Analysis of Recombinant H7N9 Wild-Type and Mutant Viruses in Pigs Shows that the Q226L Mutation in HA Is Important for Transmission

Abstract

The fact that there have been more than 300 human infections with a novel avian H7N9 virus in China indicates that this emerging strain has pandemic potential. Furthermore, many of the H7N9 viruses circulating in animal reservoirs contain putative mammalian signatures in the HA and PB2 genes that are believed to be important in the adaptation of other avian strains to humans. To date, the definitive roles of these mammalian-signature substitutions in transmission and pathogenesis of H7N9 viruses remain unclear. To address this we analyzed the biological characteristics, pathogenicity, and transmissibility of A/Anhui/ 1/2013 (H7N9) virus and variants in vitro and in vivo using a synthetically created wild-type virus (rAnhui-WT) and two mutants (rAnhui-HA-226Q and rAnhui-PB2-627E). All three viruses replicated in lungs of intratracheally inoculated pigs, yet nasal shedding was limited. The rAnhui-WT and rAnhui-PB2-627E viruses were transmitted to contact animals. In contrast, the rAnhui-HA-226Q virus was not transmitted to sentinel pigs. Deep sequencing of viruses from the lungs of infected pigs identified substitutions arising in the viral population (e.g., PB2-T271A, PB2-D701N, HA-V195I, and PB2-E627K reversion) that may enhance viral replication in pigs. Collectively, the results demonstrate that critical mutations (i.e., HA-Q226L) enable the H7N9 viruses to be transmitted in a mammalian host and suggest that the myriad H7N9 genotypes circulating in avian species in China and closely related strains (e.g., H7N7) have the potential for further adaptation to human or other mammalian hosts (e.g., pigs), leading to strains capable of sustained human-to-human transmission. IMPORTANCEThe genomes of the zoonotic avian H7N9 viruses emerging in China have mutations in critical genes (PB2-E627K and HA-Q226L) that may be important in their pandemic potential. This study shows that (i) HA-226L of zoonotic H7N9 strains is critical for binding the ␣-2,6-linked receptor and enables transmission in pigs; (ii) wild-type A/Anhui/1/2013 (H7N9) shows modest replication, virulence, and transmissibility in pigs, suggesting that it is not well adapted to the mammalian host; and (iii) both wild-type and variant H7N9 viruses rapidly develop additional mammalian-signature mutations in pigs, indicating that they represent an important potential intermediate host. This is the first study analyzing the phenotypic effects of specific mutations within the HA and PB2 genes of the novel H7N9 viruses created by reverse genetics in an important mammalian host model. Finally, this study illustrates that loss-of-function mutations can be used to effectively identify residues critical to zoonosis/transmission.

Reversion of Cold-Adapted Live Attenuated Influenza Vaccine into a Pathogenic Virus

“…We then synthesized the complete genome of FluMist/ pH1N1-P2 using a rapid gene synthesis technology described previously (48,49,54), cloned it into the bidirectional reverse-genetics vectors pBZ61A15 and pBZ61A18 (46,50), and rescued the recombinant virus rFluMist/pH1N1-P2. Based on the sequence analysis (Table 1), we assumed that rFluMist/pH1N1-P0 had a leucine at PA residue 366, as shown in the GenBank reference sequence, so we reintroduced PA-I366L into rFluMist/pH1N1-P2 and rescued the rFluMist/pH1N1-P0 virus.…”

“…For each virus, we subjected 1 of the 12-well plates to 33°C, 37°C, or 39°C. On each day (24,48,72, and 96 hpi), we collected supernatants from 3 wells at each temperature for virus titration. The inocula were back titrated.…”

“…cDNAs encoding the six backbone vRNAs (PB2, PB1, PA, NP, M, and NS) of caAA/60 were synthesized by enzymatic assembly of oligonucleotides approximately 60 bases in length using protocols described previously in the syntheses of H7N9 and bat influenza virus genomes (48,49). The synthetic genes were cloned into the modified bidirectional influenza virus reverse-genetics vectors pBZ61A18 (PB2, PB1, and PA) and pBZ61A15 (NP, M, and NS) using a recombination-based method (50) and transformed into Stella competent Escherichia coli cells (Clontech).…”

Pathogenicity and Transmissibility of Novel Reassortant H3N2 Influenza Viruses with 2009 Pandemic H1N1 Genes in Pigs

“…HA receptor specificity is another important factor that has been known to play a major role in influenza virus cross-species transmission (45)(46)(47). The swine H3 HA receptor binding site with the combination of 226V-228S is different from the HAs of most avian (226Q-228G) and human (226L-228S) influenza viruses and is present in 90% of North American triple-reassortant H3N2 influenza viruses in swine (48).…”

Genetic tuning of avian influenza A (H7N9) virus promotes viral fitness within different species

“…An updated biological feature analysis verified that this H7N9 virus of avian origin has an unusual affinity for attachment to human receptors in the upper and lower respiratory tracts [15,16]. In addition, HA-226L was experimentally shown to be critical for a-2,6-linked receptor binding, and it enabled H7N9 viral adaptation and transmission in a pig model [17,18]. Poultry, including chickens and quails, can be experimentally infected with the AH1 (H7N9) virus, which they then transmit efficiently and silently [19,20].…”