Impact of the H274Y Substitution on N1, N4, N5, and N8 Neuraminidase Enzymatic Properties and Expression in Reverse Genetic Influenza A Viruses.

The H274Y substitution (N2 numbering) in neuraminidase (NA) N1 confers oseltamivir resistance to A(H1N1) influenza viruses. This resistance has been associated with reduced N1 expression using transfected cells, but the effect of this substitution on the enzymatic properties and on the expression of other group-1-NA subtypes is unknown. The aim of the present study was to evaluate the antiviral resistance, enzymatic properties, and expression of wild-type (WT) and H274Y-substituted NA for each group-1-NA. To this end, viruses with WT or H274Y-substituted NA (N1pdm09 or avian N4, N5 or N8) were generated by reverse genetics, and for each reverse-genetic virus, antiviral susceptibility, NA affinity (Km), and maximum velocity (Vm) were measured. The enzymatic properties were coupled with NA quantification on concentrated reverse genetic viruses using mass spectrometry. The H274Y-NA substitution resulted in highly reduced inhibition by oseltamivir and normal inhibition by zanamivir and laninamivir. This resistance was associated with a reduced affinity for MUNANA substrate and a conserved Vm in all viruses. NA quantification was not significantly different between viruses carrying WT or H274Y-N1, N4 or N8, but was lower for viruses carrying H274Y-N5 compared to those carrying a WT-N5. In conclusion, the H274Y-NA substitution of different group-1-NAs systematically reduced their affinity for MUNANA substrate without a significant impact on NA Vm. The impact of the H274Y-NA substitution on viral NA expression was different according to the studied NA.

Level of maternal antibodies against respiratory syncytial virus (RSV) nucleoprotein at birth and risk of RSV very severe lower respiratory tract infection.

BACKGROUND: The nucleoprotein (N protein) of respiratory syncytial virus (RSV) is a candidate antigen for new RSV vaccine development. The aim of the present study was to investigate the association between maternal antibody titers against the RSV N protein at birth and the newborns' risk of developing very severe lower respiratory tract infection (VS-LRTI). METHODS: In this single-center prospective cohort study, 578 infants born during the RSV epidemic season in France were included. Among these, 36 were hospitalized for RSV VS-LRTI. A generalized linear model was used to test the occurrence of a VS-LRTI in function of sex, mode of delivery, parity of the mother, type of pregnancy, date of birth in relation to the peak of the epidemic, and antibody titer against N protein. RESULTS: All cord blood samples had detectable antibodies against N protein. The mean titers were significantly lower in newborns with risk factors for RSV severe LRTI (preterm infants, birth before the peak epidemic, multiparous mother). There was no association between antibody titer against the N protein and a protection against VS-LRTI. CONCLUSIONS: Further studies are needed to support the hypothesis that transfer of maternal antibodies against the RSV N protein can provide a significant immune protection early in infancy and that N protein candidate vaccine may be a suitable target for maternal vaccine.

[These viruses that inhabit and visit us: The human virome]. / Ces virus qui nous habitent et qui nous visitent : le virome humain.

Recent advances in new sequencing technologies have opened the way to the deciphering of human virome. So far, human virome is defined as the complete list of viruses found in human body. Those viruses could be endogenous, prokaryotic, archaeal and eukaryotic. In addition, each compartment of the human body constitutes a different microenvironment with its own virome. Viral infections can be categorized according to the outcome of the acute phase and until recently, only symptomatic and pathological infections were studied. It is now well established that a healthy person has an extremely diverse virome. This review summarizes the current state of our knowledge and also proposes another classification of the human virome based on principles of ecology.

Title: Ces virus qui nous habitent et qui nous visitent : le virome humain. Abstract: Les progrès récents des nouvelles techniques de séquençage ont ouvert la voie au décryptage du virome humain qui peut être défini comme l'ensemble de tous les virus présents dans le corps humain. Ces virus sont de différents types : endogènes, procaryotes, archéaux et eucaryotes1. Chaque partie du corps humain constitue un microenvironnement différent et possède donc un virome qui lui est propre. Les infections virales peuvent être catégorisées selon l'issue de la phase aiguë. Jusqu'à récemment, seules les infections symptomatiques étaient étudiées. Cette revue résume l'état actuel de nos connaissances et propose une définition du virome humain sous l'angle des principes de l'écologie, en considérant l'être humain comme un écosystème.

The NS Segment of H1N1pdm09 Enhances H5N1 Pathogenicity in a Mouse Model of Influenza Virus Infections.

In 2009, the co-circulation of H5N1 and H1N1pdm09 raised concerns that a reassortment event may lead to highly pathogenic influenza strains. H1N1pdm09 and H5N1 are able to infect the same target cells of the lower respiratory tract. To investigate the capacity of the emergence of reassortant viruses, we characterized viruses obtained from the co-infection of cells with H5N1 (A/Turkey/13/2006) and H1N1pdm09 (A/Lyon/969/2009 H1N1). In our analysis, all the screened reassortants possessed the PB2, HA, and NP segments from H5N1 and acquired one or two of the H1N1pdm09 segments. Moreover, the in vivo infections showed that the acquisition of the NS segment from H1N1pdm09 increased the virulence of H5N1 in mice. We conclude, therefore, that reassortment can occur between these two viruses, even if this process has never been detected in nature.

Synergistic Effects of Influenza and Staphylococcus aureus Toxins on Inflammation Activation and Cytotoxicity in Human Monocytic Cell Lines.

In patients with influenza, morbidity and mortality are strongly influenced by infections with Staphylococcus aureus producing high amounts of certain toxins. Here we tested the impact of influenza virus on the pro-inflammatory and cytotoxic actions of a panel of S. aureus virulence factors, including Panton-Valentine Leucocidin (PVL), phenol-soluble modulin α1 (PSMα1) and 3 (PSMα3), α-hemolysin (Hla), and cell wall components, i.e., heat-killed S. aureus (HKSA) and protein A. We initially screened for potential synergic interactions using a standardized in vitro model in influenza-infected continuous human monocytic cell lines. Then we tested the identified associations using an ex vivo model in influenza-infected human monocytes freshly isolated from blood. Co-exposure to influenza virus and HKSA, PVL, PSMα1, and PSMα3 increased NF-κB/AP-1 pathway activation in THP1-XBlue cells, and co-exposure to influenza virus and PVL increased cytotoxicity in U937 cells. In monocytes isolated from blood, the synergy between influenza virus and HKSA was confirmed based on cytokine production (TNF-α, IL-1ß, IL-6), and co-exposure to influenza virus and Hla-increased cytotoxicity. Our findings suggest that influenza virus potentiates the pro-inflammatory action of HKSA and contributes to the cytotoxicity of Hla on monocytes. Synergic interactions identified in the cell-line model must be cautiously interpreted since few were relevant in the ex vivo model.

Immune signatures of protective spleen memory CD8 T cells.

Memory CD8 T lymphocyte populations are remarkably heterogeneous and differ in their ability to protect the host. In order to identify the whole range of qualities uniquely associated with protective memory cells we compared the gene expression signatures of two qualities of memory CD8 T cells sharing the same antigenic-specificity: protective (Influenza-induced, Flu-TM) and non-protective (peptide-induced, TIM) spleen memory CD8 T cells. Although Flu-TM and TIM express classical phenotypic memory markers and are polyfunctional, only Flu-TM protects against a lethal viral challenge. Protective memory CD8 T cells express a unique set of genes involved in migration and survival that correlate with their unique capacity to rapidly migrate within the infected lung parenchyma in response to influenza infection. We also enlighten a new set of poised genes expressed by protective cells that is strongly enriched in cytokines and chemokines such as Ccl1, Ccl9 and Gm-csf. CCL1 and GM-CSF genes are also poised in human memory CD8 T cells. These immune signatures are also induced by two other pathogens (vaccinia virus and Listeria monocytogenes). The immune signatures associated with immune protection were identified on circulating cells, i.e. those that are easily accessible for immuno-monitoring and could help predict vaccines efficacy.

Involvement of an Arginine Triplet in M1 Matrix Protein Interaction with Membranes and in M1 Recruitment into Virus-Like Particles of the Influenza A(H1N1)pdm09 Virus.

The influenza A(H1N1)pdm09 virus caused the first influenza pandemic of the 21st century. In this study, we wanted to decipher the role of conserved basic residues of the viral M1 matrix protein in virus assembly and release. M1 plays many roles in the influenza virus replication cycle. Specifically, it participates in viral particle assembly, can associate with the viral ribonucleoprotein complexes and can bind to the cell plasma membrane and/or the cytoplasmic tail of viral transmembrane proteins. M1 contains an N-terminal domain of 164 amino acids with two basic domains: the nuclear localization signal on helix 6 and an arginine triplet (R76/77/78) on helix 5. To investigate the role of these two M1 basic domains in influenza A(H1N1)pdm09 virus molecular assembly, we analyzed M1 attachment to membranes, virus-like particle (VLP) production and virus infectivity. In vitro, M1 binding to large unilamellar vesicles (LUVs), which contain negatively charged lipids, decreased significantly when the M1 R76/77/78 motif was mutated. In cells, M1 alone was mainly observed in the nucleus (47%) and in the cytosol (42%). Conversely, when co-expressed with the viral proteins NS1/NEP and M2, M1 was relocated to the cell membranes (55%), as shown by subcellular fractionation experiments. This minimal system allowed the production of M1 containing-VLPs. However, M1 with mutations in the arginine triplet accumulated in intracellular clusters and its incorporation in VLPs was strongly diminished. M2 over-expression was essential for M1 membrane localization and VLP production, whereas the viral trans-membrane proteins HA and NA seemed dispensable. These results suggest that the M1 arginine triplet participates in M1 interaction with membranes. This R76/77/78 motif is essential for M1 incorporation in virus particles and the importance of this motif was confirmed by reverse genetic demonstrating that its mutation is lethal for the virus. These results highlight the molecular mechanism of M1-membrane interaction during the formation of influenza A(H1N1)pdm09 virus particles which is essential for infectivity.

Impact on antiviral resistance of E119V, I222L and R292K substitutions in influenza A viruses bearing a group 2 neuraminidase (N2, N3, N6, N7 and N9).

OBJECTIVES: While subtype-specific substitutions linked to neuraminidase (NA) inhibitor resistance are well described in human N1 and N2 influenza NAs, little is known about other NA subtypes. The aim of this study was to determine whether the R292K and E119V ±â€ŠI222L substitutions could be associated with oseltamivir resistance in all group 2 NAs and had an impact on virus fitness. METHODS: Reassortant viruses with WT NA or variant N2, N3, N6, N7 or N9 NAs, bearing R292K or E119V ±â€ŠI222L substitutions, were produced by reverse genetics. The antiviral susceptibility, activity, Km of the NA, mutation stability and in vitro virus fitness in MDCK cells were determined. RESULTS: NA activities could be ranked as follows regardless of the substitution: N3 ≥ N6 > N2 ≥ N9 > N7. Using NA inhibitor resistance interpretation criteria used for human N1 or N2, the NA-R292K substitution conferred highly reduced inhibition by oseltamivir and the N6- or N9-R292K substitution conferred reduced inhibition by zanamivir and laninamivir. Viruses with the N3- or N6-E119V substitution showed normal inhibition by oseltamivir, while those with the N2-, N7- or N9-E119V substitution showed reduced inhibition by oseltamivir. Viruses with NA-E119V + I222L substitutions showed reduced inhibition (N3 and N6) or highly reduced inhibition (N2, N7 and N9) by oseltamivir. Viruses bearing the NA-R292K substitution had lower affinity and viruses bearing the NA-E119V substitution had higher affinity for the MUNANA substrate than viruses with corresponding WT NA. CONCLUSIONS: NA-R292K and E119V + I222L substitutions conferred reduced inhibition by oseltamivir for all group 2 NAs. Surveillance of NA inhibitor resistance for zoonotic and human influenza viruses and the development of novel antiviral agents with different targets should be continued.

La balance HA-NA des virus influenza A(H1N1).

Hemagglutinin (HA) and neuraminidase (NA) are major glycoproteins expressed on the surface of influenza virus. They have complementary and antagonistic functions that facilitate in the life cycle of the virus. The functional equilibrium generated between HA and NA can impact the evolution and adaptation of influenza virus strains within the human reservoir. This functional equilibrium is referred to as the "HA-NA balance". An imbalanced HA-NA can restrict the multiplication and transmission capacity of influenza viruses. Moreover, this equilibrium is likely a limiting factor against species crossover for the virus. In light of such considerations, the HA-NA balance should be precisely studied to gain a better understanding of the emergence of pandemic and seasonal influenza virus strains. This review describes the concept of the HA-NA balance, the methods used to study it, plus a discussion of the HA-NA balance in the evolution of the pandemic influenza A H1N1 strains that plagued the world in 1918 and 2009.

Functional balance between the hemagglutinin and neuraminidase of influenza A(H1N1)pdm09 HA D222 variants.

D222G/N substitutions in A(H1N1)pdm09 hemagglutinin may be associated with increased binding of viruses causing low respiratory tract infections and human pathogenesis. We assessed the impact of such substitutions on the balance between hemagglutinin binding and neuraminidase cleavage, viral growth and in vivo virulence.Seven viruses with differing polymorphisms at codon 222 (2 with D, 3 G, 1 N and 1 E) were isolated from patients and characterized with regards hemagglutinin binding affinity (Kd) to α-2,6 sialic acid (SAα-2,6) and SAα-2,3 and neuraminidase enzymatic properties (Km, Ki and Vmax). The hemagglutination assay was used to quantitatively assess the balance between hemagglutinin binding and neuraminidase cleavage. Viral growth properties were compared in vitro in MDCK-SIAT1 cells and in vivo in BALB/c mice. Compared with D222 variants, the binding affinity of G222 variants was greater for SAα-2,3 and lower for SAα-2,6, whereas that of both E222 and N222 variants was greater for both SAα-2,3 and SAα-2,6. Mean neuraminidase activity of D222 variants (16.0 nmol/h/10(6)) was higher than that of G222 (1.7 nmol/h/10(6) viruses) and E/N222 variants (4.4 nmol/h/10(6) viruses). The hemagglutination assay demonstrated a deviation from functional balance by E222 and N222 variants that displayed strong hemagglutinin binding but weak neuraminidase activity. This deviation impaired viral growth in MDCK-SIAT1 cells but not infectivity in mice. All strains but one exhibited low infectious dose in mice (MID50) and replicated to high titers in the lung; this D222 strain exhibited a ten-fold higher MID50 and replicated to low titers. Hemagglutinin-neuraminidase balance status had a greater impact on viral replication than hemagglutinin affinity strength, at least in vitro, thus emphasizing the importance of an optimal balance for influenza virus fitness. The mouse model is effective in assessing binding to SAα-2,3 but cannot differentiate SAα-2,3- from SAα-2,6- preference, nor estimate the hemagglutinin-neuraminidase balance in A(H1N1)pdm09 strains.

A novel I221L substitution in neuraminidase confers high-level resistance to oseltamivir in influenza B viruses.

UNLABELLED: Influenza B viruses with a novel I221L substitution in neuraminidase (NA) conferring high-level resistance to oseltamivir were isolated from an immunocompromised patient after prolonged oseltamivir treatment. METHODS: Enzymatic characterization of the NAs (Km, Ki) and the in vitro fitness of viruses carrying wild-type or mutated (I221L) NA genes were evaluated. Proportions of wild-type and mutated NA genes were directly quantified in the patient samples. Structural characterizations by X-ray crystallography of a wild-type and I221L variant NA were performed. RESULTS: The Km and Ki revealed that the I221L variant NA had approximately 84 and 51 times lower affinity for oseltamivir carboxylate and zanamivir, respectively, compared with wild-type NA. Viruses with a wild-type or I221L variant NA had similar growth kinetics in Madin-Darby canine kidney (MDCK) cells, and 5 passages in MDCK cells revealed no reversion of the I221L substitution. The crystal structure of the I221L NA and oseltamivir complex showed that the leucine side chain protrudes into the hydrophobic pocket of the active site that accommodates the pentyloxy substituent of oseltamivir. CONCLUSIONS: Enzyme kinetic and NA structural analyses provide an explanation for the high level of resistance to oseltamivir while retaining good fitness of viruses carrying I221L variant NA.

Marburgvirus hijacks nrf2-dependent pathway by targeting nrf2-negative regulator keap1.

Marburg virus (MARV) has a high fatality rate in humans, causing hemorrhagic fever characterized by massive viral replication and dysregulated inflammation. Here, we demonstrate that VP24 of MARV binds Kelch-like ECH-associated protein 1 (Keap1), a negative regulator of nuclear transcription factor erythroid-derived 2 (Nrf2). Binding of VP24 to Keap1 Kelch domain releases Nrf2 from Keap1-mediated inhibition promoting persistent activation of a panoply of cytoprotective genes implicated in cellular responses to oxidative stress and regulation of inflammatory responses. Increased expression of Nrf2-dependent genes was demonstrated both during MARV infection and upon ectopic expression of MARV VP24. We also show that Nrf2-deficient mice can control MARV infection when compared to lethal infection in wild-type animals, indicating that Nrf2 is critical for MARV infection. We conclude that VP24-driven activation of the Nrf2-dependent pathway is likely to contribute to dysregulation of host antiviral inflammatory responses and that it ensures survival of MARV-infected cells despite these responses.

H1N1 influenza A virus neuraminidase modulates infectivity in mice.

In the 2years since the onset of the H1N1 2009 pandemic virus (H1N1pdm09), sporadic cases of oseltamivir-resistant viruses have been reported. We investigated the impact of oseltamivir-resistant neuraminidase from H1N1 Brisbane-like (seasonal) and H1N1pdm09 viruses on viral pathogenicity in mice. Reassortant viruses with the neuraminidase from seasonal H1N1 virus were obtained by co-infection of a H1N1pdm09 virus and an oseltamivir-resistant H1N1 Brisbane-like virus. Oseltamivir-resistant H1N1pdm09 viruses were also isolated from patients. After biochemical characterization, the pathogenicity of these viruses was assessed in a murine model. We confirmed a higher infectivity, in mice, of the H1N1pdm09 virus compared to seasonal viruses. Surprisingly, the oseltamivir-resistant H1N1pdm09 virus was more infectious than its sensitive counterpart. Moreover, the association of H1N1pdm09 hemagglutinin and an oseltamivir-resistant neuraminidase improved the infectivity of reassortant viruses in mice, regardless of the NA origin: seasonal (Brisbane-like) or pandemic strain. This study highlights the need to closely monitor the emergence of oseltamivir-resistant viruses.

[Persistence of influenza A viruses in relation with environmental factors]. / Persistance des virus influenza A en fonction des paramètres environnementaux.

Wild waterfowl represents the natural reservoir of influenza A viruses. Transmission within bird populations occurs through an indirect fecal-oral route implying contaminated water. Within human populations, influenza A viruses can be transmitted through large droplets, aerosols, or direct contact with secretions. Thus, in the human compartment as in the avian one, influenza A viruses have to experiment a free living stage. The knowledge of factors influencing viral persistence during that key step is needed to understand their transmission dynamic. Data gathered here describe the major role played by temperature, pH and salinity on viral persistence in aquatic environment and the importance of UV radiations, humidity and temperature in mid air. We discuss mechanisms underlying these roles and the potential influence of other factors. We point out the need to develop researches to improve our understanding on influenza A virus stability and thus transmission.

[Modelling the spread of influenza A]. / Circulation des virus influenza A : apports de la modélisation.

The mathematical models used to produce a simplified description of reality appear as important modern epidemiology tools. Indeed, a goal in epidemiology is to understand how pathogens are transmitted between individuals. This would predict epidemics and their extent in time and space. The models can provide a guide to public health practitioners, through comparison of different strategies in disease management. Also, it improves our understanding of pathogen transmission. Here, we focused on some of these models to emphasize their practical value in understanding the influenza viruses' circulation. First, we present a simple epidemiological model that provides an understandable approach to follow in order to build a model. Second, we show how to use such simple models to include the epidemic process in a more global scale. Finally, we explain how modeling can help investigate the extent to which a pathogen can trigger an epidemic or the effects of public health measures on evolving influenza viruses.

Genetic content of Influenza H3N2 vaccine seeds.

Influenza vaccine seeds produced in chicken eggs are selected through HA and NA surface glycoproteins antigenicity, as well as through high replicative ability. Here we characterize the genetic content of recently used thirteen H3N2 influenza vaccine seeds. Interestingly, sequence analysis of the vaccine seeds shows reassortment events leading to PR8:H3N2 segment constellations, ranging from the 6:2 to 2:6 constellations. This study shows that the H3N2 PB1 is the most frequent internal segment incorporated in the tested vaccines seeds.

Novel influenza A(H1N1) 2009 in vitro reassortant viruses with oseltamivir resistance.

BACKGROUND: With the recent emergence of the novel A(H1N1) virus in 2009, the efficacy of available drugs, such as neuraminidase (NA) inhibitors, is of great concern for good patient care. Influenza viruses are known to be able to acquire resistance. In 2007, A(H1N1) viruses related to A/Brisbane/59/2007 (H1N1) (A[H1N1] Brisbane-like virus), which are naturally resistant to oseltamivir, emerged. Resistance to oseltamivir can be acquired either by spontaneous mutation in the NA (H275Y in N1), or by reassortment with a mutated NA. It is therefore crucial to determine the risk of pandemic A(H1N1) 2009 virus acquiring resistance against oseltamivir by reassortment. METHODS: We estimated the capacity of reassortment between the A(H1N1) 2009 virus and an oseltamivir-resistant A(H1N1) Brisbane-like virus by in vitro coinfections of influenza-permissive cells. The screening and the analysis of reassortant viruses was performed by specific reverse transcriptase PCRs and by sequencing. RESULTS: Out of 50 analysed reassortant viruses, two harboured the haemagglutinin (HA) segment from the pandemic A(H1N1) 2009 virus and the mutated NA originated from the A(H1N1) Brisbane-like virus. The replicating capacities of these viruses were measured, showing no difference as compared to the two parental strains, suggesting that acquisition of the mutated NA segment did not impair viral fitness in vitro. CONCLUSIONS: Our results suggest that the novel A(H1N1) 2009 virus can acquire by in vitro genetic reassortment the H275Y mutated NA segment conferring resistance to oseltamivir.

Ebola virus glycoprotein GP is not cytotoxic when expressed constitutively at a moderate level.

Transient expression of Ebola virus (EBOV) glycoprotein GP causes downregulation of surface proteins, cell rounding and detachment, a phenomenon believed to play a central role in the pathogenicity of the virus. In this study, evidence that moderate expression of GP does not result in such morphological changes was provided. It was shown that GP continuously produced in 293T cells from the Kunjin virus replicon was correctly processed and transported to the plasma membrane without affecting the surface expression of beta1 and alpha5 integrins and major histocompatibility complex I molecules. The level of GP expression in Kunjin replicon GP-expressing cells was similar to that observed in cells infected with EBOV early in infection and lower than that produced in cells transfected with plasmid DNA, phCMV-GP, expressing GP from a strong promoter. Importantly, transient transfection of Kunjin replicon GP-expressing cells with GP-coding plasmid DNA resulted in overexpression of GP, which lead to the downregulation of surface molecules and massive rounding and detachment of transfected cells. Here, it was also demonstrated that cell rounding and downregulation of the surface markers are the late events in EBOV infection, whereas synthesis and massive release of virus particles occur at early steps and do not cause significant cytotoxic effects. These findings indicate that the synthesis of EBOV GP in virus-infected cells is controlled well by several mechanisms that do not allow GP overexpression and hence the early appearance of its cytotoxic properties.