Vanessa Escuret

Rhinoviruses delayed the circulation of the pandemic influenza A (H1N1) 2009 virus in France

In contrast to the experience in other European countries, the onset of the A(H1N1)2009 influenza virus epidemic was unexpectedly slow in France during the first part of autumn 2009. Our objective was to test the hypothesis that intense circulation of rhinoviruses might have reduced the probability of infection by A(H1N1)2009 virus at the beginning of autumn 2009. Systematic analysis for the detection of A(H1N1)2009 (H1N1) and human rhinovirus (HRV) was performed by RT-PCR from week 36 to week 48 on respiratory samples sent to the diagnostic laboratory by the paediatric hospital (n = 2121). Retrospective analysis of the obtained data, using 2 x 2 contingency tables with Fishers exact test, revealed evidence of an inverse relationship between HRV and H1N1 detection. Between weeks 36 and 48 of 2009, both HRV and H1N1 were detected but in different time frames. HRV dispersed widely during early September, peaking at the end of the month, whereas the H1N1 epidemic began during mid-October and was still active at the end of this survey. During the co-circulation period of these two respiratory viruses (weeks 43-46), HRV detection appeared to reduce the likelihood of H1N1 detection in the same sample (OR = 0.08-0.24 p <0.0001). These results support the hypothesis that HRV infections can reduce the probability of A(H1N1) infection. This viral interference between respiratory viruses could have affected the spread of the H1N1 viruses and delayed the influenza pandemic at the beginning of autumn in France.

Pandemic A(H1N1)2009 influenza virus detection by real time RT-PCR : is viral quantification useful?

The emergence of the influenza A(H1N1) 2009 virus prompted the development of sensitive RT-PCR detection methods. Most are real time RT-PCRs which can provide viral quantification. In this manuscript, we describe a universal influenza A RT-PCR targeting the matrix (M) gene, combined with an RNaseP RT-PCR. These PCRs allow the detection of all influenza A virus subtypes, including A(H1N1)2009, together with a real-time assessment of the quality of the specimens tested. These PCR procedures were evaluated on 209 samples collected from paediatric patients. Viral loads determined through Ct values were corrected according to the RNaseP Ct value. The mean viral load in the collected samples was estimated to be 6.84 log RNA copies/mL. For poor quality samples (RNaseP Ct > 27), corrections resulted in +3 to +8 Ct values for the M gene RT-PCR. Corrected influenza Ct values were lower in late samples. No correlation was established between viral loads and clinical severity or duration of disease.This study shows that real time RT-PCR targeting the matrix gene is a reliable tool for quantification of type A influenza virus but emphasises the need for sample quality control assessment through cellular gene quantification for reliable estimation of the viral load. This method would be useful for disease management when repeated specimens are collected from an infected individual.

Risk of Influenza-Like Illness in an Acute Health Care Setting During Community Influenza Epidemics in 2004-2005, 2005-2006, and 2006-2007: A Prospective Study

BACKGROUND The person-to-person transmission of influenza-like illness (ILI) and influenza has been described mostly in long-term care units. Studies in acute hospital settings are rare and mostly retrospective. METHODS We prospectively estimated the relative risk (RR) of hospital-acquired (HA) ILI during hospitalization according to in-hospital exposures to contagious individuals. Surveillance of ILI and laboratory-confirmed influenza was undertaken at Edouard Herriot Hospital (1100 beds) during 3 influenza seasons. A total of 21 519 patients and 2153 health care workers (HCWs) from 2004 to 2007 were included. The RR of HA-ILI in patients was calculated according to exposure to other contagious patients and HCWs. RESULTS For patients exposed to at least 1 contagious HCW compared with those with no documented exposure in the hospital, the RR of HA-ILI was 5.48 (95% confidence interval [CI], 2.09-14.37); for patients exposed to at least 1 contagious patient, the RR was 17.96 (95% CI, 10.07-32.03); and for patients exposed to at least 1 contagious patient and 1 contagious HCW, the RR was 34.75 (95% CI, 17.70-68.25). CONCLUSIONS Hospitalized patients exposed to potentially infectious patients and HCWs with ILI inside the hospital are at greater risk for HA-ILI. Such results identify priorities regarding preventive measures for seasonal or pandemic influenza.

Oseltamivir–zanamivir bitherapy compared to oseltamivir monotherapy in the treatment of pandemic 2009 influenza A(H1N1) virus infections

BACKGROUND The emergence of oseltamivir resistance in 2007 highlighted the need for alternative strategies against influenza. To limit the putative emergence of resistant viruses this clinical trial aimed to evaluate the antiviral efficacy and tolerability of oseltamivir-zanamivir (O+Z) bitherapy compared to oseltamivir monotherapy (O). This clinical trial was designed in 2008-2009 and was conducted during the A(H1N1) influenza virus pandemic in 2009-2010. The A(H1N1)pdm09 viruses were reported to be sensitive to oseltamivir and zanamivir but resistant to amantadine. METHODS During the pandemic phase in France, adults with influenza-like illness for less than 42h and who tested positive to influenza A were randomised into treatment groups: (O+Z) or (O). Patients had a nasal wash at day 0, before the beginning of treatment and daily at days 1 to 4. They also had a nasal swab at days 5 and 7 to check for the negativation of viral excretion. Virological response was assessed using the GAPDH adjusted M gene quantification. RESULTS Analysis was possible for 24 patients, 12 in the (O+Z) arm and 12 in the (O) arm. The mean viral load decreased at around 1 log(10)cgeq/μl per day regardless of allocated treatment group. We could not detect any significant difference between treatment groups in the duration needed to alleviate symptoms. All treatments were well tolerated. No oseltamivir-resistant H275Y NA mutated virus has been detected in patients of both treatment groups. CONCLUSIONS The sample size of our study is too limited to be fully informative and we could not detect whether combination therapy (O+Z) improves or reduces the effectiveness of oseltamivir in the treatment of influenza A(H1N1)pdm09 virus infection in community patients. Additional studies are needed to improve the antiviral treatment of patients infected with influenza virus.

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/106) was higher than that of G222 (1.7 nmol/h/106 viruses) and E/N222 variants (4.4 nmol/h/106 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.

Surveillance and oseltamivir resistance of human influenza a virus in Turkey during the 2007–2008 season

Monitoring the activity of influenza viruses is important for establishing the circulating types and for detection of the emergence of novel sub‐types and antiviral resistant strains. This is the first report from Turkey on the surveillance and oseltamivir resistance of influenza viruses in 2007–2008. Five hundred twenty‐four nasal swabs were tested from different geographical regions in Turkey during November 2007–April 2008. One hundred sixty‐three (31%) samples were positive for influenza viruses of which 111 (68%) were influenza A, 52 (31%) influenza B using an immuno‐capture ELISA. Forty isolates were selected at random from influenza A positive samples and grown in MDCK cell cultures. The supernatant of the cell cultures was used for RNA extraction followed by RT‐PCR to detect the sub‐types. Sub‐typing revealed all samples as A/H1N1. The N1 gene segment of 30 A/H1N1 samples was sequenced in part, from the 201st to 365th residue, which included the critical region for oseltamivir resistance. Then resulting sequences were analyzed with oseltamivir sensitive and resistant strains obtained from National Center for Biotechnology Information (NCBI) GenBank by CLC Main Workbench Software. H275Y (H274Y according to N2 numbering) mutation, which is known to confer resistance to oseltamivir, was detected in 6 out of 30 (20%) H1N1 isolates from four cities (Istanbul, Bursa, Ankara, and Izmir). The D354G mutation was observed in all oseltamivir resistant H1N1 isolates but not in the oseltamivir sensitive isolates. Assay of neuraminidase activity revealed that these isolates were resistant to oseltamivir, but sensitive to zanamivir. J. Med. Virol. 81:1645–1651, 2009.

Functional balance between neuraminidase and haemagglutinin in influenza viruses

Seasonal influenza A and B viruses are important human pathogens responsible for significant morbidity and mortality worldwide. In addition, influenza A zoonotic viruses are a constant pandemic threat. These viruses present two major surface glycoproteins: the haemagglutinin (HA) and the neuraminidase (NA). These two glycoproteins both recognize the sialic acid and have complementary activities, the HA binds the sialic acid through its receptor-binding site, the NA is a receptor-destroying enzyme that cleaves α2-3 and α2-6-linked sialic acids. Therefore, the functional HA/NA balance is a critical factor for a good viral fitness and plays a major role in overcoming the host barrier and the efficiency of sustained human-to-human transmission. Although the two glycoproteins are in constant evolution, the HA/NA balance seems to remain stable in human viruses because an optimal balance is required to maintain good viral fitness. Understanding the evolution of influenza viruses requires an in-depth exploration of the HA/NA balance.

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.

Intérêts des inhibiteurs de la neuraminidase dans la prise en charge des infections dues aux virus influenza

Oseltamivir and zanamivir are two neuraminidase inhibitors (NAIs) active on A and B influenza viruses. These analogues have been developed from the structure of sialic acid, the neuraminidase (NA) substrate. Resistance to NAIs have been detected. They are mainly associated to mutations located on the NA gene. The use of these antiviral drugs remains low in the context of seasonal flu, even the duration of symptoms can be reduced of one day if an antiviral treatment is started within 48 hours after disease onset. NAIs also present a significant effect when used in postexposition prophylaxis. Resistance, mainly to oseltamivir, have been detected but remained rare until the spontaneous emergence in 2007-2008 winter of a seasonal A(H1N1) variant resistant to this drug. NAIs are also interesting for the treatment of severe flu infections, specially those associated to A(H5N1). Finally, because of the pandemic A(H1N1)2009 virus, NAIs use has largely increased for prophylactic and therapeutic treatment of severe and non severe infections. This large use may be associated to an increased risk of selection of resistant viruses. Up to now, this phenomenon remains fortunately limited but has to be closely monitored.

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.