1887

Abstract

H5N1 avian influenza viruses demonstrate different phenotypes, such as pathogenicity after one or serial passages in mammalian hosts or cells. To establish the molecular basis of these phenotypes, we cloned isolates from the lungs of mice infected with human A/Vietnam/1194/2004 (H5N1) influenza virus. Large-plaque isolates were less pathogenic to mice than small-plaque isolates. Genome sequencing revealed that the small-plaque and large-plaque isolates differed in several amino acids. In order to assess their effects on pathogenicity in mice, two amino acid changes common to attenuated isolates, one in PB2 (I63T) and the other in PB1 (T677M), were inserted into a wild-type recombinant virus construct. The PB2 (I63T) or PB1 (T677M) mutations alone did not alter the phenotype of H5N1 virus, whereas recombinant virus with both mutations was less pathogenic than the wild-type recombinant virus. Furthermore, the PB1 (T677M) mutation showed a lower replication efficiency, although it had higher polymerase activity. The recombinant virus with the PB2 (63T) mutation replicated as well as the wild-type recombinant virus. These results suggest that the C terminus of PB1 of H5N1 influenza virus mediates virulence attenuation of H5N1 influenza virus in mice, associating with the N terminus of PB2. However, the role of the N terminus of PB2 in virulence attenuation in mice remains unclear.

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2011-06-01
2019-10-24
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Plaque formation assay of recombinant viruses. Confluent monolayers of MDCK cells were inoculated with 10-fold serial dilutions of recombinant viruses and overlaid with 1 % hypo-Tm-solved agarose. After 2 days of incubation at 35 °C, cells were stained with neutral red and plaque morphology was evaluated.

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Relative amounts of PB2 (a) and PB1 (b) in each group. Levels were determined by real-time quantitative PCR, as follows. At 24 h post-transfection with pHW2000 plasmid constructs encoding PB2 or PB2 , PB1 or PB1 , PA, NP and pPol1-NP-Luc (1 µg each) with Lipofectamine 2000 (as described in Methods), 293T cells were harvested and lysed for total RNA extraction. As described by Liang (2005), total RNA was extracted from transfected cells using a Qiagen RNeasy Mini kit according to the instructions of the manufacturer. Total RNA was cleared of plasmid DNA contamination by incubation for 30 min at 37 °C with DNase I, which was inactivated by incubation at 85 °C for 15 min. Reverse transcription was conducted using oligo(dT) for mRNA. A glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific primer (5′-GAAGATGGTGATGGGATTTC-3′) was also included in the reverse transcription reaction. Quantitative real-time PCR was carried out in a 20 µl reaction mixture containing primers specific for PB2 (5′-CCAAAGCTGCCAGAGGTCAA-3′ and 5′-GGGGCTGCTGCAAATGGTAG-3′) and PB1 (5′-CAGGGATCTTTGAAAACTCGTGTC-3′ and 5′-AGTTGCAGCCGGTTGGTTTCTATT-3′) or for GAPDH RNA (5′-GAAGGTGAAGGTCGGAGTC-3′ and 5′-GAAGATGGTGATGGGATTTC-3′) by using SYBR Premix Ex kit (Takara). The PCR conditions were 95 °C for 30 s, 40 cycles of 95 °C for 5 s, 60 °C for 20 s and 95 °C for 0 s followed by 65 °C for 15 s and 95 °C for 0 s. Levels of PB2 and PB1 RNA in each group were expressed relative to the level of GAPDH RNA.

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