Eefje J. A. Schrauwen

Airborne Transmission of Influenza A/H5N1 Virus Between Ferrets

Avian flu can acquire the capacity for airborne transmission between mammals without recombination in an intermediate host. Highly pathogenic avian influenza A/H5N1 virus can cause morbidity and mortality in humans but thus far has not acquired the ability to be transmitted by aerosol or respiratory droplet (“airborne transmission”) between humans. To address the concern that the virus could acquire this ability under natural conditions, we genetically modified A/H5N1 virus by site-directed mutagenesis and subsequent serial passage in ferrets. The genetically modified A/H5N1 virus acquired mutations during passage in ferrets, ultimately becoming airborne transmissible in ferrets. None of the recipient ferrets died after airborne infection with the mutant A/H5N1 viruses. Four amino acid substitutions in the host receptor-binding protein hemagglutinin, and one in the polymerase complex protein basic polymerase 2, were consistently present in airborne-transmitted viruses. The transmissible viruses were sensitive to the antiviral drug oseltamivir and reacted well with antisera raised against H5 influenza vaccine strains. Thus, avian A/H5N1 influenza viruses can acquire the capacity for airborne transmission between mammals without recombination in an intermediate host and therefore constitute a risk for human pandemic influenza.

Pathogenesis and transmission of swine-origin 2009 A(H1N1) influenza virus in ferrets

“Swine Flu” Pathology The clinical spectrum of disease caused by the swine-origin 2009 A(H1N1) influenza virus and its transmissibility are not completely understood. Munster et al. (p. 481; published online 2 July) and Maines et al. (p. 484; published online 2 July) used ferrets, an established model for human influenza, to evaluate the pathogenesis and transmissibility of a selection of 2009 A(H1N1) virus isolates compared with representative seasonal H1N1 viruses. The results help explain the atypical symptoms seen so far, including the gastrointestinal distress and vomiting observed in many patients. Although results were variable, it seems that the 2009 A(H1N1) virus may be less efficiently transmitted by respiratory droplets in comparison to the highly transmissible seasonal H1N1 virus, suggesting that additional virus adaptation in mammals may be required before we see phenotypes observed in earlier pandemics. Animal experiments compare the dynamics and effects of the virus causing the 2009 flu outbreak to those of seasonal H1N1 flu. The swine-origin A(H1N1) influenza virus that has emerged in humans in early 2009 has raised concerns about pandemic developments. In a ferret pathogenesis and transmission model, the 2009 A(H1N1) influenza virus was found to be more pathogenic than a seasonal A(H1N1) virus, with more extensive virus replication occurring in the respiratory tract. Replication of seasonal A(H1N1) virus was confined to the nasal cavity of ferrets, but the 2009 A(H1N1) influenza virus also replicated in the trachea, bronchi, and bronchioles. Virus shedding was more abundant from the upper respiratory tract for 2009 A(H1N1) influenza virus as compared with seasonal virus, and transmission via aerosol or respiratory droplets was equally efficient. These data suggest that the 2009 A(H1N1) influenza virus has the ability to persist in the human population, potentially with more severe clinical consequences.

The Potential for Respiratory Droplet–Transmissible A/H5N1 Influenza Virus to Evolve in a Mammalian Host

Some natural influenza viruses need only three amino acid substitutions to acquire airborne transmissibility between mammals. Avian A/H5N1 influenza viruses pose a pandemic threat. As few as five amino acid substitutions, or four with reassortment, might be sufficient for mammal-to-mammal transmission through respiratory droplets. From surveillance data, we found that two of these substitutions are common in A/H5N1 viruses, and thus, some viruses might require only three additional substitutions to become transmissible via respiratory droplets between mammals. We used a mathematical model of within-host virus evolution to study factors that could increase and decrease the probability of the remaining substitutions evolving after the virus has infected a mammalian host. These factors, combined with the presence of some of these substitutions in circulating strains, make a virus evolving in nature a potentially serious threat. These results highlight critical areas in which more data are needed for assessing, and potentially averting, this threat.

Virulence-Associated Substitution D222G in the Hemagglutinin of 2009 Pandemic Influenza A(H1N1) Virus Affects Receptor Binding

ABSTRACT The clinical impact of the 2009 pandemic influenza A(H1N1) virus (pdmH1N1) has been relatively low. However, amino acid substitution D222G in the hemagglutinin of pdmH1N1 has been associated with cases of severe disease and fatalities. D222G was introduced in a prototype pdmH1N1 by reverse genetics, and the effect on virus receptor binding, replication, antigenic properties, and pathogenesis and transmission in animal models was investigated. pdmH1N1 with D222G caused ocular disease in mice without further indications of enhanced virulence in mice and ferrets. pdmH1N1 with D222G retained transmissibility via aerosols or respiratory droplets in ferrets and guinea pigs. The virus displayed changes in attachment to human respiratory tissues in vitro, in particular increased binding to macrophages and type II pneumocytes in the alveoli and to tracheal and bronchial submucosal glands. Virus attachment studies further indicated that pdmH1N1 with D222G acquired dual receptor specificity for complex α2,3- and α2,6-linked sialic acids. Molecular dynamics modeling of the hemagglutinin structure provided an explanation for the retention of α2,6 binding. Altered receptor specificity of the virus with D222G thus affected interaction with cells of the human lower respiratory tract, possibly explaining the observed association with enhanced disease in humans.

Limited airborne transmission of H7N9 influenza A virus between ferrets

Wild waterfowl form the main reservoir of influenza A viruses, from which transmission occurs directly or indirectly to various secondary hosts, including humans. Direct avian-to-human transmission has been observed for viruses of subtypes A(H5N1), A(H7N2), A(H7N3), A(H7N7), A(H9N2) and A(H10N7) upon human exposure to poultry, but a lack of sustained human-to-human transmission has prevented these viruses from causing new pandemics. Recently, avian A(H7N9) viruses were transmitted to humans, causing severe respiratory disease and deaths in China. Because transmission via respiratory droplets and aerosols (hereafter referred to as airborne transmission) is the main route for efficient transmission between humans, it is important to gain an insight into airborne transmission of the A(H7N9) virus. Here we show that although the A/Anhui/1/2013 A(H7N9) virus harbours determinants associated with human adaptation and transmissibility between mammals, its airborne transmissibility in ferrets is limited, and it is intermediate between that of typical human and avian influenza viruses. Multiple A(H7N9) virus genetic variants were transmitted. Upon ferret passage, variants with higher avian receptor binding, higher pH of fusion, and lower thermostability were selected, potentially resulting in reduced transmissibility. This A(H7N9) virus outbreak highlights the need for increased understanding of the determinants of efficient airborne transmission of avian influenza viruses between mammals.

Identification, Characterization, and Natural Selection of Mutations Driving Airborne Transmission of A/H5N1 Virus

Recently, A/H5N1 influenza viruses were shown to acquire airborne transmissibility between ferrets upon targeted mutagenesis and virus passage. The critical genetic changes in airborne A/Indonesia/5/05 were not yet identified. Here, five substitutions proved to be sufficient to determine this airborne transmission phenotype. Substitutions in PB1 and PB2 collectively caused enhanced transcription and virus replication. One substitution increased HA thermostability and lowered the pH of membrane fusion. Two substitutions independently changed HA binding preference from α2,3-linked to α2,6-linked sialic acid receptors. The loss of a glycosylation site in HA enhanced overall binding to receptors. The acquired substitutions emerged early during ferret passage as minor variants and became dominant rapidly. Identification of substitutions that are essential for airborne transmission of avian influenza viruses between ferrets and their associated phenotypes advances our fundamental understanding of virus transmission and will increase the value of future surveillance programs and public health risk assessments.

Introduction of Virulence Markers in PB2 of Pandemic Swine-Origin Influenza Virus Does Not Result in Enhanced Virulence or Transmission

ABSTRACT In the first 6 months of the H1N1 swine-origin influenza virus (S-OIV) pandemic, the vast majority of infections were relatively mild. It has been postulated that mutations in the viral genome could result in more virulent viruses, leading to a more severe pandemic. Mutations E627K and D701N in the PB2 protein have previously been identified as determinants of avian and pandemic influenza virus virulence in mammals. These mutations were absent in S-OIVs detected early in the 2009 pandemic. Here, using reverse genetics, mutations E627K, D701N, and E677G were introduced into the prototype S-OIV A/Netherlands/602/2009, and their effects on virus replication, virulence, and transmission were investigated. Mutations E627K and D701N caused increased reporter gene expression driven by the S-OIV polymerase complex. None of the three mutations affected virus replication in vitro. The mutations had no major impact on virus replication in the respiratory tracts of mice and ferrets or on pathogenesis. All three mutant viruses were transmitted via aerosols or respiratory droplets in ferrets. Thus, the impact of key known virulence markers in PB2 in the context of current S-OIVs was surprisingly small. This study does not exclude the possibility of emergence of S-OIVs with other virulence-associated mutations in the future. We conclude that surveillance studies aimed at detecting S-OIVs with increased virulence or transmission should not rely solely on virulence markers identified in the past but should include detailed characterization of virus phenotypes, guided by genetic signatures of viruses detected in severe cases of disease in humans.

The Multibasic Cleavage Site in H5N1 Virus Is Critical for Systemic Spread along the Olfactory and Hematogenous Routes in Ferrets

ABSTRACT The route by which highly pathogenic avian influenza (HPAI) H5N1 virus spreads systemically, including the central nervous system (CNS), is largely unknown in mammals. Especially, the olfactory route, which could be a route of entry into the CNS, has not been studied in detail. Although the multibasic cleavage site (MBCS) in the hemagglutinin (HA) of HPAI H5N1 viruses is a major determinant of systemic spread in poultry, the association between the MBCS and systemic spread in mammals is less clear. Here we determined the virus distribution of HPAI H5N1 virus in ferrets in time and space—including along the olfactory route—and the role of the MBCS in systemic replication. Intranasal inoculation with wild-type H5N1 virus revealed extensive replication in the olfactory mucosa, from which it spread to the olfactory bulb and the rest of the CNS, including the cerebrospinal fluid (CSF). Virus spread to the heart, liver, pancreas, and colon was also detected, indicating hematogenous spread. Ferrets inoculated intranasally with H5N1 virus lacking an MBCS demonstrated respiratory tract infection only. In conclusion, HPAI H5N1 virus can spread systemically via two different routes, olfactory and hematogenous, in ferrets. This systemic spread was dependent on the presence of the MBCS in HA.

Host adaptation and transmission of influenza A viruses in mammals

A wide range of influenza A viruses of pigs and birds have infected humans in the last decade, sometimes with severe clinical consequences. Each of these so-called zoonotic infections provides an opportunity for virus adaptation to the new host. Fortunately, most of these human infections do not yield viruses with the ability of sustained human-to-human transmission. However, animal influenza viruses have acquired the ability of sustained transmission between humans to cause pandemics on rare occasions in the past, and therefore, influenza virus zoonoses continue to represent threats to public health. Numerous recent studies have shed new light on the mechanisms of adaptation and transmission of avian and swine influenza A viruses in mammals. In particular, several studies provided insights into the genetic and phenotypic traits of influenza A viruses that may determine airborne transmission. Here, we summarize recent studies on molecular determinants of virulence and adaptation of animal influenza A virus and discuss the phenotypic traits associated with airborne transmission of newly emerging influenza A viruses. Increased understanding of the determinants and mechanisms of virulence and transmission may aid in assessing the risks posed by animal influenza viruses to human health, and preparedness for such risks.

Possible increased pathogenicity of pandemic (H1N1) 2009 influenza virus upon reassortment

Since emergence of the pandemic (H1N1) 2009 virus in April 2009, three influenza A viruses—seasonal (H3N2), seasonal (H1N1), and pandemic (H1N1) 2009—have circulated in humans. Genetic reassortment between these viruses could result in enhanced pathogenicity. We compared 4 reassortant viruses with favorable in vitro replication properties with the wild-type pandemic (H1N1) 2009 virus with respect to replication kinetics in vitro and pathogenicity and transmission in ferrets. Pandemic (H1N1) 2009 viruses containing basic polymerase 2 alone or in combination with acidic polymerase of seasonal (H1N1) virus were attenuated in ferrets. In contrast, pandemic (H1N1) 2009 with neuraminidase of seasonal (H3N2) virus resulted in increased virus replication and more severe pulmonary lesions. The data show that pandemic (H1N1) 2009 virus has the potential to reassort with seasonal influenza viruses, which may result in increased pathogenicity while it maintains the capacity of transmission through aerosols or respiratory droplets.