Segment 2 from influenza A(H1N1)pdm09 viruses confers temperature sensitive HA yield on candidate vaccine virus growth in eggs that is complemented by PB2 701D

Candidate vaccine viruses (CVVs) for seasonal influenza A virus are made by reassortment of the antigenic virus with a high-yielding egg-adapted strain, typically A/Puerto Rico/8/34 (PR8). Many 2009 H1N1 pandemic (pdm09) high-growth reassortants (HGRs) selected by this process contain pdm09 segment 2 in addition to the antigenic genes. To investigate this, we made CVV mimics by reverse genetics (RG) that were either 6:2 or 5:3 reassortants between PR8 and two pdm09 strains, A/California/7/2009 (Cal7) and A/England/195/2009, differing in the source of segment 2. The 5:3 viruses replicated better in MDCK-SIAT1 cells than the 6:2 viruses, but the 6:2 CVVs gave higher HA antigen yields from eggs. This unexpected phenomenon reflected temperature sensitivity conferred by pdm09 segment 2, as HA yields from eggs for the 5:3 viruses improved substantially when viruses were grown at 35°C compared with 37.5°C, whereas 6:2 virus yield did not. Authentic 5:3 pdm09 HGRs, X-179A and X-181, were not markedly temperature-sensitive however, despite their PB1 sequences being identical to that of Cal7, suggestive of compensatory mutations elsewhere in the genome. Sequence comparisons of the PR8-derived backbone genes identified single changes in PB2 and NP, 5 in NS1, and 1 in NS2. PB2 N701D but not NP T130A affected the temperature dependency of viral transcription. Furthermore, introducing the PB2 701D change into a 5:3 CVV mimic improved and drastically reduced the temperature sensitivity of HA yield. We conclude that RG PR8 backbones used for vaccine manufacture in eggs should contain PB2 701D to maximise virus yield.


Introduction
R e s u l t s 1 3 and western blotting either before or after treatment with PNGaseF to remove 2 9 6 glycosylation. This gave the expected alternating pattern of slow and faster-migrating 2 9 7 HA polypeptide species ( Figure 3A, top row). The amount of HA 1 fluctuated between 2 9 8 samples but for both Cal7 and Eng195 reassortants, yield was generally higher from 2 9 9 viruses grown at 35°C than 37.5°C and highest from the 6:2 reassortants. To test the 3 0 0 reproducibility of this, de-glycosylated HA 1 was quantified from the western blots of

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To test to what extent the varying HA1 yields reflected difference in virus virus showed a significantly higher NP:HA 1 ratio than the 6:2 virus when grown at 3 1 8 37.5°C but not at 35°C ( Figure 3D). Therefore, the inclusion of the pdm09-derived Following the observation of temperature sensitivity of our four viruses, RG Cal7, RG Eng195, X-179A and X181, were identical (Table 2A).

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The HA polypeptides of the Cal7, X-179A and X-181 viruses were very similar,

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differing only with a T209K in the Cal7 sequence and a N129D substitution in the X- virus was temperature sensitive, giving significantly lower titres at 37.5°C ( Figure 6A).

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The  virus sample by TCID 50 assay and used to calculate genome copy:infectivity ratios,

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Altering the backbone of our PR8 strain to contain PB2 701D did not completely 4 4 9 convert the phenotype of our RG 5:3 CVV mimic to that of its closest authentic HGR 4 5 0 counterpart, X-179A in terms of growth in eggs ( Figure 6C). It may be that one or more

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However, any effects of these precise amino acid differences in NS1 and NS2 are not well documented.

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The exact mechanism of how PB2 N701D reduces temperature sensitivity of our     U. K. for their support during experiments performed in their lab. We also thank Dr.

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