Antiviral susceptibility of variant influenza A(H3N2)v viruses isolated in the United States from 2011 to 2013.

Since 2011, outbreaks caused by influenza A(H3N2) variant [A(H3N2)v] viruses have become a public health concern in the United States. The A(H3N2)v viruses share the A(H1N1)pdm09 M gene containing the marker of M2 blocker resistance, S31N, but do not contain any known molecular markers associated with resistance to neuraminidase (NA) inhibitors (NAIs). Using a fluorescent NA inhibition (NI) assay, the susceptibilities of recovered A(H3N2)v viruses (n=168) to FDA-approved (oseltamivir and zanamivir) and other (peramivir, laninamivir, and A-315675) NAIs were assessed. All A(H3N2)v viruses tested, with the exception of a single virus strain, A/Ohio/88/2012, isolated from an untreated patient, were susceptible to the NAIs tested. The A/Ohio/88/2012 virus contained two rare substitutions, S245N and S247P, in the NA and demonstrated reduced inhibition by oseltamivir (31-fold) and zanamivir (66-fold) in the NI assay. Using recombinant NA (recNA) proteins, S247P was shown to be responsible for the observed altered NAI susceptibility, in addition to an approximately 60% reduction in NA enzymatic activity. The S247P substitution has not been previously reported as a molecular marker of reduced susceptibility to the NAIs. Using cell culture assays, the investigational antiviral drugs nitazoxanide, favipiravir, and fludase were shown to inhibit the replication of A(H3N2)v viruses, including the virus with the S247P substitution in the NA. This report demonstrates the importance of continuous monitoring of susceptibility of zoonotic influenza viruses to available and investigational antiviral drugs.

Standardizing the influenza neuraminidase inhibition assay among United States public health laboratories conducting virological surveillance.

BACKGROUND: Monitoring influenza virus susceptibility to neuraminidase (NA) inhibitors (NAIs) is vital for detecting drug-resistant variants, and is primarily assessed using NA inhibition (NI) assays, supplemented by NA sequence analysis. However, differences in NI testing methodologies between surveillance laboratories results in variability of 50% inhibitory concentration (IC50) values, which impacts data sharing, reporting and interpretation. In 2011, the Centers for Disease Control and Prevention (CDC), in collaboration with the Association for Public Health Laboratories (APHL) spearheaded efforts to standardize fluorescence-based NI assay testing in the United States (U.S.), with the goal of achieving consistency of IC50 data. METHODS: For the standardization process, three participating state public health laboratories (PHLs), designated as National Surveillance Reference Centers for Influenza (NSRC-Is), assessed the NAI susceptibility of the 2011-12 CDC reference virus panel using stepwise procedures, with support from the CDC reference laboratory. Next, the NSRC-Is assessed the NAI susceptibility of season 2011-12 U.S. influenza surveillance isolates (n = 940), with a large subset (n = 742) tested in parallel by CDC. Subsequently, U.S. influenza surveillance isolates (n = 9629) circulating during the next three influenza seasons (2012-15), were independently tested by the three NSRC-Is (n = 7331) and CDC (n = 2298). RESULTS: The NI assay IC50s generated by respective NSRC-Is using viruses and drugs prepared by CDC were similar to those obtained with viruses and drugs prepared in-house, and were uniform between laboratories. IC50s for U.S. surveillance isolates tested during four consecutive influenza seasons (2011-15) were consistent from season to season, within and between laboratories. CONCLUSION: These results show that the NI assay is robust enough to be standardized, marking the first time IC50 data have been normalized across multiple laboratories, and used for U.S. national NAI susceptibility surveillance.

[Change in regulation of influenza virus vRNA synthesis during adaptation to various hosts]. / Izmeneniia reguliatsii sinteza vRNK virusa grippa pri adaptatsii k raznym khoziaevam.

To evaluate the effect of laboratory passaging of influenza virus A/USSR/90/77 (H1N1) on the pattern of vRNA synthesis regulation in course of the one step infection cycle, we have used the viral variants adapted to growth in the continuous cell line MDCK or to the reproduction in the mice lungs in vivo. Enhancement of regulation was registered in the adapted variants as compared to the original virus strain. The results are discussed in connection with possible significance of the vRNA synthesis regulation for the efficiency of viral reproduction under natural conditions or in laboratory passaging.

[Synthesis of virus-specific RNA in cells infected with influenza B virus]. / Sintez virusspetsificheskikh RNK v kletkakh, zarazhennykh virusom grippa B.

Synthesis of virus-specific RNA in cells infected with influenza B virus was studied by means of PAGE analysis of RNA hybrid duplexes or nucleocapsid-associated RNA. A switch from an "early" to "late" pattern was registered in the relative rates of synthesis of the corresponding mRNA as well as in the synthesis of vRNA segments 7 and 8. Contrary to the pattern described earlier for influenza A virus, the relative rate of synthesis of mRNA 5 was increased at the late stage of the replication cycle; besides, the efficiency of transcription of the polymerase genes was higher. Partial suppression of protein synthesis with moderate concentration of cycloheximide at a late stage of infection resulted in a differential inhibition of vRNA segments synthesis and stimulation of mRNA synthesis, leading to restoration of an "early" pattern in both cases. The results confirm the general outline of regulation as presented for influenza A virus in an earlier publication and reveal several peculiarities in regulation of influenza B viral RNA synthesis.

[Regulation of synthesis of virus-specific RNA in cells infected with influenza virus]. / Reguliatsiia sinteza virusspetsificheskikh RNK v kletkakh, zarazhennykh virusom grippa.

Interrelationships between the level of protein synthesis and the pattern of virus-specific transcription in influenza virus-infected cells have been studied with the use of a wide range of concentrations of a protein synthesis inhibitor (cycloheximide). The obtained data reveal a predominant stimulation of the transcription for some viral genes by partial suppression of protein synthesis. The data also suggest the existence of cRNA (full transcripts) synthesis regulation, in addition to the regulation of vRNA and mRNA synthesis described earlier.

[Role of the NS gene in regulating the synthesis of RNA-segments of the influenza A virus]. / Uchastie gena NS v reguliatsii sinteza RNK-segmentov virusa grippa A.

To find the role of any influenza virus gene in regulation of the RNA-segments replication the transfer of ts-mutants to nonpermissive temperature on the late step of infection has been used (shift-up). The mutants having impaired the NS or NP-genes have been obtained and studied. The transfer of mutants to partially nonpermissive conditions (when the amount of replication is decreased, but it still continues) results in the distinct return to the early mode of replication in ts-mutant with the mutation in NS-gene. This suggests the NS-gene role in replication of viral RNA-segments, in particular, in the switch from the "early" stage of replication to the "late" one. In NP-gene mutant only the decrease in general replication takes place without the shift to "early" replication mode.

[The role of polymerase protein genes of influenza A virus in the transition from the early to late stage of replication]. / Rol' genov polimeraznykh belkov virusa grippa A v perekhode ot rannei stadii replikatsii k pozdnei.

Regulation of influenza virus RNA replication was studied with the use of A/FPV/Rostock/34 strain ts-mutants. Mutants C44, C15, C45 possessing the ts-defects in the PB2, PB1 and PA genes respectively were used for the infection of chick embryo cultured cells and H-uridine-labelled nucleocapsid-associated RNA was analysed in polyacrylamide gel electrophoresis to assess the kinetics of vRNA synthesis. A typical early-late transition of the pattern of vRNA synthesis was observed in the cells infected with C44, whereas the other two mutants exhibited a slightly changed (C15) or strongly distorted (C45) pattern. In shift up experiments after the transfer to non-permissive temperature all the mutants exhibited partial reversion to an early pattern of vRNA synthesis. The results are discussed in connection with the mechanism of the early-late transition of influenza virus-specific RNA synthesis.

[Analysis of the reassorted influenza virus clone genome: data for use of ordered selection of RNA segments]. / Analiz genoma reassortantnykh klonov virusa grippa: dannye v pol'zu uporiadochennogo podbora RNK-segmentov.

Chicken embryonated eggs were coinfected with influenza A/FPV/Rostock and A/FPV/Weybridge strains. 25 plaque isolates were obtained from the mixed yield and their genetic content was analysed by polyacrylamide gel electrophoresis of H3-uridine-labelled vRNA in a modified gel system. At least 18 clones out of 25 plaque isolates proved to be reassortants; however, only one among them contained the homologous RNA-segments belonging to both parents. The results are in agreement with the concept of an ordered recruitment of vRNA-segments into virions.

[Changes in natural killer activity in the influenza virus infection of mice and in treating lymphocytes with the influenza virus and its proteins in vitro]. / Izmenenie aktivnosti estestvennykh killerov pri zarazhenii myshei virusom grippa i pri obrabotke limfotsitov virusom grippa i ego belkami in vitro.

Changes in the activity of natural cytotoxic lymphocytes (natural killers, NK) were studied in the course of infection of CBA mice with a mouse-adapted or unadapted (epidemic) variants of influenza A/USSR/90/77 virus. The activity of NK against K562 target cells in the peripheral blood, lungs, and spleens was found to increase considerably 18 hours after intranasal inoculation with any of the variants and to persist at a high level for up to 72 hours. Virus replication in the lungs and peripheral blood was observed only in mice infected with the adapted virus variant. A conclusion was drawn that the increase in NK activity in the organs of influenza virus-infected mice was nonspecific providing no protection against a fatal outcome after infection with the mouse-adapted (pathogenic) virus. Treatment of lymphocytes recovered from the peripheral blood, lungs, and spleens of infected and non-infected mice in vitro with preparations of intact viruses and isolated viral proteins exerted different effects on the NK activity: enhancement after treatment with intact viruses and inhibition by isolated viral proteins. The effect did not depend on the virulence of the strain from which viral proteins were isolated. The importance of the phenomenon of different modulating effect of influenza virus and its structural proteins on the NK activity is discussed.