Filament-producing mutants of influenza A/Puerto Rico/8/1934 (H1N1) virus have higher neuraminidase activities than the spherical wild-type.

Influenza virus exhibits two morphologies - spherical and filamentous. Strains that have been grown extensively in laboratory substrates are comprised predominantly of spherical virions while clinical or low passage isolates produce a mixture of spheres and filamentous virions of varying lengths. The filamentous morphology can be lost upon continued passage in embryonated chicken eggs, a common laboratory substrate for influenza viruses. The fact that the filamentous morphology is maintained in nature but lost in favor of a spherical morphology in ovo suggests that filaments confer a selective advantage within the infected host that is not necessary for growth in laboratory substrates. Indeed, we have recently shown that filament-producing variant viruses are selected upon passage of the spherical laboratory strain A/Puerto Rico/8/1934 (H1N1) [PR8] in guinea pigs. Toward determining the nature of the selective advantage conferred by filaments, we sought to identify functional differences between spherical and filamentous particles. We compared the wild-type PR8 virus to two previously characterized recombinant PR8 viruses in which single point mutations within M1 confer a filamentous morphology. Our results indicate that these filamentous PR8 mutants have higher neuraminidase activities than the spherical PR8 virus. Conversely, no differences were observed in HAU:PFU or HAU:RNA ratios, binding avidity, sensitivity to immune serum in hemagglutination inhibition assays, or virion stability at elevated temperatures. Based on these results, we propose that the pleomorphic nature of influenza virus particles is important for the optimization of neuraminidase functions in vivo.

Residue 41 of the Eurasian avian-like swine influenza a virus matrix protein modulates virion filament length and efficiency of contact transmission.

UNLABELLED: Position 41 of the influenza A virus matrix protein encodes a highly conserved alanine in human and avian lineages. Nonetheless, strains of the Eurasian avian-like swine (Easw) lineage contain a change at this position: position 41 of A/swine/Spain/53207/04 (H1N1) (SPN04) encodes a proline. To assess the impact of this naturally occurring polymorphism on viral fitness, we utilized reverse genetics to produce recombinant viruses encoding wild-type M1 41P (rSPN04-P) and consensus 41A (rSPN04-A) residues. Relative to rSPN04-A, rSPN04-P virus displayed reduced growth in vitro. In the guinea pig model, rSPN04-P was transmitted to fewer contact animals than rSPN04-A and failed to infect guinea pigs that received a low-dose inoculum. Moreover, the P41A change altered virion morphology, reducing the number and length of filamentous virions, as well as reducing the neuraminidase activity of virions. The lab-adapted human isolate, A/PR/8/34 (H1N1) (PR8), is nontransmissible in the guinea pig model, making it a useful background in which to identify certain viral factors that enhance transmissibility. We assessed transmission in the context of single-, double-, and triple-reassortant viruses between PR8 and SPN04; PR8/SPN04 M, PR8/SPN04 M+NA, and PR8/SPN04 M+NA+HA, encoding either matrix 41 A or P, were generated. In each case, the virus possessing 41P transmitted less well than the corresponding 41A-encoding virus. In summary, we have identified a naturally occurring mutation in the influenza A virus matrix protein that impacts transmission efficiency and can alter virion morphology and neuraminidase activity. IMPORTANCE: We have developed a practical model for examining the genetics underlying transmissibility of the Eurasian avian-like swine lineage viruses, which contributed M and NA segments to the 2009 pandemic strain. Here, we use our system to investigate the impact on viral fitness of a naturally occurring polymorphism at matrix (M1) position 41 in an Easw isolate. Position 41 has been implicated previously in adaptation to laboratory substrates and to mice. Here we show that the polymorphism at M1 41 has a limited effect on growth in vitro but changes the morphology of the virus and impacts growth and transmission in the guinea pig model.

Spherical influenza viruses have a fitness advantage in embryonated eggs, while filament-producing strains are selected in vivo.

Influenza viruses can take on two distinct morphologies: filamentous or spherical. While the functional significance of each virion type is unclear, filaments are generally observed in low-passage-number isolates, while an exclusively spherical morphology is seen in strains grown extensively in laboratory substrates. Previous studies have shown that filamentous morphology is lost upon passage in eggs. The fact that the filamentous morphology is maintained in nature but not in the laboratory suggests that filaments provide an advantage in the host that is not necessary for growth in laboratory substrates. To test this hypothesis and identify naturally occurring mutations that alter morphology, we examined the effect of serial adaptation in eggs, MDCK cells, and guinea pigs. Two filamentous strains, A/Netherlands/602/2009 (H1N1) and A/Georgia/M5081/2012 (H1N1), were passaged in eggs and MDCK cells. Conversely, the spherical laboratory strain A/Puerto Rico/8/1934 (H1N1) was passaged in guinea pigs. We found that although passage in eggs and MDCK cells can lead to a loss of filaments, an exclusively spherical morphology is not required for highly efficient growth in either substrate. We did, however, identify two point mutations in the matrix of egg passage 10 isolates that confer spherical morphology and increased growth in eggs. In contrast, serial passage in guinea pigs resulted in the selection of filament-forming variants. Sequencing revealed point mutations to the PR8 matrix that, when introduced individually, yielded filaments. These findings suggest a functional role for filaments in the infected host and expand the breadth of mutations known to affect influenza virus shape.

Characterization of virulence-associated determinants in the envelope glycoprotein of Pichinde virus.

We use a small animal model, based on guinea pigs infected with a non-pathogenic Pichinde virus (PICV), to understand the virulence mechanisms of arenavirus infections in the hosts. PICV P2 strain causes a mild febrile reaction in guinea pigs, while P18 causes severe disease with clinical and pathological features reminiscent of Lassa hemorrhagic fever in humans. The envelope glycoproteins (GPC) of P2 and P18 viruses differ at positions 119, 140, and 164, all localized to the receptor-binding G1 subunit. We found that lentiviral pseudotyped virions (VLPs) bearing P18 GPC show more efficient cell entry than those with P2 GPC, and that the E140 residue plays a critical role in this process. Infection of guinea pigs with the recombinant viruses containing the E140K change demonstrated that E140 of GPC is a necessary virulence determinant of P18 infections, possibly by enhancing the ability of virus to enter target cells.