10.1080/21645515.2016.1231261
PMCID: PMC5287297

Reassortment of high-yield influenza viruses in vero cells and safety assessment as candidate vaccine strains


Vaccination is the practiced and accessible measure for preventing influenza infection. Because chicken embryos used for vaccine production have various insufficiencies, more efficient methods are needed. African green monkey kidney (Vero) cells are recommended by the World Health Organization (WHO) as a safe substitute for influenza vaccine production for humans. However, the influenza virus usually had low-yield in Vero cells, which limits the usage of Vero cellular vaccines. This study used 2 high-yield influenza viruses in Vero cells: A/Yunnan/1/2005Va (H3N2) and B/Yunnan/2/2005Va (B) as donor viruses. It used 3 wild strain viruses to reassort new adaptation viruses, including: A/Tianjin/15/2009(H1N1), A/Fujian/196/2009(H3N2), and B/Chongqing/1384/2010(B). These three new viruses could maintain the characteristic of high-yield in Vero cells. Furthermore, they could keep the immunogenic characteristics of the original wild influenza viruses. Importantly, these viruses were shown as safe in chicken embryo and guinea pigs assessment systems. These results provide an alternative method to produce influenza vaccine based on Vero cells.

Keywords: influenza virus, reassortant, safety assessment, Vero cells


Influenza A and B viruses could infect 5–15% people in the whole word every year, causing half a million deaths.1. Annual vaccination remains the best way to prevent or reduce the clinical severity of influenza infection.2 Currently, most commercial vaccine companies mainly produce vaccines in chicken embryos.3 Although chicken embryos have been used since the 1940s, there are several limitations of this system in influenza vaccine production.2,4 First, vaccines protect people from a certain range of influenza viruses worldwide, but not all influenza viruses.4 Second, the current production cycles of the seasonal influenza vaccine usually require a 6-month preparation period before usage. On the other hand, the inadequate supply of eggs is considered a big challenge in the short term.4 Third, the egg adaptation system could cause an antigenic drift between the vaccine and the original virus, which might cause litter range in mammalian cells.5

In 1995, the World Health Organization (WHO) recommended development of alternative influenza virus cultivation systems, specifically, to explore promising mammalian cell culture lines (cell culture as a substrate for the production of influenza vaccines: Memorandum from a WHO meeting in 1995). Several cell lines are currently approved for cell culture-based vaccine production, such as, the African green monkey (Vero) cell line (e.g., used for polio and rabies vaccines).5,6 Therefore, the primate origin of Vero cells might be a positive choice in the retention of the biological properties of human influenza viruses. Recently, we have screened 2 main donor strains for influenza A and B viruses which have high-yield characteristics in Vero cell. They were A/Yunnan/1/2005Va(H3N2) and B/Yunnan/2/2005Va(B). However, these 2 Vero cells adaptation strains had different antigenic epitopes with annual epidemic influenza viruses. In this study, we generated 2 new influenza A and one influenza B reassortant viruses that carried HA and NA epitopes from original wild type influenza viruses. They were isolated from China during 2009 to 2010. These viruses were A/Tianjin/15/2009(H1N1), A/Fujian /196/2009(H3N2) and B/Chongqing /1384/2010(CQ/10). Another antigen of these new reassortment influenza viruses were from these donor strains: A/Yunnan/1/2005Va or B/Yunnan/2/2005Va. A systematic safety evaluation among the reassortant viruses was completed by calculating growth rate, completing immunogenicity testing, and using an animal experiment evaluation system.


Identified the 3 reassortant IV FJ/09Va, TJ/09Va, and CQ/10Va

After coinfection with original virus and donor virus in same Vero cells, following antibody selection, 2 Vero-adapted (Va) influenza A viruses and one influenza B virus were generated. According to the gene sequencing results, the reassortant FJ/09Va and TJ/09Va carried the HA and NA from original viruses TJ/09 (H1N1) and FJ/09 (H3N2), respectively. But, they maintained the backbone of YN/05A with 6 other gene fragments. As for the influenza B virus, the same trend was seen with the reassortant virus (CQ/10Va had backbone gene fragments of YN/05B).

Growth curve of 3 reassortant viruses in Vero cells

To draw the growth curves of these 3 viruses—FJ/09Va, TJ/09Va, and CQ/10Va—in Vero cells, they were cultured in Vero cells. Simultaneously, their original wild type viruses (FJ/09, TJ/09, and CQ/10) were used as negative controls. The donor strains of YN/05A and YN/05B were used as positive controls. Fig 1 illustrates that the HA titer of these 3 reassortment was at a high level. But their original wild stains were at a lower level from first generation (Fig. 1A for FJ/09 Va, Fig. 1B for TJ/09 Va, and Fig. 1C for CQ/10 Va). These results indicate that the reassortment virus could reach high-yield characteristics in Vero cells, similar to donor strains.

Figure 1.
Growth profiles of 2 master donor YN/05A, YN/05B, and reassortant viruses FJ/09Va, TJ/09Va, and CQ/10Va and 3 wild type parental viruses. Wild type and reassortant viruses grown in Vero cell incubated at 33°C for 3 d. Viral titers were determined ...

Plaque assay on CEF cells with trypsin

To detect viral activity and infectivity of these reassortment viruses, plaque assays were used on fourth generation chick embryo fibroblast (CEF) cells, performed with 0.1 µg/mL TPCK-trypsin. After calculation, the live influenza virus content measured as follows: FJ/09Va with 108.9 PFU/mL, TJ/09Va with 108.4 PFU/mL, and CQ/10Va with 107.2 PFU/mL, using the tenth passage level of viruses. Additionally, the results of trypsin-dependent testing showed that these reassortant viruses could not grow without TPCK-trypsin.

Biological safety evaluation by chicken embryo

Because highly virulent influenza viruses could be observed in the chicken embryos as early as 24 hours after infection, the study used the chicken embryo to assess the biological safety of vaccine strains at the first stage. If these reassortment viruses had high pathogenicity, they would not be considered as vaccine candidates. The results of the safety evaluation with 10-day-old chicken embryos showed that they did not have higher pathogenicity than the original viruses. After calculating the 50% chicken embryo lethal dose (CELD50), these reassortment viruses were all under 106.2 PFU/mL, which was similar to the original virus. There was no significant difference in CELD50 between them, respectively (Table 1). Furthermore, the study also used 50% tissue culture infective dose (TCID50) by chicken embryo fibroblast (CEF) cells for the reassortment virus assessment. The results of TCID50 were consistent with results of CELD50.

Table 1.
Virulence assessment in chicken embryos.

Biological safety evaluation by mice

Then mice were used to assess the biological safety for live animals, because mice are usually considered as model animals for drug interventions for influenza virus infection. After intraperitoneal injection, 3 reassortant viruses did not kill any mice at the maximum permissible dose. However, the original wild type viruses (TJ/09 and FJ/09) killed 2 and 3 mice, respectively, in each positive control group. Therefore, these reassortant viruses were safe for live animals. Diethyl ether was used to euthanize the animals.

After pathological anatomy, the reassortant viruses FJ/09Va, TJ/09Va, and CQ/10Va had lower titers than the original wild type influenza viruses in the lungs and nasal turbinate of mice. No virus replication was detected in their brains.

Biological evaluation of horizontal transmission by guinea pigs

Because the guinea pig is a transmission model for human influenza viruses, the study used guinea pigs to assess the horizontal transmission safety. First, the original wild type influenza viruses with TJ/09 killed one guinea pig. However, there was no death in any of the reassortant virus groups. Second, the other 9 guinea pigs got secondary infections in each original wild type virus group, which had high occurrence of horizontal transmission. As for the reassortant virus groups, there were some differences, depending on the specific strains. By observation, 5 guinea pigs got secondary infections in TJ/09Va group, and 4 animals got secondary infections in the FJ/09Va and CQ/10Va groups, respectively.

Biological evaluation for virus re-activity with temperature inactivation by ferrets

As known, the ferret is a typical sensitive animal model for influenza virus. After temperature inactivation of all reassortant viruses in 56°C for 30 min, infection was spread intranasally and virus re-activity was not detected among all reassortant virus groups. All the ferrets were in good condition, and their weight grew steadily.

Detection of its immunogenicity of reassortment by HI

In order to detect the immunogenicity of FJ/09Va, TJ/09Va, and CQ/10Va, 6-week-old female BALB/c mice were immunized with dose of 0.1 mL, 7.5 ug through subcutaneous injection. Simultaneously, physiological saline was used as negative control. Four weeks after immunization, blood was collected and serum then separated. Immunogenicity was detected by hemagglutination inhibition (HI) testing. These results showed that there were no significant differences between the reassortant viruses and wild type original viruses with HI testing. That demonstrated that FJ/09Va, TJ/09Va, and CQ/10Va have safe and effective immunogenicity as vaccine candidates.

Mice weight and behavior were recorded and observed daily for 14 d (Fig. 2A). All mice survived in the experiment, and their body weights did not decrease in the groups of reassortant viruses, as similar to the negative control group.

Figure 2.
Immunogenicity of reassortant viruses FJ/09Va, TJ/09Va, and CQ/10Va and 3 wild type parental viruses. (A) Group of mice were immunized with one dose of 0.2 mL, 5 ug HA formalin-inactivated reassortant viruses and wild type parental viruses by ...


More and more researchers are concerned about the inadequate supply of chicken embryos and that the antigenic variation for influenza viruses could cause some shortcomings of the influenza virus vaccine. There is insufficient immune protection for the population. WHO suggested that the vaccine manufacturers should explore more suitable vaccine production technologies.2 Among these new solutions, the cell culture–based influenza vaccine had some significant advantages.7,8 Vaccine from a cell culture medium would overcome many insufficiencies of egg-based production, including the long cycle production time and complex preparation chain. Cell-based influenza vaccine enables better control of raw materials and production processes, even under influenza pandemic. However, most wild type influenza viruses are high-yield when grown in chicken embryo or MDCK cells but not Vero cells.1,9 In several recent publications, we and other researcher have described screening methods of highly productive vaccine strains in Vero cell.2,10,11 In this study, we used 2 donors of high-yield influenza viruses in Vero cells: A/Yunnan/1/2005Va(H3N2) (YN/05A) and B/Yunnan/2/2005Va(B) (YN/05B). But, A/Tianjin/15/2009(H1N1), A/Fujian/196/2009(H3N2), and B/Chongqin /1384/2010, were isolated in China during the 2009–2010 influenza epidemic season. However, as original wild type viruses, they had low yield in Vero cell. After reassortment experiments, the series of experiment results demonstrated that 3 new reassortant viruses have high-yield capability in Vero cells, as similar as donors. Meanwhile, all the reassortant viruses had low virulence according to the results of biological safety assessments using chicken embryo, mice, guinea pigs, and ferrets.

HA epitope is the most important protective antigen on the surface of the influenza virus.3,12 After the influenza virus has been grown in chicken embryos, variability of epitope could be conducted, which reduced the protective effect of the vaccine.6,12 Three new strains of reassortant influenza viruses could maintain original immunogenicity, which stimulated the mice body to produce high titers of specific antibodies. Even after whole gene sequencing, the HA gene could remain the same with the original wild type virus.

In summary, 3 reassortant viruses had significant high yield in Vero cells and retained immunogenicity similar to original wild viruses. Furthermore, the virulence of the reassortant viruses was low. The reassortant viruses were prepared from wild type strains that were isolated in mainland China from clinical trials. These methods could be used for other strains' vaccine preparation in each year's pandemic. These methods could save time for screening the adaption reassortment viruses, when faced with influenza pandemic or epidemics. Our work might break some barriers of Vero cell-based inactivated vaccine use.

However, there are some limitations of the current study. In order to detect the vaccine protective efficacy, virus challenge experiments should to be conducted with ferrets, and the changes in the animals' antibody levels should be analyzed by neutralize testing.

Materials and methods

Cells and virus culture media

Vero cells were obtained from a European collection of cell cultures (ECACC; code: 03129010). The fourth passage transformed chicken embryo fibroblasts (CEFs) were obtained from the Chinese CDC. All cells were maintained in Dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and 2 mM L-glutamine.

As for the virus culture medium, DMEM/F12 (pH 7.0–7.2) was used, containing 1% bovine serum albumin (BSA), 100 U/mL penicillin, 100 mg/mL streptomycin, 2 mM L-glutamine, and 1 μg/ml L-1-tosylamide-2-phenylethyl-chloromethyl-ketone (TPCK) trypsin.5,6


Three original wild type (wt) influenza viruses—A/Tianjin/15/2009(H1N1) (TJ/09), A/Fujian /196/2009(H3N2) ( FJ/09), and B/Chongqing/1384/2010 (CQ/10)—were from the Chinese national influenza center (Beijing, China). Two main donor strains for reassortment—A/Yunnan/1/2005Va (H3N2) (YN /05A) (CCTCC No: V200514) and B/Yunnan/2/2005Va(B) (YN/05B) (CGMCC No.2931)—were stored at the Institute of Medical Biology, Chinese Academy of Medical Sciences.


Goat anti-influenza virus serum was prepared by immunizing with YN/05A, YN/05B, and FJ/09 individually, by the Institute of Medical Biology. After three months, booster immunizations occurred, and the serum was treated with a receptor destroying enzyme (RDE, Denka Seiken, Japan) for deleting the nonspecific agglutination inhibitors and heated at 56°C for 1 hour to inactivate the complement. After calculating, the serum hemagglutination inhibition (HI) titer was 1:2048 with YN/05A, 1:1024 with YN/05B, and 1:1024 with FJ/09, respectively.

Reassortment by co-infection

All experiments were carried out under biosafety level (BSL) 3+ containment procedures. In order to ensure the reassortant viruses retained the HA and NA genes of the original viruses, classical traditional reassortment methods were used. Briefly, 108TCID50/mL of the TJ/09 virus and 109TCID50/mL of the YN/05A were mixed in 150 uL phosphate-buffered saline (PBS). Then the same Vero cell was co-infected. The mixed virus solution was harvested after 72-hour incubation at 33°C. To screen the target virus without the surface antigen of HA and NA proteins of the YN/05A donor virus, 100 uL harvesting was mixed with 100 uL anti-YN/05A goat anti-serum and incubated at 30°C for 30 min before infecting the Vero cell again. The specific antibody combination experiments were repeated 3 times. At last, hemagglutination inhibition (HI) testing was undertaken to identify its surface antigen.

As for the FJ/09 Va with donor of A/Yunnan/1/2005Va (H3N2) and CQ/10 Va with donor of B/Yunnan/2/2005Va (B), these used the same method, according to the above description. Before establishment of microbial culture collections, sequencing for each reassortment virus at the tenth generation was performed, to analyze their full genetic background.

As for accounting the passage level, “passage 1” represented the original wild type viruses and new reassortant viruses at the first generation in Vero cells. A bank of donor viruses was constructed with adaption of the Vero cell, which were called stock viruses. It used the 57th (A/Yunnan/1/2005Va (H3N2)) and 64th (B/Yunnan/2/2005Va (B)) passages for depth of study of reassortment. But when used for the first time in this study, it was marked as passage 1.

Sequenced and blasted the virus gene

There were 3 wild type influenza viruses, 3 new reassortment Vero adaption viruses, and 2 donor virus, resulting in 64 gene fragments to analyze. The whole genome for each virus was sequenced. These gene fragments were sequenced by Shanghai Biological Engineering Technology Services Ltd. Blasting the gene difference between the original virus and the reassortment Vero adaption virus by Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College.

Hemagglutination assay

Virus samples were diluted in twofold steps in PBS, mixed with an equal volume of a 1% erythrocyte suspension (chicken), and incubated in a U-shaped microtiter plate for 30 min at room temperature. The calculation method of virus hemagglutinin quantitation was described previously.11,13

Plaque assay TCID50

Cells passage involves transferring a small number of cells into a new vessel. It avoids the senescence associated with prolonged high cell density. Cells first need to be detached; this is commonly done with a mixture of trypsin-EDTA. A small number of detached cells can then be used to seed a new culture.

Before the assessment preparation, the TCID50 for all viruses was determined by infecting CEF cells by serial 10-fold dilutions of samples inoculated into 96-well microtiter plates as described in other publications.7,8

As for plaque assay on CEF cells, the same method according to the previous study was used. The transformed chicken embryo fibroblasts (CEFs) were obtained from the Chinese CDC. It was used the CEF cells at fourth passage level in this study. CEF cells were briefly challenged by each virus strain in 6-well plates with 10-fold serial dilutions at 30°C for 2 hours, then all solutions were removed and the cell surfaces washed with PBS. The cells' surfaces were overlaid with MEM containing 1% low melting agarose and 1 ug/mL of TPCK-trypsin. After 72 hours, samples were stained with 0.5% crystal violet and the plaque calculated.

MOI was tested from the tenth generation by plaques, when these reassortant viruses could adapt to Vero cells stably. However, the HA titers were tested at each generation.

Biological safety evaluation by chicken embryo

All animal experiments were according to the guidelines for animal experiments, which were approved by the Institute of Medical Biology, Chinese Academy of Medical Sciences. All experiments were implemented under BSL3+ conditions.

There were 10 chicken embryos, 10 mice, 10 guinea pigs, and 6 ferrets used for biological safety evaluation experiments. The mortality of each animal group was calculated.

Ten-day-old chicken embryos were inoculated in the allantoic sac with 0.1 mL of log10 dilutions of each virus preparation, according to the plaque testing. The embryo survival rate was observed after infection for 48 hours. A 50% death rate of embryos was calculated by the method of Reed and Muench and reported as the Median Chicken Embryo Lethal Dose (CELD50).

Biological safety evaluation by mice

The immunization programs for mice were carried out, according to WHO guidelines (Cell culture as a substrate for the production of influenza vaccines: Memorandum from a WHO meeting in 1995).

Testing of 50% lethal dose of mice (MLD50) of 3 reassortant viruses was taken by intraperitoneal injection, for ten 8-week-old female BALB/c mice with serial 10-fold dilutions with 50 µ viruses (TCID50 from107 to 103). All mice were observed and their condition and their death rates were recorded. The MLD50 values are calculated by the method of Reed and Muench.1,14

To investigate the degree of the viruses' safety in different organs, mice were inoculated with 106 TCID50 of the each reassortant virus. After infection for 72 hours, histopathological detection for the nasal turbinate, lungs, and brains was undertaken in the Basic Medical College, Kunming Medical University.

Biological evaluation by guinea pigs and ferrets

Biological evaluation was also carried out by studying horizontal transmission by guinea pigs. The viral load was according to the above described with plaque testing. It was to highlight a difference in route of immunization—via intranasal infection for guinea pigs and ferrets.

There were differences in viral inactivity, with inactive viruses in 56°C for 30 min for the experiment with ferrets. A new method of attenuated vaccine by temperature-inactivation should be analyzed. Because these reassortant viruses had low pathogenicity according to a previous study, an attempt was made to inactivate influenza vaccine viruses by changing the temperature.

Antigenicity of reassortant vaccine in mice

To evaluate the immunogenicity and antigenicity of the 3 reassortant viruses, 6-week-old female BALB/c mice (n = 10/group) were immunized with one dose of 100 uL 7.5 ug HA unit of formalin-inactivated FJ/09Va, TJ/09Va, and CQ/10Va reassortant virus by subcutaneous injection.

Weight changes were recorded daily. If they were sick after infection, their weight could not grow stably. Their behavior could not be active, which might be observed via a swim test within a half-hour. Observation method was used to analyze the differences between the 2 groups. In addition, Beijing Institute of Microbiology and Epidemiology (302 hospital) had reported that if the mice were infected highly virulent influenza viruses, they exhibited a rapid decrease in body weight at 5 days, which was published in Plos One in April 2015.13

Serum was collected after 28 d of infection. Then hemagglutination inhibition was measured.

Hemagglutination inhibition (HI) assay

Sample serum was treated with RDE to remove nonspecific agglutination inhibitors and heated at 56°C for 1 hour. The serum was diluted with PBS in serial twofold dilutions in 96-well plates. Equal volumes (25 uL) of influenza virus (8 hemagglutination units) were then added to the diluted serum, mixed well, and incubated at 37°C for 1 h. After incubation, equal volume of 1% chicken erythrocytes was added and incubated at 25°C for 1 h. The HI antibody titer of each serum was expressed as the reciprocal of the highest dilution of the sample that completely inhibited hemagglutination.

Statistical analysis

All data were expressed as means ± SD unless otherwise stated. Analysis of variance was used to compare the experiment group means within a time point and to compare mean responses between time points. The statistics tests were performed in SPSS software (Version 15.0, SPSS Inc., Chicago, IL). The ANOVA F-statistics were used to assess significance at the 5% level. The values plotted represent the antibody titers for each group and error bars extend to 95% confidence upper limits.


World Health Organization
Influenza virus
chick embryo fibroblast
Technological Pedagogical Content Knowledge
chicken embryo lethal dose
tissue culture infective dose
hemagglutination inhibition

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.


This work was supported by International Cooperation Project (2011DFR30420); Innovation team project in Yunnan Province(2015HC027); Science and technology innovation strong province project in Yunnan Province (2014AE008); Scientific Research Projects of Health Care (200802023); National 863 Program (2012AA02A404); National Science and Technology Major Project (2013ZX10004003-003-002).


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