Virulence of influenza a virus for mouse lung

Robert A. Author information Article notes Copyright and License information Disclaimer. E-mail: ude. Received Dec This article has been cited by other articles in PMC. Abstract The virulence of influenza virus is a multigenic trait.

Open in a separate window. Table 1. Mouse lethal dose MLD 50 in pfu for wt and mutant influenza viruses. Materials and Methods Cells and Viruses. Viral Growth Kinetics. Mouse Experiments. Supplementary Material Supporting Information: Click here to view. Footnotes The authors declare no conflict of interest.

References 1. In: Fields Virology. Kash JC, et al. Genomic analysis of increased host immune and cell death responses induced by influenza virus. Taubenberger JK. Proc Am Philos Soc. Glaser L, et al. A single amino acid substitution in influenza virus hemagglutinin changes receptor binding specificity.

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The genetic basis for virulence in influenza virus is largely unknown. Genomic sequencing provided the first demonstration, to our knowledge, that a group of 11 mutations can convert an avirulent virus to a virulent variant that can kill at a minimal dose.

Mutations in virulent variants repeatedly involved nuclear localization signals and sites of protein and RNA interaction, implicating them as novel modulators of virulence. Mouse-adapted variants with the same hemagglutinin mutations possessed different pH optima of fusion, indicating that fusion activity of hemagglutinin can be modulated by other viral genes. Analysis of viral adaptation by serial passage appears to provide the identification of biologically relevant mutations.

Virulence is the measure of the ability of a pathogen to damage its host. Human influenza A virus infection typically causes tracheobronchitis with a low incidence of fatal pneumonia. In , a virulent influenza A virus variant arose, causing a devastating pandemic killing 50 million people 1. Although this virus was not isolated, it must have possessed mutations that increased its virulence. The genomic sequence of viruses are being determined from archival tissues and, whereas the sequence of the hemagglutinin HA and neuraminidase NA genes are now available 2 , we do not yet have the understanding of the molecular basis for virulence needed to interpret this information.

Subsequent human infections by related avian H9N2 viruses indicate a continued threat to the human population 1. There is thus a need to understand the genetic basis for virulence in influenza virus variants, with the hope that specific mutations will be indicators and thus predictors of virulence. No clinical isolates of human influenza virus are known to differ in virulence 5 , therefore necessitating the analysis of infection in animals.

Influenza virus is partially host restricted, where virus from one host does not normally transmit or cause disease in other hosts. Adaptation of human influenza virus to mice by serial passage results in the selection of highly virulent variants that have acquired mutations in multiple genes 7 — 9.

Analyses of the genetic basis for virulence by using reassortants that possess mixtures of genes from virulent and avirulent strains have identified various groupings of genes, which in aggregate implicate all eight genome segments 8. These data have led to the untested assumption that virulence cannot be genetically predicted, because there are too many degrees of freedom in the control of virulence.

A goal of the study of influenza pathogenesis is to define the roles of each viral gene in disease production. This process identified single amino acid substitutions in 5 of its 10 genes 9. Reintroduction of each of these mutations into the parental FM strain confirmed their roles not only in increasing virulence but also in replicative fitness for the mouse 9. These findings are compelling, because they show a clear relationship between replicative fitness and the ability to damage the host.

It was also surprising that the MA variant did not possess unselected mutations that typically accumulate in clinical isolates. This, however, would be predicted from studies of viral adaptation in cell culture, where genetic variation is a function of virus population size Serial passage of large populations of virus under novel conditions permits competition among all possible mutants with the selection of optimal genotypes.

In contrast, the transfer of small populations, typical of normal disease transmission, leads to the fixation of unselected and deleterious mutations because of stochastic effects, a process termed Muller's ratchet The primary feature of organisms with adaptive mutations is that they increase in prevalence in the population because of improved replicative fitness.

A strong indicator of adaptive change at the molecular level is convergent evolution, characterized by the repeated and independent occurrence of common mutations in adapted variants. The occurrence of identical mutations is termed parallel evolution, and mutation at the same sites but with different amino acids is termed directional evolution, both of which are characteristic of instances of convergent evolution This is the standard criterion for identifying mutations responsible for drug and inhibitor resistance, providing evidence for convergent evolution in many organisms, including influenza virus The objective of this analysis was to identify and characterize the complexity and nature of the mutations that control virulence.

Genomic sequencing of a highly virulent MA variant identified 11 mutations that were acquired on mouse adaptation. Sequencing of clonal variants showed that most of these mutations were positively selected in the population and affected specific regions of individual genes that identify functional themes for regulating virulence.

Experimental evolution may have recapitulated natural evolution, at least in part, because the Hong Kong H5N1 lineage of viruses possessed several mutations in common with the MA strains, suggesting their instrumental operation in virulence. Viruses were clonally purified by two plaque isolations in Madin—Darby canine kidney cell MDCK monolayer followed by stock preparation in the allantoic cavity of 9-day-old chicken embryos.

MA viruses were clonally isolated and titrated by plaque assays on MDCK cell monolayer, as described Viral RNA was purified by phenol extraction from stock virus, as previously described Sequencing primers sequences available on request were complementary to related H3N2 viruses GenBank accession nos. Labeled virus was purified by adsorption to guinea pig erythrocytes.

Virulence was measured as the lethal dose in CD1 strain Swiss—Webster mice. The median lethal dose LD 50 in plaque-forming units pfu was measured by intranasal infection of five groups of five mice each with serial fold dilutions of virus, as described previously Mortality from influenza virus infection occurs primarily before day 8 and was thus monitored for 10 days.

LD 50 values were determined by using the Karber method The significance of differences in virulence and growth values was determined by using the Z statistic for a standard normal distribution. The probability of multiple independent mutations in the same clonal isolate was predicted by the Poisson distribution by using the observed mutation frequency.

Supernatants were collected over a h period, and plaque assays were performed in duplicate for each sample. Over a day period, lungs were removed from groups of three mice, pooled, and sonicated for quantification by plaque assay on MDCK monolayers, as described above. Hemagglutination and hemagglutination inhibition assays were performed as described previously Several HKMA clonal isolates from each passage level were similar in virulence to their respective populations and were thus representative of these populations; however, some were less virulent, indicating genetic heterogeneity within these populations.

To identify the mutations responsible for the increased ability to cause fatal lung infection, we initially sequenced the genome of the most virulent clonal isolate, HKMA20C, as well as the HK parent for comparison. Although it is clear that these mutations as a group must account for the difference in biology of the HKMA20C variant, it is not clear that they are all instrumental in adaptation to increased virulence.

Because adaptive changes increase replicative fitness, viruses that possess these changes will be present at a greater frequency in the virus population than their rate of formation predicted from the mutation frequency. To detect mutations that were positively selected on mouse adaptation, we tested for the null hypothesis that the mutations in individual virus clones were randomly and thus independently generated. In mammals, the association between the presence of an MBCS and systemic spread is less obvious.

In the ferret model, incorporation of an MBCS in the HA of a human influenza H3N2 virus did not resulted in increased virulence or a change in tissue tropism. These findings suggest that, in addition to an MBCS, other factors are involved to result in systemic spread in ferrets.

The influenza virus polymerase proteins, and in particular PB2, have been shown to be important determinants of virulence. Amino acid substitution lysine K to glutamic acid E at position in PB2 has been studied extensively in the context of mammalian adaptation [ 53 ]. This substitution is suggested to occur in order to adapt to physiological constraints, e.

Most avian influenza A viruses, which preferentially replicate at a relatively high temperature of around 41 degrees Celsius in the digestive tract of birds, have a E residue at position In contrast, human influenza A viruses replicate at the lower temperature of around 33 degrees Celsius, which is the temperature in the human URT.

These viruses typically have a K residue at this position. The EK substitution was acquired rapidly when avian viruses were pass-aged in mice [ 54 ]. In the absence of the EK mutation, an aspartate D to asparagine N substitution at position of H5N1 PB2 was found to increase virulence and to expand the host range of avian H5N1 virus to mammalian hosts [ 54 — 56 ].

The adaptive mutation DN caused enhancement of binding of PB2 to importin alpha1 in mammalian cells resulting in increased transport of PB2 into the nucleus [ 57 ]. When the mammalian adaptation substitutions EK or DN were introduced in pH1N1, no increase in virulence was observed [ 58 ]. Recently, numerous other mutations in PB1, PA, NP and NEP have been described, that can overcome the poor polymerase activity of avian influenza viruses in human cells [ 60 ].

PB1-F2 contributes to the virulence of influenza A viruses by inducing apoptosis of infected cells [ 61 ]. Moreover, PB1-F2 promotes and increases severity of secondary pneumonia [ 62 ]. In contrast to previous pandemic influenza viruses, pH1N1 does not encode a PB1-F2, because of three premature stop co-dons.

This protein modulates the host response by repressing cellular gene expression, i. PA-X deficient influenza viruses cause more severe disease in mice, as a result of an accelerated host response.

Moreover, influenza viruses lacking PA-X differ in host-cell shutoff compared to wild-type virus. Truncation of PA-X protein appears to be associated with influenza virus lineages circulating in particular hosts, indicating that there may be some species specificity to the evolution of PA-X [ 68 ]. To establish productive infection, influenza viruses have mechanisms to evade host immune responses, including the type-I IFN response.

NS1 has been studied extensively as a molecular determinant of virulence. H5N1 viruses, unlike other human, avian and swine influenza viruses, are relatively resistant to the antiviral effects of INFs, which result in increased levels of pro-inflammatory cytokines [ 70 ]. This effect requires an E at amino acid position 92 of the NS1 molecule and allows virus replication in the presence of IFN; this mutation was a determinant of virulence in pigs [ 71 ].

This demonstrates that NS1 can modulate virulence through different mechanisms. However, even when these functions are restored for pH1N1, they do not appear to have a significant effect on replication, virulence or transmission of pH1N1 in various animal models [ 74 ]. Two influenza viruses are known that have developed additional mechanisms that promote cleavage of HA. On the other hand, the H1N1 NA gene enables the virus to replicate in the absence of trypsin.

Additionally, this NA protein was shown to play a critical role in the high virulence of the pandemic H1N1 in mice [ 77 ]. In , an outbreak of HPAI H7N7 virus in poultry in the Netherlands resulted in the death of one person and 89 human cases of conjunctivitis. When the sequence of the virus obtained from the fatal case was compared to the sequence of a virus isolated from a patient with conjunctivitis, four amino acid substitutions in the NA gene were identified [ 21 ].

These mutations all contributed to an increased NA activity, resulting in more efficient replication in mammalian cells most likely by preventing the formation of virus aggregates [ 44 ]. When avian influenza viruses are transmitted from wild birds to poultry, genetic changes as a result of adaptation to the new host frequently occur.

One example of such a change is a deletion in the stalk region of the NA that has been reported in several viruses isolated from unrelated poultry outbreaks [ 78 ]. This shortened NA stalk region is frequently detected upon transmission of avian influenza viruses from waterfowl to domestic poultry and is associated with increased virulence [ 79 , 80 ]. It is not yet clear how this shortened NA stalk region influences virulence, however, deletion in the NA stalk does not enhance the release of progeny viruses since the active site in the head cannot efficiently access its substrate [ 81 ].

Efficient and sustained human-to-human transmission is critical for the circulation of seasonal and pandemic influenza viruses in the human population. Transmission has been studied extensively in mammalian models, in particular the ferret and guinea pig [ 82 ]. Ferrets are naturally susceptible to both human and avian influenza viruses and upon infection develop similar symptoms and pulmonary pathology as humans. Avian influenza viruses do not transmit via the airborne route in the ferret model [ 85 , 86 ].

Therefore, the ferret model is a valuable tool to study viral traits for influenza virus transmission in mammals. This is highly relevant as it is currently unclear what exactly determines transmission of influenza viruses in mammals via aerosols or respiratory droplets.

For this reason, the ferret model was used extensively to compare the transmissibility of pH1N1 with the contemporary seasonal H1N1 virus, when it first emerged in humans [ 86 ]. In addition, transmission of the H1N1 virus was studied in ferrets. These studies showed that changes in the HA receptor-binding domain and PB2 were critical to initiate transmission of an avian-derived influenza virus [ 39 , 87 ].

Similar genetic changes were required for the Asian H2N2 virus. However a Q to L at position in HA was sufficient to change its binding preference from avian to human receptors, subsequently resulting in transmission between ferrets [ 88 ].

Overall, amino acid substitutions in HA and polymerase proteins can affect host range and transmission of influenza viruses [ 56 , 59 , 87 ]. As described above, avian influenza viruses of the H5, H7, and H9 subtypes have infected humans on several occasions and are therefore considered a potential pandemic threat.

However, the requirements for a virus to become pandemic i. In order to study the determinants that could lead to a pandemic virus, an avian H9N2 virus that harbored the internal genes from a human H3N2 virus was adapted to replication in mammals by serial pass-aging in ferrets.

This indicates that avian H9N2 viruses may acquire the ability to be transmitted between humans. Early attempts to create airborne-transmissible H5 viruses by generating reassortant viruses between H5N1 and human influenza viruses did not result in H5 viruses that could be transmitted between mammals via the airborne route [ 90 — 92 ]. Nevertheless, changing the receptor binding preference alone was not sufficient to confer airborne transmission of H5N1 virus, indicating that additional adaptive changes are required for H5N1 viruses to become transmissible [ 90 , 93 ].

Herfst et al. This research proved that avian-origin H5N1 viruses with a human SA receptor binding preference can become airborne-transmissible, but that indeed additional mutations were required for this phenotype [ 93 ].

Recent transmission studies in guinea pigs demonstrate that reassortants between H5N1 and pH1N1 virus that harbor the H5N1 HA with a dual SA receptor preference, are airborne-transmissible between guinea pigs as well [ 94 ].

In addition, phenotypical analysis of airborne H5 virus demonstrated that, the stability of HA in an acidic environment is important for airborne transmission [ 95 ]. For H7N9 virus, only one case of non-sustainable transmission between humans has been reported to date [ 96 ].

However, transmission experiments in ferrets indicated that this virus has a limited ability to be transmitted via the airborne route is limited [ 97 — ]. The recent H7N9 outbreak again accentuates that increased understanding of the mechanisms and molecular determinants that facilitate avian influenza viruses to cross the species barrier and become airborne-transmissible in humans, is urgently needed.

It is still impossible to predict when a new influenza virus will emerge in humans to cause the next pandemic, and what the subtype of this virus will be [ ]. Therefore, surveillance of bird and swine influenza viruses should specifically target particular mutations that render viruses more virulent or airborne-transmissible in humans, as described above.

Detection of such genetic traits should trigger more aggressive control programs than those employed currently. Improving pandemic preparedness by developing new vaccines that induce broader and stronger immune responses than the current influenza vaccines is another research priority.

The ultimate goal of influenza vaccine research should be to design a universal vaccine that would induce protection against all influenza virus subtypes.

National Center for Biotechnology Information , U. Author manuscript; available in PMC Apr Eefje J. Rimmelzwaan , Albert D. Osterhaus , and Ron A. Author information Copyright and License information Disclaimer. Fouchier, PhD. Copyright notice. See other articles in PMC that cite the published article. Abstract Influenza A viruses cause yearly seasonal epidemics and occasional global pandemics in humans.

Keywords: Influenza A viruses, virulence factors, pandemic threat, transmission. Influenza A virus Influenza A virus is a single-stranded negative-sense segmented RNA virus, with a genome consisting of eight gene segments, that can encode up to 16 proteins [ 1 — 5 ] Fig 1a.

Open in a separate window. Figure 1. Influenza A virus particle and replication cycle A Schematic representation of influenza A virus particle and gene segments. Influenza epidemics and pandemics Influenza A viruses are the cause of recurrent epidemics and occasional pandemics. Figure 2. Pandemics threats Historically, influenza viruses of three HA subtypes H1, H2 and H3 have acquired the ability to be transmitted efficiently between humans.

Influenza A virus virulence factors HA Amino acid substitutions in HA that affect the receptor binding preference can influence the cellular host range and tissue tropism which may alter virulence. Figure 3. Cleavage site as an important virulence factor The HA0 is cleaved into two subunits HA1 and HA2 by cellular proteases which recognize either a mono basic or multi basic cleavage site. Polymerase proteins The influenza virus polymerase proteins, and in particular PB2, have been shown to be important determinants of virulence.

Footnotes Conflict of interest The authors declare that they have no conflict of interest. References 1. Palese P, Shaw ML. Fields Virology Lippincott. Identification of a novel splice variant form of the influenza a virus m2 ion channel with an antigenically distinct ectodomain. PLoS Pathog. Journal of virology. An overlapping protein-coding region in influenza A virus segment 3 modulates the host response.

Novel insights into proteolytic cleavage of influenza virus hemagglutinin. Reviews in medical virology.

Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems. Mapping the antigenic and genetic evolution of influenza virus. Molecular basis for the generation in pigs of influenza A viruses with pandemic potential. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology.