Comparative In Vitro and In Vivo Analysis of H1N1 and H1N2 Variant Influenza Viruses Isolated from Humans between 2011 and 2016

Abstract

Influenza A virus pandemics are rare events caused by novel viruses which have the ability to spread in susceptible human populations. With respect to H1 subtype viruses, swine H1N1 and H1N2 viruses occasionally cross the species barrier to cause human infection. Recently isolated from humans (termed variants), swine viruses were shown to display great genetic and antigenic diversity, hence posing considerable public health risk. Here, we utilized in vitro and in vivo approaches to provide characterization of H1 subtype variant viruses isolated since the 2009 pandemic and discuss the findings in context with previously studied H1 subtype human isolates. The variant viruses were well adapted to replicate in the human respiratory cell line Calu-3 and the respiratory tracts of mice and ferrets. However, with respect to hemagglutinin (HA) activation pH, the variant viruses had fusion pH thresholds closer to that of most classical swine and triple-reassortant H1 isolates rather than viruses that had adapted to humans. Consistent with previous observations for swine isolates, the tested variant viruses were capable of efficient transmission between cohoused ferrets but could transmit via respiratory droplets to differing degrees. Overall, this investigation demonstrates that swine H1 viruses that infected humans possess adaptations required for robust replication and, in some cases, efficient respiratory droplet transmission in a mammalian model and therefore need to be closely monitored for additional molecular changes that could facilitate transmission among humans. This work highlights the need for risk assessments of emerging H1 viruses as they continue to evolve and cause human infections.IMPORTANCE Influenza A virus is a continuously evolving respiratory pathogen. Endemic in swine, H1 and H3 subtype viruses sporadically cause human infections. As each zoonotic infection represents an opportunity for human adaptation, the emergence of a transmissible influenza virus to which there is little or no preexisting immunity is an ongoing threat to public health. Recently isolated variant H1 subtype viruses were shown to display extensive genetic diversity and in many instances were antigenically distinct from seasonal vaccine strains. In this study, we provide characterization of representative H1N1v and H1N2v viruses isolated since the 2009 pandemic. Our results show that although recent variant H1 viruses possess some adaptation markers of concern, these viruses have not fully adapted to humans and require further adaptation to present a pandemic threat. This investigation highlights the need for close monitoring of emerging variant influenza viruses for molecular changes that could facilitate efficient transmission among humans.

Keywords: H1N1; H1N2; ferret; influenza; pathogenesis; risk assessment; variant virus.

FIG 1

Replication kinetics of H1 influenza viruses in polarized human airway epithelial cells. Calu-3 cells grown on Transwell inserts were inoculated apically with virus at an MOI of 0.01. The cells were incubated at 33°C or 37°C, and culture supernatants were collected at 2, 24, 48, and 72 h postinoculation for viral titer determination by standard plaque assay. (A) Mean peak viral titers are shown as log₁₀ PFU/milliliter ± standard deviation (SD). Hours postinoculation of peak titers are shown parenthetically. (B and C) Replication kinetics curves at 33°C (B) or 37°C (C) are expressed as mean titers from three time courses and are expressed as log₁₀ PFU/milliliter ± SD. The limit of detection is 10 PFU. Statistical analysis is included in Tables S1 and S2 in the supplemental material.

FIG 2

Pathogenicity of H1 influenza viruses in mice. (A) Mice were intranasally inoculated with 5.0 log₁₀ PFU of virus, and then lung and nose tissues were collected on day 3 (n = 3) and day 6 (n = 3) p.i. for virus titer determination by plaque assay. Mean titers are expressed as log₁₀ PFU/milliliter ± SD. The limit of detection is 10 PFU. (B) The remaining mice were monitored for signs of morbidity and mortality for 14 days p.i. (n = 4 for CA/07, n = 5 for all other viruses); percent survival is shown. Statistical analysis is included in Table S3 in the supplemental material.

FIG 3

Transmissibility of H1N2v influenza viruses in ferrets. Ferrets were inoculated with 6.0 log₁₀ PFU of A/Minnesota/45/2016, A/Minnesota/19/2011 or A/Wisconsin/71/2016. After 24 h, the direct-contact transmission (DCT) model was used by adding a naive ferret to each cage housing an inoculated ferret (3 ferret pairs per virus) (A), and the respiratory droplet transmission (RDT) model was used by placing a naive ferret in an adjacent cage (3 ferret pairs per virus) (B). Virus titers in nasal wash samples collected from individual inoculated ferrets are shown on the left side of each panel, while those from individual contact ferrets are shown on the right side of each panel. The limit of detection is 10 PFU.

FIG 4

Detection of H1N2v influenza viruses in tissues of ferrets. Viral titers in tissues from individual ferrets on day 3 following inoculation with 6.0 log₁₀ PFU of A/Minnesota/45/2016 (A), A/Minnesota/19/2011 (B), and A/Wisconsin/71/2016 (C). Blood and nasal turbinate (Nasal Tur) viral titers are presented as log₁₀ PFU/milliliter, and kidney, spleen, liver, intestines (pooled duodenum, jejuno-ileal loop, and descending colon), olfactory bulb (BnOB), brain (pooled anterior and posterior brain), lungs, and trachea are presented as log₁₀ PFU/gram of tissue. The limit of detection is 10 PFU.

FIG 5

HA activation pH of selected H1 viruses. HA activation was determined by syncytium formation in infected Vero cells exposed to pH 4.8 to 7.4 fusion buffers increasing by 0.1 increments. The means and ranges from 3 or 4 independent experiments are shown. Variant H1 viruses: A/New Jersey/08/1976, A/Ohio/02/2007, A/Texas/14/2008, A/Minnesota/45/2016, A/Ohio/09/2015, A/Iowa/39/2015, A/Minnesota/19/2011, A/Wisconsin/71/2016. H1N1pdm09 viruses: A/California/07/2009, A/Texas/15/2009. Human seasonal H1N1 virus: A/Brisbane/59/2007.