Pandemic Swine H1N1 Influenza Viruses with Almost Undetectable Neuraminidase Activity Are Not Transmitted via Aerosols in Ferrets and Are Inhibited by Human Mucus but Not Swine Mucus

Abstract

A balance between the functions of the influenza virus surface proteins hemagglutinin (HA) and neuraminidase (NA) is thought to be important for the transmission of viruses between humans. Here we describe two pandemic H1N1 viruses, A/swine/Virginia/1814-1/2012 and A/swine/Virginia/1814-2/2012 (pH1N1 low -1 and -2, respectively), that were isolated from swine symptomatic for influenza. The enzymatic activity of the NA of these viruses was almost undetectable, while the HA binding affinity for ␣2,6 sialic acids was greater than that of the highly homologous pH1N1 viruses A/swine/Pennsylvania/2436/2012 and A/swine/Minnesota/2499/2012 (pH1N1-1 and -2), which exhibited better-balanced HA and NA activities. The in vitro growth kinetics of pH1N1 low and pH1N1 viruses were similar, but aerosol transmission of pH1N1 low -1 was abrogated and transmission via direct contact in ferrets was significantly impaired compared to pH1N1-1, which transmitted by direct and aerosol contact. In normal human bronchial epithelial cells, pH1N1 low -1 was significantly inhibited by mucus but pH1N1-1 was not. In Madin-Darby canine kidney cell cultures overlaid with human or swine mucus, human mucus inhibited pH1N1 low -1 but swine mucus did not. These data show that the interaction between viruses and mucus may be an important factor in viral transmissibility and could be a barrier for interspecies transmission between humans and swine for influenza viruses. IMPORTANCEA balance between the functions of the influenza virus surface proteins hemagglutinin (HA) and neuraminidase (NA) is thought to be important for transmission of viruses from swine to humans. Here we show that a swine virus with extremely functionally mismatched HA and NAs (pH1N1 low -1) cannot transmit via aerosol in ferrets, while another highly homologous virus with HA and NAs that are better matched functionally (pH1N1-1) can transmit via aerosol. These viruses show similar growth kinetics in Madin-Darby canine kidney (MDCK) cells, but pH1N1 low -1 is significantly inhibited by mucus in normal human bronchial epithelial cells whereas pH1N1-1 is not. Further, human mucus could inhibit these viruses, but swine mucus could not. These data show that the interaction between viruses and mucus may be an important factor in viral transmissibility and could be a species barrier between humans and swine for influenza viruses. H emagglutinin (HA) and neuraminidase (NA), the surface glycoproteins of influenza virus, play vital roles in the virus life cycle. HA binds to sialic acids on the cell surface, initiating fusion of the cell and viral membranes. NA enzymatically cleaves sialic acids from glycans on the host cell surface, facilitating the release of budding progeny viruses (1, 2). Thus, HA and NA have opposing roles in the viral life cycle and both are required for viral replication. A functional balance between HA and NA is important for both efficient replication and respiratory droplet transmission in humans. Four pandemic human viruses, A/California/04/2009 (H1N1) (...

The Interaction between Respiratory Pathogens and Mucus

“…Experiments in vitro demonstrated that an overlay of human mucus can inhibit the infection of underlying MDCK cells by both swine- and human-origin viruses in vitro , whilst swine mucus does not appear to inhibit infection (Cohen et al, 2013; Zanin et al, 2015) (figure 3a and b). The interaction between influenza viruses and mucus may also be a factor in viral transmissibility, particularly in the context of NA enzymatic activity.…”

“…The interaction between influenza viruses and mucus may also be a factor in viral transmissibility, particularly in the context of NA enzymatic activity. Pandemic H1N1 viruses isolated in swine with almost undetectable NA enzymatic activity were significantly inhibited by mucus in NHBE cell cultures in vitro (Zanin et al, 2015). These viruses did not transmit via aerosols in ferrets and direct contact transmission was severely attenuated (Zanin et al, 2015).…”

“…Pandemic H1N1 viruses isolated in swine with almost undetectable NA enzymatic activity were significantly inhibited by mucus in NHBE cell cultures in vitro (Zanin et al, 2015). These viruses did not transmit via aerosols in ferrets and direct contact transmission was severely attenuated (Zanin et al, 2015). Similarly, viruses expressing NA proteins with short stalks that have decreased enzymatic activity against multivalent substrates such as those found on cells are inhibited by mucus in vitro (Castrucci and Kawaoka, 1993; Matsuoka et al, 2009).…”

Three-Dimensional Rotating Wall Vessel-Derived Cell Culture Models for Studying Virus-Host Interactions

“…Tight junction proteins are also key components of the epithelial barrier function and integrity that protect against viral infection and potentially influence the susceptibility of tissue to viral infection. Mucin production at mucosal epithelial sites additionally influences virus-host interactions at the epithelial barrier and ultimately impacts viral infection and transmission [5,6,7]. While some of these features may be present in conventional monolayer cell cultures, they often lack the polarity and other topographic features that are present in vivo.…”

Quantification of Influenza Neuraminidase Activity by Ultra-High Performance Liquid Chromatography and Isotope Dilution Mass Spectrometry

“…While swine to human transmission was observed in both precursor pH1N1 viruses containing the North American-origin NA, no secondary human cases were observed and pre-2009 isolates do not appear to be transmissible to ferrets via respiratory droplets. 28–31 Only after attaining the Eurasian-origin M segment and Eurasian-origin NA, of increased activity and similarity to HA, did the virus become transmissible. 28,32 A comprehensive study by Kobasa et al of N2 viruses isolated from 1957 to 1987 found a time-dependent drift in substrate specificity.…”