The Temperature-Sensitive and Attenuation Phenotypes Conferred by Mutations in the Influenza Virus PB2, PB1, and NP Genes Are Influenced by the Species of Origin of the PB2 Gene in Reassortant Viruses Derived from Influenza A/California/07/2009 and A/WSN/33 Viruses

Abstract

Live attenuated influenza vaccines in the United States are derived from a human virus that is temperature sensitive (ts), characterized by restricted (>100-fold) replication at 39°C. The ts genetic signature (ts sig) has been mapped to 5 loci in 3 genes: PB1 (391E, 581G, and 661T), PB2 (265S), and NP (34G). However, when transferred into avian and swine influenza viruses, only partial ts and attenuation phenotypes occur. ts sig was restricted >100-fold compared to that at 33°C. Reassortant CA07 ts sig viruses were generated with individual polymerase gene segments from WSN, and vice versa. Only ts sig viruses with a PB2 gene segment derived from WSN were restricted in replication >100-fold at 39°C. In ferrets, the CA07 ts sig virus replicated in the upper and lower respiratory tract, but the replication of a reassortant CA07 ts sig virus with a WSN PB2 gene was severely restricted in the lungs. Taken together, these data suggest that the origin of the PB2 gene segment influences the ts phenotype in vitro and attenuation in vivo. This could have implications for the design of novel live vaccines against animal origin influenza viruses. IMPORTANCELive attenuated influenza vaccines (LAIVs) on temperature-sensitive (ts) backbones derived from animal origin influenza viruses are being sought for use in the poultry and swine industries and to protect people against animal origin influenza. However, inserting the ts genetic signature from a licensed LAIV backbone fails to fully attenuate these viruses. Our data indicate this is associated with the presence of a PB2 gene segment derived from an avian influenza virus. We show that a reassortant 2009 pandemic H1N1 virus with the ts signature from a licensed LAIV donor virus is ts in vitro and attenuated in vivo when the PB2 gene is derived from a human origin virus but not from an avian virus. Our study provides information that could benefit the rational design of alternative LAIV backbones against animal origin influenza viruses.

Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis

“…S7 in the supplemental material). Interestingly, for those PB1 mutant versions, the nuclear import of PA protein was also delayed, which is consistent with the notion that PA and PB1 are imported into the nucleus as a complex (32, 83, 90, 91) (see Fig. S7).…”

Avian influenza viruses that cause highly virulent infections in humans exhibit distinct replicative properties in contrast to human H1N1 viruses

“…Therefore, a strain exhibiting high levels of replication at 33 o C may infect humans more easily. In a study focusing on temperature-sensitive mutants used for live attenuated vaccine production, Broadbent et al 51 compared the growth of lab-adapted H1N1, 2009 pandemic H1N1 (pH1N1) and HPAI H5N1. Endpoint titres were measured after 72 h of incubation in MDCK cells infected at a MOI of 0.01 and incubated at temperatures ranging from 33 o C to 42 o C. The lab-adapted H1N1 strain exhibited optimal growth at 37 o C with approximately one log reduction at 33 o C. Conversely, the pH1N1 strain showed optimal titers at 33 o C with one log reduction at 37 o C. The H5N1 strain showed similar titers at both temperatures.…”

“…Over 72 h, the human viruses showed similar growth at both 32 o C and 37 o C, the avian H5N3 grew well at 37 o C but did not grow at 32 o C and the H5N1 HPAI strain exhibited reduced growth at 32 o C compared to 37 o C. In differentiated primary human bronchial and tracheal epithelial cells (HTE) cultured in transwells under air-liquid interface, infection with sH1N1, HPAI H5N1 and novel H7N9 strains exhibited significant differences between the strains when grown at 33 and 37 o C 56 . The sH1N1 strain had identical growth at both temperatures while the H5N1 and, to a lesser extent the H7N9 showed reduced growth at 33 o C. All the studies mentioned above used traditional TCID 50 infectivity measure as their end-point and, in some cases 51 54 qRT-PCR methods. Solely using qRT-PCR, Kasloff et al 57 assessed the replication of 11 IAV.…”

Structural studies of influenza virus RNPs by electron microscopy indicate molecular contortions within NP supra-structures

“…For example, mutations that allowed the escape of viruses from cellular restriction factors and adaption to new hosts were mapped to NP (Brooke et al, 2014; Gotz et al, 2016; Ince et al, 2013). Moreover, NP can acquire temperature-sensitive and attenuating mutations, which now form the basis for commercial live-attenuated influenza vaccines (Broadbent et al, 2014; Murphy et al, 1982). Some drugs directly target NP oligomerization and can abrogate virus infection (Amorim et al, 2013; Gerritz et al, 2011; Kao et al, 2010).…”