Influenza virus PB1 and neuraminidase gene segments can cosegregate during vaccine reassortment driven by interactions in the PB1 coding region.

UNLABELLED: Egg-grown influenza vaccine yields are maximized by infection with a seed virus produced by "classical reassortment" of a seasonal isolate with a highly egg-adapted strain. Seed viruses are selected based on a high-growth phenotype and the presence of the seasonal hemagglutinin (HA) and neuraminidase (NA) surface antigens. Retrospective analysis of H3N2 vaccine seed viruses indicated that, unlike other internal proteins that were predominantly derived from the high-growth parent A/Puerto Rico/8/34 (PR8), the polymerase subunit PB1 could be derived from either parent depending on the seasonal strain. We have recently shown that A/Udorn/307/72 (Udorn) models a seasonal isolate that yields reassortants bearing the seasonal PB1 gene. This is despite the fact that the reverse genetics-derived virus that includes Udorn PB1 with Udorn HA and NA on a PR8 background has inferior growth compared to the corresponding virus with PR8 PB1. Here we use competitive plasmid transfections to investigate the mechanisms driving selection of a less fit virus and show that the Udorn PB1 gene segment cosegregates with the Udorn NA gene segment. Analysis of chimeric PB1 genes revealed that the coselection of NA and PB1 segments was not directed through the previously identified packaging sequences but through interactions involving the internal coding region of the PB1 gene. This study identifies associations between viral genes that can direct selection in classical reassortment for vaccine production and which may also be of relevance to the gene constellations observed in past antigenic shift events where creation of a pandemic virus has involved reassortment. IMPORTANCE: Influenza vaccine must be produced and administered in a timely manner in order to provide protection during the winter season, and poor-growing vaccine seed viruses can compromise this process. To maximize vaccine yields, manufacturers create hybrid influenza viruses with gene segments encoding the surface antigens from a seasonal virus isolate, important for immunity, and others from a virus with high growth properties. This involves coinfection of cells with both parent viruses and selection of dominant progeny bearing the seasonal antigens. We show that this method of creating hybrid viruses does not necessarily select for the best yielding virus because preferential pairing of gene segments when progeny viruses are produced determines the genetic makeup of the hybrids. This not only has implications for how hybrid viruses are selected for vaccine production but also sheds light on what drives and limits hybrid gene combinations that arise in nature, leading to pandemics.

The source of the PB1 gene in influenza vaccine reassortants selectively alters the hemagglutinin content of the resulting seed virus.

The yields of egg-grown influenza vaccines are maximized by the production of a seed strain using a reassortment of the seasonal influenza virus isolate with a highly egg-adapted strain. The seed virus is selected based on high yields of viral hemagglutinin (HA) and expression of the surface antigens from the seasonal isolate. The remaining proteins are usually derived from the high-growth parent. However, a retrospective analysis of vaccine seeds revealed that the seasonal PB1 gene was selected in more than 50% of reassortment events. Using the model seasonal H3N2 virus A/Udorn/307/72 (Udorn) virus and the high-growth A/Puerto Rico/8/34 (PR8) virus, we assessed the influence of the source of the PB1 gene on virus growth and vaccine yield. Classical reassortment of these two strains led to the selection of viruses that predominantly had the Udorn PB1 gene. The presence of Udorn PB1 in the seed virus, however, did not result in higher yields of virus or HA compared to the yields in the corresponding seed virus with PR8 PB1. The 8-fold-fewer virions produced with the seed virus containing the Udorn PB1 were somewhat compensated for by a 4-fold increase in HA per virion. A higher HA/nucleoprotein (NP) ratio was found in past vaccine preparations when the seasonal PB1 was present, also indicative of a higher HA density in these vaccine viruses. As the HA viral RNA (vRNA) and mRNA levels in infected cells were similar, we propose that PB1 selectively alters the translation of viral mRNA. This study helps to explain the variability of vaccine seeds with respect to HA yield.

A mouse model for the study of contact-dependent transmission of influenza A virus and the factors that govern transmissibility.

Influenza A virus transmission by direct contact is not well characterized. Here, we describe a mouse model for investigation of factors regulating contact-dependent transmission. Strains within the H3N2 but not H1N1 subtype of influenza virus were transmissible, and reverse-engineered viruses representing hybrids of these subtypes showed that the viral hemagglutinin is a determinant of the transmissible phenotype. Transmission to contact mice occurred within the first 6 to 54 h after cohousing with directly infected index mice, and the proportion of contacts infected within this period was reduced if the index mice had been preinfected with a heterologous subtype virus. A threshold level of virus present in the saliva of the index mice was identified, above which the likelihood of transmission was greatly increased. There was no correlation with transmission and viral loads in the nose or lung. This model could be useful for preclinical evaluation of antiviral and vaccine efficacy in combating contact-dependent transmission of influenza.

Influenza Virus Neuraminidase Structure and Functions.

With the constant threat of emergence of a novel influenza virus pandemic, there must be continued evaluation of the molecular mechanisms that contribute to virulence. Although the influenza A virus surface glycoprotein neuraminidase (NA) has been studied mainly in the context of its role in viral release from cells, accumulating evidence suggests it plays an important, multifunctional role in virus infection and fitness. This review investigates the various structural features of NA, linking these with functional outcomes in viral replication. The contribution of evolving NA activity to viral attachment, entry and release of virions from infected cells, and maintenance of functional balance with the viral hemagglutinin are also discussed. Greater insight into the role of this important antiviral drug target is warranted.

Recombinant influenza virus expressing HIV-1 p24 capsid protein induces mucosal HIV-specific CD8 T-cell responses.

Influenza viruses are promising mucosal vaccine vectors for HIV but their use has been limited by difficulties in engineering the expression of large amounts of foreign protein. We developed recombinant influenza viruses incorporating the HIV-1 p24 gag capsid into the NS-segment of PR8 (H1N1) and X31 (H3N2) influenza viruses with the use of multiple 2A ribosomal skip sequences. Despite the insertion of a sizable HIV-1 gene into the influenza genome, recombinant viruses were readily rescued to high titers. Intracellular expression of p24 capsid was confirmed by in vitro infection assays. The recombinant influenza viruses were subsequently tested as mucosal vaccines in BALB/c mice. Recombinant viruses were attenuated and safe in immunized mice. Systemic and mucosal HIV-specific CD8 T-cell responses were elicited in mice that were immunized via intranasal route with a prime-boost regimen. Isolated HIV-specific CD8 T-cells displayed polyfunctional cytokine and degranulation profiles. Mice boosted via intravaginal route induced recall responses from the distal lung mucosa and developed heightened HIV-specific CD8 T-cell responses in the vaginal mucosa. These findings demonstrate the potential utility of recombinant influenza viruses as vaccines for mucosal immunity against HIV-1 infection.