Cloning of the canine RNA polymerase I promoter and establishment of reverse genetics for influenza A and B in MDCK cells
© Wang and Duke; licensee BioMed Central Ltd. 2007
Received: 13 September 2007
Accepted: 23 October 2007
Published: 23 October 2007
Recent incidents where highly pathogenic influenza A H5N1 viruses have spread from avian species into humans have prompted the development of cell-based production of influenza vaccines as an alternative to or replacement of current egg-based production. Madin-Darby canine kidney (MDCK) cells are the primary cell-substrate candidate for influenza virus production but an efficient system for the direct rescue of influenza virus from cloned influenza cDNAs in MDCK cells did not exist. The objective of this study was to develop a highly efficient method for direct rescue of influenza virus in MDCK cells.
The eight-plasmid DNA transfection system for the rescue of influenza virus from cloned influenza cDNAs was adapted such that virus can be generated directly from MDCK cells. This was accomplished by cloning the canine RNA polymerase I (pol I) promoter from MDCK cells and exchanging it for the human RNA pol I promoter in the eight plasmid rescue system. The adapted system retains bi-directional transcription of the viral cDNA template into both RNA pol I transcribed negative-sense viral RNA and RNA pol II transcribed positive-sense viral mRNA. The utility of this system was demonstrated by rescue in MDCK cells of 6:2 genetic reassortants composed of the six internal gene segments (PB1, PB2, PA, NP, M and NS) from either the cold-adapted (ca) influenza A vaccine strain (ca A/Ann Arbor/1/60) or the ca influenza B vaccine strain (ca B/Ann Arbor/1/66) and HA and NA gene segments from wild type influenza A and B strains. Representative 6:2 reassortants were generated for influenza A (H1N1, H3N2, H5N1, H6N1, H7N3 and H9N2) and for both the Victoria and Yamagata lineages of influenza B. The yield of infectious virus in the supernatant of transfected MDCK cells was 106 to 107 plaque forming units per ml by 5 to 7 days post-transfection.
This rescue system will enable efficient production of both influenza A and influenza B vaccines exclusively in MDCK cells and therefore provides a tool for influenza pandemic preparedness.
The type A and B influenza viruses have genomes consisting of eight negative-sense single-stranded viral RNAs (vRNAs), each of which contains a coding region and terminal 5' and 3' noncoding regions. Within the virion, the vRNAs are associated with nucleoprotein (NP) and the three polymerase subunits (PB1, PB2, PA) to form ribonucleoprotein (RNP) complexes. Upon infection of cells, the RNPs are released into the cytoplasm and subsequently enter the nucleus where replication of the vRNA results in the production of both mRNA and complementary RNA (cRNA), the template for synthesis of more vRNA. Errors generated in the viral genome during replication by a low fidelity viral RNA polymerase combined with the segmented arrangement of the influenza genome has resulted in the generation of reassortants in nature with new genetic characteristics. Natural influenza variants have emerged in the past for which humans have little to no immunity and world wide influenza pandemics have ensued.
One aspect of preparation for an influenza pandemic is to create adequate production facilities for vaccine manufacture. Although current production of influenza vaccines for human vaccination is in most cases an egg-base process, many vaccine manufacturers are actively developing cell-based influenza vaccine capabilities. Cell-based influenza vaccine production is potentially less susceptible to biological contamination and more adaptable to large scale production than current egg-based vaccine production. The ability to quickly increase the scale of influenza vaccine production is especially critical in response to an incipient pandemic influenza outbreak, where global vaccination could be an important defence against the development of a full blown pandemic. To this end, MDCK cells are being developed by many of the influenza vaccine manufacturers as a cell-substrate for influenza vaccine production because of the capacity for high virus yields of both A and B strains in this cell line.
In addition to the cell substrate used for influenza virus production, response time to pandemic influenza may be impacted by the need to generate a pandemic vaccine from properly constructed influenza reassortants. These may be reassortants with high growth properties for inactivated vaccine production or reassortants which carry influenza segments that confer attenuation to the resulting pandemic vaccine, either to function as a live vaccine or to reduce risk to production personnel when producing an inactivated vaccine. For any desired reassortant, influenza reverse genetics systems based on rescue of virus from transfected plasmids encoding the viral genome increases the quality, speed, accuracy and reliability of obtaining a desired reassortant as compared to classical reassortment techniques based on dual strain infections followed by selection. Currently, Vero cells are the only cell substrate licensed for plasmid-based rescue of human vaccines. Yet, efficient rescue in Vero cells is hampered by their low productivity for many influenza strains. This prompted us to develop an efficient influenza plasmid-based rescue system in MDCK cells. Although direct influenza A rescue in MDCK cells from a plasmid system based on a T7 RNA polymerase vector has been reported, this system has not been documented to work for type B influenza strains and is less efficient than the eight plasmid bidirectional system which utilizes cellular RNA polymerase I (pol I) and RNA pol II (pol II) to synthesize vRNA and viral mRNA, respectively, from plasmids transfected into susceptible cells . Although efficient rescue occurs in the bidirectional system, the species specificity of the RNA pol I promoter limits its utility to cells from species within the same taxonomic order . The result, which we have confirmed, is that the bidirectional human RNA pol I based plasmid rescue system which functions in primate cells such as Vero, does not support influenza rescue in MDCK cells (data not shown).
For these reasons, we chose to develop a bidirectional influenza reverse genetics system which utilizes a canine RNA pol I promoter derived from MDCK cells as a refinement of the human RNA pol I system developed by Hoffmann and co-workers .
Results and discussion
Cloning the canine RNA polymerase I promoter from MDCK DNA
In higher eukaryotes, ribosomal RNA (rRNA) genes are transcribed by RNA pol I into a large 45S pre-rRNA which is subsequently processed into mature 18S, 5.8S and 28S rRNAs. Although the mature rRNA sequences are conserved among higher eukaryotes, the 5' non-transcribed region directly upstream of the transcription start site, which contains the RNA pol I promoter and other regulatory sequence elements, has diverged significantly and is conserved only among species within the same taxonomic order [2, 4]. Functionally this results in the RNA pol I promoter from a species of one order not being recognized by the RNA pol I and transcription factors from species belonging to other orders. The currently described pol I promoters used in the rescue of recombinant influenza will function only in primate or avian cells [5–7]. In order to rescue influenza using the RNA pol I transcription machinery in canine cells, cloning the canine RNA pol I promoter was necessary.
In developing an approach to cloning the canine RNA pol I promoter, we examined the known features of the RNA pol I promoters and rRNA genes from other mammalian species. In the genomes of human, mouse and rat, the distance from the beginning of the 18S rRNA sequences to the RNA pol I transcription initiation site is 3676, 4026 and 4264 bp, respectively. A BLAST analysis of the pre-rRNA sequences upstream of the 18S rRNA of these species revealed no significant similarities among the sequences (data not shown). Functional assays using cloned regions of DNA have demonstrated that the region immediately upstream of a transcription initiation site has RNA pol I promoter activity in vitro and the sizes of these cloned promoter elements have been narrowed to 225 bp and 169 bp for human and mouse genomes, respectively [5, 8]. Based on these data, we hypothesized that a functional RNA pol I promoter may be present within a region from 3000 to 5000 bp upstream of the beginning of the canine 18S rRNA gene. Thus, our strategy for isolating the canine RNA pol I promoter was to clone a MDCK genomic DNA fragment that contained sequences which extended at least 5 kb upstream from the start of the 18S rRNA gene sequence. The resulting MDCK clone then would be tested for RNA pol I promoter activity in vitro.
In order to evaluate the presence of pol I promoter activity in cloned DNA fragments, an MDCK-based assay for replication of an artificial influenza vRNA containing a reporter gene was developed based on an analogous assay used to evaluate the human RNA pol I promoter . The DNA sequences to be tested for RNA pol I activity are cloned upstream of a negative sense reporter gene which has 5' and 3' terminal noncoding sequences derived from influenza vRNA. These noncoding regions of the vRNA in turn enable the antisense reporter transcript to be recognized by influenza replication proteins expressed by co-transfected plasmids and converted into a positive sense transcript which is subsequently translated into a reporter protein, such as enhanced green fluorescence protein (EGFP) or chloramphenicol acetyltransferase (CAT). Additionally, due to the nature of the influenza replication machinery, the transcription initiation and termination sites of this vRNA reporter are critical for functionality, addition of even one extra nucleotide at the 5' end of the negative sense vRNA abrogates the function of this molecule. Therefore, if no pol I promoter element is present in the cloned DNA fragment or if the transcription initiation site is not accurate, no vRNA will result and no reporter signal will be measured.
Generation of FluMist®strains from eight plasmids
FluMist is a licensed live attenuated influenza vaccine which currently contains two A virus strains (H1N1 and H3N2) and one influenza B strain. Each vaccine strain component of FluMist is a 6:2 reassortant, composed of the six internal gene segments (PB1, PB2, PA, NP, M, and NS) from an attenuated master donor virus (MDV) strain and the HA and NA gene segments from a wild type (wt) strain. The FluMist MDV strains are ca A/Ann Arbor/6/60 and ca B/Ann Arbor/1/66, originally developed by serial passage at successively reduced temperatures in primary chick kidney cells [11, 12].
Kinetics of MDVA and MDVB generation after transfectiona
Virus titer (pfu/ml)
2.8 × 104
1.5 × 103
2.2 × 102
2.8 × 107
6.6 × 105
9.8 × 104
1.3 × 108
2.3 × 107
5. 2 × 106
3.8 × 107
1.9 × 107
1.8 × 107
1.2 × 107
3.6 × 106
3. 2 × 106
1.2 × 107
2.6 × 106
3. 0 × 106
As a control to demonstrate the rescued virus was derived from the genomic sequences carried in the plasmids, MDV-B NS and PB1 genes containing the silent coding mutations NS (416 A/G) and PB1 (561 T/C, 924 A/G) were also inserted into pAD4000 and substituted for their MDV-B counterparts in an eight plasmid MDV-B mix. Electroporation of MDCK cells with this mix resulted in an accumulation of supernatant virus with kinetics and titer similar to that of the nonmutated MDV-B rescue (Table 1). Subsequent sequence analysis of supernatant virus from the MDV-B NS/PB1 mutant electroporation confirmed the presence of the mutations in the rescued virus and demonstrated that this virus was plasmid derived.
Plasimid Rescued influenza A & B Viruses In MDCK cellsa
Virus titer (pfu/ml)
ca A/New Caledonia/20/1999
7. 0 × 106
ca A/Solomon Island/3/2006
5.4 × 107
MDV-A (ca A/Ann Arbor/6/1960)
1.5 × 108
8.4 × 106
2.6 × 106
9. 0 × 106
ca A/Hong Kong/213/2003
1.3 × 107
ca A/Hong Kong/1997(491 H5/486 N1)
1.7 × 107
4.6 × 107
6.0 × 106
3.9 × 106
1.4 × 107
MDV-B (B/Ann Arbor/1/1966)
6.0 × 107
MDV-B (B/Ann Arbor/1/1966)-Mutant
1.5 × 107
ca B/Hong Kong/330/2001
1.7 × 107
1.6 × 107
5.3 × 107
2.6 × 107
To further test the MDCK based system, 6:2 reassortants were rescued in which the HA and NA were derived from isolates of the H6N1, H7N3 and H9N2 subtypes (Table 2). The wt virus which was the source of the HA and NA for the H6N1 reassortant was isolated from teal and contains seven segments (NA, PB1, PB2, PA, NP, NS and M) which are very similar to their counterparts in the human H5N1 virus A/Hong Kong/156/97 and it may represent a derivative or precursor of the H5N1 viruses . The H7N3 wt virus (A/CK/BC-CN/2004) exhibits low pathogenicity in avian species although it was isolated during an outbreak of a related highly pathogenic H7N3 strain which contained a multibasic amino acid insertion near the HA0 cleavage site . Finally, the H9N2 virus (A/chicken/HK/G9/1997) is representative of one of the three H9N2 subgroups which were isolated from poultry in Hong Kong during the 1997 H5N1 outbreak in humans .
Using the MDCK RNA pol I plasmid-based system, we have demonstrated the rescue of a wide variety of influenza A subtypes as well both lineages of influenza B. These results indicate that influenza reverse genetic performed exclusively in MDCK cells can efficiently result in the rescue of seasonal FluMist vaccine strains as well as prototype attenuated vaccines for wt strains which may harbor the potential for becoming pandemic. The application of the rescue system utilizing the MDCK RNA pol I promoter reported here is focused on the rescue of FluMist influenza vaccine strains in MDCK cells, although our expectation is that it should be applicable to other influenza strains too. As such, this refinement of the plasmid-based rescue system for influenza virus may be a useful tool in the development of vaccines as a response to an imminent pandemic.
Nucleic acid extraction and Southern hybridization
Total DNA and RNA was recovered from MDCK cells (passage 64, ATCC) using MasterPure™ DNA Purification Kit and MasterPure™ RNA Purification Kit, respectively, according to the manufacturer's instructions (Epicentre Biotechnologies). For Southern hybridization experiments, MDCK DNA (20 μg) was digested overnight at 37°C with the indicated restriction enzyme and subjected to electrophoresis on 0.7 % agarose gels. DNA was transferred to Hybond-N+ membranes (Amersham Corp.) and immobilized with a UV Crosslinker 10000 (Hoeffer Scientific Instruments).
Probe DNA was prepared by PCR amplification of sequences from the 5' end of the 18S rRNA gene using MDCK DNA as the template and forward and reverse primers (5'-CTTGTCTCAAAGATTAAGCCATGCATG-3' and 5'-CAGGGCCTCGAAAGAGTCCTGTATTG-3', respectively). The PCR products were labeled using a BrightStar Psoralen-Biotin Nonisotopic Labeling Kit according to the manufacturer's instructions (Ambion) and hybridizations were performed as described previously . Detection of hybridized probe DNA was performed using a BrightStar BioDetect TM Nonisotopic Detection Kit (Ambion).
Cloning MDCK DNA and plasmid construction
All cloning and PCR reactions were performed according to standard protocols. To clone the 7.1 kb MDCK EcoR I fragment which hybridized to 18S rRNA sequences, 100 μg of MDCK DNA was digested with 100 units of EcoR I overnight at 37°C and subjected to electrophoresis on a 0.7 % agarose gel along with a 1 kb ladder size marker. Using the marker as a guide, the 7 kb region of the EcoR I digested MDCK DNA lane was excised from the gel followed by recovery of the DNA from the gel sample. The recovered DNA was ligated to EcoR I digested pGEM 7 vector (Promega) and the ligation mixture was used to transform E. coli TOP10 cells (Invitrogen). DNA preparations from the resulting ampicillin resistant colonies were used as templates in PCR reactions containing the same forward and reverse primers to the canine 18S rRNA gene that were used to prepare the probe for the Southern hybridizations. PCR products then were analyzed on agarose gels to identify colonies which produced 500 bp products, the size predicted from the 18S rRNA gene sequence. One such clone, designated pK9PolI, was determined by nucleotide sequencing and restriction enzyme analysis to have a 7.1 kb insert which contained canine 18S rRNA sequences. Plasmid pK9Pol I EB was constructed by subcloning the 3.5 kb EcoRI BamHI fragment from the insert contained in pK9Pol I into pGEM 7.
Reporter plasmids pK9GFP 1–1802(T), pK9GFP 1–1803(G), and pK9GFP 1–1804(C) were derived from pHW72-EGFP  by replacing the human RNA pol I promoter sequences in pHW72-EGFP with bases 1–1802, 1–1803 and 1–1804, respectively, from the MDCK EcoRI BamHI insert in pK9Pol I EB. Reporter plasmid pK9CAT(1803) was derived from pK9GFP 1–1803(G) by replacing the EGFP gene with a CAT gene. The plasmids pK9CAT(469), pK9CAT(230), pK9CAT(88) and pK9CAT(77) were made by deleting, respectively, the MDCK sequences 1–1334, 1–1573, 1–1715 and 1–1726 from the 1803 bp MDCK-derived sequence in pK9CAT(1803), where position 1803 is at -1 with respect to the RNA pol I transcription start site. The plasmid pΔHW72-EGFP is a derivative of pHW72-EGFP in which the human pol I promoter has been deleted.
The plasmid for expression of influenza segments in MDCK cells, pAD4000, was derived from pAD3000  by replacing human RNA pol I promoter sequences in pAD3000 with the MDCK RNA pol I promoter sequences from pK9CAT (469). In addition, the cloning sites between the RNA pol I promoter and the RNA pol I terminator were changed from BsmB I-Kpn I-BsmB I (pAD3000) to Bbs I-Xho I-Bbs I (pAD4000).
Influenza segments, previously cloned into pAD3000 and shown to be virus rescue competent [3, 10, 19] were amplified with Accu Prime Pfx DNA Polymerase (Invitrogen) using forward and reverse primers containing, respectively, segment specific 5' or 3' sequences and a restriction site appropriate for cloning between the Bbs I sites of pAD4000. For influenza A strains, the restriction sites were BsmBI (PB1, PA, NP, M, and NS) or AarI (PB2, HA and NA). For influenza B strains, the restriction sites were BsmBI (PB1, PB2, PA, HA, NA, M, and NS) or AarI (NP). The HA and NA segments subcloned from pAD3000 into pAD4000 were originally derived from the following wt strains: A/New Caledonia/20/1999 (H1N1), A/Solomon Island/3/2006 (H1N1), A/Wisconsin/67/2005 (H3N2), A/California/7/2004 (H3N2), A/Panama/2007/1999 (H3N2), A/Hong Kong/213/2003 (H5N1), A/Hong Kong/1997(491 H5/486 N1), A/Vietnam/1203/2004 (H5N1), A/Teal/HK/W312/1997 (H6N1), A/BC-CN/04 (H7N3), A/chicken/HK/G9/1997 (H9N2), B/Malaysia/2506/2004, B/Jiangsu/10/2003, B/Hong Kong/330/2001 and B/Florida/07/2004.
Primer extension reactions and M13 sequencing
Primers for primer extention reactions (PrimerEx1: 5'-CGCGGCACGGAGCCTTGC-3' and PrimerEx2: 5'-GCCACCTGGAGTCCAGCA-3'), M13 (-40) sequencing primer and dephoshorylated, Hinf I digested φX174 size marker DNAs were labeled at their 5' ends with [γ-32P] ATP using T4 polynucleotide kinase for 30 min at 37°C. After labeling, 32P PrimerEx1 and 32P PrimerEx2 were used to direct cDNA synthesis from MDCK whole cell RNA utilizing a Primer Extension System-AMV Reverse Transcriptase kit (Promega). Primer extension products and 32P labeled φX174 size marker DNAs were subjected to electrophoresis on 6% polyacrylamide, 7 M urea gels followed by detection of the radioisotope in the gel with a BioRad Molecular Imager Fx. PrimerEx2 primer extension products were also subjected to electrophoresis adjacent to a M13 sequencing ladder on a 6% polyacrylamide, 7 M urea sequencing gel followed by imaging of the radioisotope in the gel. Sequencing reactions using 32P labeled M13 (-40) sequencing primer and M13mp18 ssDNA template were performed utilizing a Sequenase Version 2.0 DNA sequencing kit (USB).
Cell culture and transfection
MDCK cells were originally obtained from the American Type Culture Collection and were maintained in MEM supplemented with 10% fetal bovine serum (FBS). One day prior to electroporation, subconfluent MDCK cells were detached from their growth flasks by treatment with trypsin and seeded at 5 × 104 cells/cm2. The following day, cells were detached from their growth flask by treatment with trypsin, pelleted by centrifugation and resuspended in Opti MEM I (Invitrogen). For each electroporation, 5 × 106 cells were mixed with Opti-MEM I to a final volume of 300 ul in a 0.4 cm electroporation cuvette (BioRAD). DNA (3 μg of each individual plasmid in no more than 25 μl) was added to the cells in the cuvette followed by electroporation at 220 volts, 950 microFarads (BioRad Gene Pulser II). Opti-MEM I (0.7 ml) was added to the cuvette about 2 min after electroporation, gently mixed with the DNA-cell mixture and transferred to a well of a 6 well plate which contains 2 ml Opti-MEM I. After incubation at 33°C for 16–20 hrs, cells were washed with 2 ml of Opti-MEM I to remove unattached cell debris and overlayed with 2 ml of Opti-MEM I containing 1 ug/ml TPCK-trypsin. On days 2–7 post-electroporation, a 1 ml sample of media was collected from the electroporated cells and replaced with 1 ml Opti-MEM I containing 1 ug/ml TPCK-trypsin. Virus in the collected samples was titrated by plaque assay on MDCK cells followed by immunostaining with an influenza virus-specific polyclonal antibody and plaques were visualized using a secondary antibody conjugated with horseradish peroxidase.
Transfections using PromoFectin (PromoKine, Heidelberg, Germany) were performed according to the manufacturer's protocol with slight modification. Briefly, MDCK cells (1.5 × 105 cells/ml) in MEM supplemented with 10% FBS were seeded at 2 ml per well of a 6 well plate approximately 24 hrs before transfection. DNA (3 ug total) was diluted into 100 ul of Opti-MEM I. A mix composed of 8 ul of PromoFectin in 100 ul of Opti-MEM I was added to the diluted DNA and immediately mixed using a vortex mixer. After 30 min at room temperature, the PromoFectin/DNA mixture was added drop-wise to one well of MDCK cells (50 to 60% confluent) and the mixture was distributed by gently swirling. The plate then was subjected to centrifugation at 280 g for 5 min at room temperature and incubated at 33°C. After 4 to 5 hrs of incubation, the transfection mix was removed and the cells were washed with 2 ml/well Opti-MEM followed by the addition of 1.5 ml/well of Opti-MEM I containing 0.5 ug/ml TPCK-trypsin. Incubation was continued at 33°C with the addition 1 ml of Opti-MEM I containing 1 ug/ml TPCK-trypsin on days 2 and 4 after transfection. Microscopic examination of the transfected cells was performed daily to check for the appearance of CPE (characteristic virus induced cytopathic effect). The media from wells exhibiting 50 to 100% CPE (usually 5 -7 days after transfection) was collected and virus titrated by plaque assay on MDCK cells followed by immunostaining, as described above, to confirm the influenza virus infection.
Cell lysates were prepared from transfected cells approximately 44 hours post-transfection and assayed for the synthesis of CAT using CAT ELISA kits (Roche Applied Science) according to the manufacturer's protocol.
We thank Hong Jin, Bin Lu, Zhongying Chen and Helen Zhou for providing plasmid clones of influenza segments and Chin-Fen Yang for providing primers for sequencing. We thank Xing Cheng and Kathy Wang for helpful discussions, Qing Yan and Indira Kottayil for technical assistance and Yang He for supplying MDCK cells. We also thank George Kemble and Richard Spaete for reviewing the manuscript and providing helpful suggestions.
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