Erhard van der Vries

Influenza Virus Inactivation for Studies of Antigenicity and Phenotypic Neuraminidase Inhibitor Resistance Profiling

ABSTRACT Introduction of a new influenza virus in humans urges quick analysis of its virological and immunological characteristics to determine the impact on public health and to develop protective measures for the human population. At present, however, the necessity of executing pandemic influenza virus research under biosafety level 3 (BSL-3) high-containment conditions severely hampers timely characterization of such viruses. We tested heat, formalin, Triton X-100, and β-propiolactone treatments for their potencies in inactivating human influenza A(H3N2) and avian A(H7N3) viruses, as well as seasonal and pandemic A(H1N1) virus isolates, while allowing the specimens to retain their virological and immunological properties. Successful heat inactivation coincided with the loss of hemagglutinin (HA) and neuraminidase (NA) characteristics, and β-propiolactone inactivation reduced the hemagglutination titer and NA activity of the human influenza virus 10-fold or more. Although Triton X-100 treatment resulted in inconsistent HA activity, the NA activities in culture supernatants were enhanced consistently. Nonetheless, formalin treatment permitted the best retention of HA and NA properties. Triton X-100 treatment proved to be the easiest-to-use influenza virus inactivation protocol for application in combination with phenotypic NA inhibitor susceptibility assays, while formalin treatment preserved B-cell and T-cell epitope antigenicity, allowing the detection of both humoral and cellular immune responses. In conclusion, we demonstrated successful influenza virus characterization using formalin- and Triton X-100-inactivated virus samples. Application of these inactivation protocols limits work under BSL-3 conditions to virus culture, thus enabling more timely determination of public health impact and development of protective measures when a new influenza virus, e.g., pandemic A(H1N1)v virus, is introduced in humans.

Fatal Oseltamivir-Resistant Influenza Virus Infection

The incidence of influenza A (H1N1) viruses that carry the neuraminidase H274Y mutation has increased by 30% this year in the Netherlands. This case suggests that this oseltamivir-resistant virus can be pathogenic, at least in an immunocompromised patient.

H1N1 2009 Pandemic Influenza Virus: Resistance of the I223R Neuraminidase Mutant Explained by Kinetic and Structural Analysis

Two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that antiviral resistant viruses emerge and spread in the human population. The 2009 pandemic H1N1 virus is already resistant to adamantanes. Recently, a novel neuraminidase inhibitor resistance mutation I223R was identified in the neuraminidase of this subtype. To understand the resistance mechanism of this mutation, the enzymatic properties of the I223R mutant, together with the most frequently observed resistance mutation, H275Y, and the double mutant I223R/H275Y were compared. Relative to wild type, KM values for MUNANA increased only 2-fold for the single I223R mutant and up to 8-fold for the double mutant. Oseltamivir inhibition constants (KI) increased 48-fold in the single I223R mutant and 7500-fold in the double mutant. In both cases the change was largely accounted for by an increased dissociation rate constant for oseltamivir, but the inhibition constants for zanamivir were less increased. We have used X-ray crystallography to better understand the effect of mutation I223R on drug binding. We find that there is shrinkage of a hydrophobic pocket in the active site as a result of the I223R change. Furthermore, R223 interacts with S247 which changes the rotamer it adopts and, consequently, binding of the pentoxyl substituent of oseltamivir is not as favorable as in the wild type. However, the polar glycerol substituent present in zanamivir, which mimics the natural substrate, is accommodated in the I223R mutant structure in a similar way to wild type, thus explaining the kinetic data. Our structural data also show that, in contrast to a recently reported structure, the active site of 2009 pandemic neuraminidase can adopt an open conformation.

Prolonged Influenza Virus Shedding and Emergence of Antiviral Resistance in Immunocompromised Patients and Ferrets

Immunocompromised individuals tend to suffer from influenza longer with more serious complications than otherwise healthy patients. Little is known about the impact of prolonged infection and the efficacy of antiviral therapy in these patients. Among all 189 influenza A virus infected immunocompromised patients admitted to ErasmusMC, 71 were hospitalized, since the start of the 2009 H1N1 pandemic. We identified 11 (15%) cases with prolonged 2009 pandemic virus replication (longer than 14 days), despite antiviral therapy. In 5 out of these 11 (45%) cases oseltamivir resistant H275Y viruses emerged. Given the inherent difficulties in studying antiviral efficacy in immunocompromised patients, we have infected immunocompromised ferrets with either wild-type, or oseltamivir-resistant (H275Y) 2009 pandemic virus. All ferrets showed prolonged virus shedding. In wild-type virus infected animals treated with oseltamivir, H275Y resistant variants emerged within a week after infection. Unexpectedly, oseltamivir therapy still proved to be partially protective in animals infected with resistant virus. Immunocompromised ferrets offer an attractive alternative to study efficacy of novel antiviral therapies.

Multidrug resistant 2009 a/h1n1 influenza clinical isolate with a neuraminidase i223r mutation retains its virulence and transmissibility in ferrets

Only two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that influenza virus becomes resistant to these antiviral drugs and spreads in the human population. The 2009 pandemic A/H1N1 influenza virus is naturally resistant to adamantanes. Recently a novel neuraminidase I223R mutation was identified in an A/H1N1 virus showing cross-resistance to the neuraminidase inhibitors oseltamivir, zanamivir and peramivir. However, the ability of this virus to cause disease and spread in the human population is unknown. Therefore, this clinical isolate (NL/2631-R223) was compared with a well-characterized reference virus (NL/602). In vitro experiments showed that NL/2631-I223R replicated as well as NL/602 in MDCK cells. In a ferret pathogenesis model, body weight loss was similar in animals inoculated with NL/2631-R223 or NL/602. In addition, pulmonary lesions were similar at day 4 post inoculation. However, at day 7 post inoculation, NL/2631-R223 caused milder pulmonary lesions and degree of alveolitis than NL/602. This indicated that the mutant virus was less pathogenic. Both NL/2631-R223 and a recombinant virus with a single I223R change (recNL/602-I223R), transmitted among ferrets by aerosols, despite observed attenuation of recNL/602-I223R in vitro. In conclusion, the I223R mutated virus isolate has comparable replicative ability and transmissibility, but lower pathogenicity than the reference virus based on these in vivo studies. This implies that the 2009 pandemic influenza A/H1N1 virus subtype with an isoleucine to arginine change at position 223 in the neuraminidase has the potential to spread in the human population. It is important to be vigilant for this mutation in influenza surveillance and to continue efforts to increase the arsenal of antiviral drugs to combat influenza.

Influenza Virus Resistance to Antiviral Therapy

Antiviral drugs for influenza therapy and prophylaxis are either of the adamantane or neuraminidase inhibitor (NAI) class. However, the NAIs are mainly prescribed nowadays, because of widespread adamantane resistance among influenza A viruses and ineffectiveness of adamantanes against influenza B. Emergence and spread of NAI resistance would further limit our therapeutic options. Taking into account the previous spread of oseltamivir-resistant viruses during the 2007/2008 season preceding the last pandemic, emergence of yet another naturally NAI-resistant influenza virus may not be an unlikely event. This previous incident also underlines the importance of resistance surveillance and asks for a better understanding of the mechanisms underlying primary resistance development. We provide an overview of the major influenza antiviral resistance mechanisms and future therapies for influenza. Here, we call for a better understanding of the effect of virus mutations upon antiviral treatment and for a tailored antiviral approach to severe influenza virus infections.

Avian influenza a(H10n7) virus–associated mass deaths among harbor seals

To the Editor: Avian influenza A viruses occasionally cross the species barrier; influenza A(H5N1) virus and the recently emerged influenza A(H7N9) virus are prime examples of bird-to-human transmission (1,2). In addition, avian influenza A viruses can cross to various other mammalian species, including pinnipeds (e.g., seals) (3,4). Recently, mass deaths have occurred among harbor seals (Phoca vitulina); hundreds of carcasses washed up the shores of Sweden (March 2014), Denmark (July 2014), and Germany (October 2014). Approximately 1,400 dead harbor seals were seen in the coastal waters of Schleswig-Holstein in Germany alone, where the population is ≈12,000 animals. We report the investigation of the deaths of 17 seals from different age groups that were found dead on the islands of Helgoland and Sylt, Germany, during the second week of October 2014. Complete necropsies were performed on the carcasses, which were in variable nutritional conditions, ranging from very poor to good. Necropsy results showed consistently poorly retracted lungs with severe congestion, occasional diffuse consolidation, and multifocal firm nodular areas of gray-yellow discoloration with varying numbers of metazoic parasites. Histologic examinations showed acute necrotizing bronchitis and adenitis of bronchial glands with sloughing of epithelial cells (Figure, panel A). Occasionally, mild interstitial pneumonia was found. Multifocal pyogranulomatous to necrotizing pneumonia was associated with parasite infestation. A few animals had suppurative to necrotizing or nonsuppurative rhinitis and tracheitis. Figure Histopathologic and phylogenetic analyses of necropsy samples from harbor seals infected with avian influenza A(H10N7) virus, Germany, 2014. A) Lung of harbor seal showing marked necrosis and sloughing of epithelial cells in bronchial glands (arrows); ... Because mass deaths among seals were caused by phocine distemper virus in the same area in 1988 and 2002, we tested lung and throat swab samples for morbillivirus using reverse transcription PCR (RT-PCR) and immunohistochemical analysis (5). In addition, real-time RT-PCR targeting the influenza A virus matrix gene was performed (6). No indications for the morbillivirus were detected by RT-PCR and immunohistochemistry; however, in lung lesions and throat swab samples of 11 animals, a positive signal was observed by the influenza A matrix gene real-time RT-PCR (cycle threshold values 15.0–33.9). Influenza A virus (A/harbor seal/Germany/1/2014) was subsequently isolated from lung and throat swab samples; the virus replicated to high titers in 11-day-old embryonated chicken eggs and on MDCK cells. By PCR using specific primers and subsequent Sanger sequencing of the hemagglutinin and neuraminidase genes, this virus was characterized as an influenza A virus of the H10N7 subtype, commonly found in migratory waterfowl (6). In addition, genetic analyses of all other gene segments indicated that the influenza virus A/harbor seal/Germany/1/2014 was most closely related to various influenza A viruses detected in wild birds. Specifically, the hemagglutinin and neuraminidase genes were genetically most closely related to subtype H10N7 viruses recently found in migratory ducks in Georgia, Egypt, and the Netherlands (Figure, panel C) (7). Genetic analyses were based on BLAST analyses using public databases available as of October 17, 2014 (http://www.ncbi.nlm.nih.gov, http://www.gisaid.com) and supplemented with H10 and N7 sequences from the international wild bird surveillance program of Erasmus Medical Center (Technical Appendix Table). A maximum-likelihood phylogenetic tree of the hemagglutinin gene was generated by using PhyML version 3.1 (8) with the general time reversible +I+Γ model of nucleotide substitution; a full heuristic search and subtree pruning and regrafting searches were performed. The tree was visualized by using Figtree version 1.4.0 (http://tree.bio.ed.ac.uk/software/figtree). To further elucidate the role of influenza A(H10N7) virus in the pathogenesis of the disease causing deaths among the seals, we conducted immunohistochemical analysis on the lungs using an influenza A virus nucleoprotein-specific monoclonal antibody (9). Evaluation of the lung tissues of the dead seals showed viral antigen in cytoplasm and nuclei of epithelial cells of bronchi and bronchial glands of affected lung areas (Figure, panel B), which suggests that this virus played a major role in the deaths. Immunohistochemical analysis performed on various organs (including brain and olfactory bulb) indicated that viral antigen was restricted to the respiratory tract. Although avian influenza A virus infections previously have caused mass deaths in seals (3,4,10), subtype H10N7 has not been associated with such events. We can speculate that the ongoing deaths could eventually affect all harbor seal populations of northwestern Europe and have consequences for wildlife management and seal rehabilitation activities. In addition, preliminary analysis of the hemagglutinin sequence of the influenza A(H10N7) virus suggests the presence of molecular determinants that indicate mammalian adaptation. Various analyses are ongoing to answer questions about the route of transmission among seals and possible transmissibility to humans. Note added in proof: Zohari et al. also recently reported the involvement of avian influenza A(H10N7) virus in mass deaths of harbor seals in Sweden (Euro Surveill. 2014;19:pii: 20967). Technical Appendix: Details of hemagglutinin sequences shown in the Figure. Click here to view.(22K, pdf)

Molecular assays for quantitative and qualitative detection of influenza virus and oseltamivir resistance mutations.

Sensitive and reproducible molecular assays are essential for influenza virus diagnostics. This manuscript describes the design, validation, and evaluation of a set of real-time RT-PCR assays for quantification and subtyping of human influenza viruses from patient respiratory material. Four assays are included for detection of oseltamivir resistance mutations H275Y in prepandemic and pandemic influenza A/H1N1 and E119V and R292K in influenza A/H3N2 neuraminidase. The lower limits of detection of the quantification assay were determined to be 1.7 log(10) virus particles per milliliter (vp/mL) for influenza A and 2.2 log(10) vp/mL for influenza B virus. The lower limits of quantification were 2.1 and 2.3 log(10) vp/mL, respectively. The RT-PCR efficiencies and lower limits of detection of the quantification assays were only marginally affected when tested on the most dissimilar target sequences found in the GenBank database. Finally, the resistance RT-PCR assays detected at least 5% mutant viruses present in mixtures containing both wild-type and mutant viruses with approximated limits of detection of 2.4 log(10) vp/mL. Overall, this set of RT-PCR assays is a powerful tool for enhanced influenza virus surveillance.

Evidence for influenza virus CNS invasion along the olfactory route in an immunocompromised infant

Central nervous system (CNS) disease is the most common extrarespiratory complication of influenza in humans. However, the pathogenesis, including the route of virus entry, is largely unknown. Here we present, for the first time, evidence of influenza virus entry into the CNS via the olfactory route in an immune-compromised infant. Since the nasal cavity is a primary site of influenza virus replication and is directly connected to the CNS via the olfactory nerve, these results imply that influenza virus invasion of the CNS may occur more often than previously believed.

The potential for multidrug-resistant influenza.

Purpose of review The 2009 influenza pandemic introduced a new influenza A/H1N1 subtype in the human population. This pandemic 2009 influenza A/H1N1 virus has natural resistance to the adamantanes class and has a low threshold to become resistant to the neuraminidase class of antiviral drugs. This review describes recent findings on influenza antiviral resistance in pandemic 2009 influenza A/H1N1 virus. Recent findings Pandemic 2009 viruses have emerged with novel resistance patterns to the neuraminidase inhibitors. In addition, the identification of mutations that facilitated oseltamivir resistance in prepandemic influenza emphasizes the ability of influenza to become resistant to antiviral drugs without significant loss of fitness. Summary Novel initiatives are required to find and develop high genetic barrier influenza therapeutic regimens for effective treatment of severe influenza virus infections.