Influenza virus surveillance in Argentina during the 2012 season: antigenic characterization, genetic analysis and antiviral susceptibility.

The activity and circulation of influenza viruses in Argentina was studied during 2012 as part of the Argentinean Surveillance for Influenza and other Respiratory Viruses, in the context of Global Influenza Surveillance. The antigenicity and molecular characteristics of haemagglutinins (HA) of circulating influenza A and B viruses were analysed to assess the emergence of virus variants. Susceptibility to oseltamivir and zanamivir was evaluated by enzymatic assay and results were backed-up by sequencing of the neuraminidase (NA) genes. During the 2012 season, influenza virus circulation in Argentina was detected from weeks 24 to 51. The HA sequences of the studied A(H1N1)pdm09 subtype viruses segregated in a different genetic group compared to those identified during the 2009 pandemic, although they were still closely related antigenically to the vaccine virus A/California/07/2009. The HA sequences of the A(H3N2) viruses analysed fell into the A/Victoria/208/2009 clade, genetic group 3C. A mixed circulation of virus variants belonging to B/Victoria and B/Yamagata lineages was detected, with B/Victoria being dominant. All viruses tested were sensitive to oseltamivir and zanamivir except one. This isolate, an A(H1N1)pdm09 virus possessing the substitution NA-N295S, showed highly reduced inhibition by oseltamivir and reduced inhibition by zanamivir. Virological and epidemiological surveillance remains critical for detection of evolving influenza viruses.

Strain-specific antiviral activity of iminosugars against human influenza A viruses.

OBJECTIVES: Drugs that target host cell processes can be employed to complement drugs that specifically target viruses, and iminosugar compounds that inhibit host α-glucosidases have been reported to show antiviral activity against multiple viruses. Here the effect and mechanism of two iminosugar α-glucosidase inhibitors, N-butyl-deoxynojirimycin (NB-DNJ) and N-nonyl-deoxynojirimycin (NN-DNJ), on human influenza A viruses was examined. METHODS: The viruses examined were a recently circulating seasonal influenza A(H3N2) virus strain A/Brisbane/10/2007, an older H3N2 strain A/Udorn/307/72, and A/Lviv/N6/2009, a strain representative of the currently circulating pandemic influenza A(H1N1)pdm09 virus. RESULTS: The inhibitors had the strongest effect on Brisbane/10 and NN-DNJ was more potent than NB-DNJ. Both compounds showed antiviral activity in cell culture against three human influenza A viruses in a strain-specific manner. Consistent with its action as an α-glucosidase inhibitor, NN-DNJ treatment resulted in an altered glycan processing of influenza haemagglutinin (HA) and neuraminidase (NA), confirmed by MS. NN-DNJ treatment was found to reduce the cell surface expression of the H3 subtype HA. The level of sialidase activity of NA was reduced in infected cells, but the addition of exogenous sialidase to the cells did not complement the NN-DNJ-mediated inhibition of virus replication. Using reassortant viruses, the drug susceptibility profile was determined to correlate with the origin of the HA. CONCLUSIONS: NN-DNJ inhibits influenza A virus replication in a strain-specific manner that is dependent on the HA.

Estimating reassortment rates in co-circulating Eurasian swine influenza viruses.

Swine have often been considered as a mixing vessel for different influenza strains. In order to assess their role in more detail, we undertook a retrospective sequencing study to detect and characterize the reassortants present in European swine and to estimate the rate of reassortment between H1N1, H1N2 and H3N2 subtypes with Eurasian (avian-like) internal protein-coding segments. We analysed 69 newly obtained whole genome sequences of subtypes H1N1-H3N2 from swine influenza viruses sampled between 1982 and 2008, using Illumina and 454 platforms. Analyses of these genomes, together with previously published genomes, revealed a large monophyletic clade of Eurasian swine-lineage polymerase segments containing H1N1, H1N2 and H3N2 subtypes. We subsequently examined reassortments between the haemagglutinin and neuraminidase segments and estimated the reassortment rates between lineages using a recently developed evolutionary analysis method. High rates of reassortment between H1N2 and H1N1 Eurasian swine lineages were detected in European strains, with an average of one reassortment every 2-3 years. This rapid reassortment results from co-circulating lineages in swine, and in consequence we should expect further reassortments between currently circulating swine strains and the recent swine-origin H1N1v pandemic strain.

The glycosylation pattern of baculovirus expressed envelope protein E2 affects its ability to prevent infection with bovine viral diarrhoea virus.

We have investigated the role of glycosylation of the envelope glycoprotein E2 of bovine viral diarrhoea virus (BVDV), produced in insect cells, in BVDV infection. When amino acids predicated to code for the C-terminal N-linked glycosylation site were mutated the resulting protein was less efficient than wild type protein at preventing infection of susceptible cells with BVDV. In addition, mutational analysis showed that a further two predicted N-terminal N-linked glycosylation sites of E2 are required for efficient production of recombinant protein.

Glycosylation of haemagglutinin and stalk-length of neuraminidase combine to regulate the growth of avian influenza viruses in tissue culture.

The influence on virus replication in culture of the presence and location of glycosylation sites on the haemagglutinin (HA) glycoprotein of avian influenza viruses and differences in length of the stalk region of their neuraminidase (NA) glycoprotein was examined using reassortant viruses. Plaque size was measured in the presence or absence of bacterial neuraminidase (CPNA) and/or an influenza virus NA inhibitor, zanamivir, to assess the relative contribution of the NA to replication efficiency in tissue culture. The following conclusions were drawn, (1) HA lacking glycosylation at 158 gives inefficient growth when combined with short-stalked NAs, and efficient growth when combined with long-stalked NAs. (2) Glycosylation at 158 of HA makes the virus less dependent on NA for release from its receptors. (3) HA with glycosylation at 158 gives efficient growth when combined with short-stalked NAs but, when combined with long-stalked NAs, growth is very efficient and excess NA activity is disadvantageous. (4) HA having glycosylation at 158 combined with short-stalked NAs, or HA lacking glycosylation at 158 combined with long-stalked NAs may represent optimal combinations. The results reinforce the importance of a balance of HA and NA activity for efficient virus exit from, and entry into cells.

The critical cut-off temperature of avian influenza viruses.

We have measured the pathogenicity for 6-week-old chicks of infection by H7 avian influenza viruses. One virus, strain S3 from A/FPV/Rostock/34(H7N1) showed a temperature sensitive phenotype at 41.5 degrees C and reduced pathogenicity. By analysis of reassortants made between virus S3 and A/FPV/Dobson/27(H7N7), a fully pathogenic virus, two conclusions arise. (1) The critical cut-off temperature for avian influenza virus in 6-week-old chicks is 41.5 degrees. (2) RNA segment 1 of virus S3 is responsible for the lack of pathogenicity in reassortant viruses. Nucleotide sequencing of RNA segment 1 from S3 and its parent, A/FPV/Rostock/34 has revealed a single mutation at nucleotide 1561. This results in a substitution of isoleucine for leucine at amino acid position 512 in the cap-binding protein, PB2.

The complete nucleotide sequence of a pathogenic swine vesicular disease virus.

The nucleotide sequence of a swine vesicular disease virus (SVDV) strain that is pathogenic for pigs has been determined and compared with that of a non-pathogenic strain of SVDV, as well as a number of other enteroviruses. It shows only 98 base changes in comparison with a non-pathogenic strain of SVDV (Inoue et al., 1989, J. Gen. Virol. 70, 919-934). Fourteen of these nucleotide differences between the pathogenic and the non-pathogenic SVDV strains occur in the 5' non-coding region which, by analogy with the other picornaviruses, has been implicated in the efficiency with which the RNA is employed as mRNA. Additional differences found throughout the coding regions are largely conservative in nature. A number of residues are discussed as candidates for determinants of pathogenicity. This sequence has been submitted to the PIR database and has accession number A30061.

A rapid method for the analysis of influenza virus genes: application to the reassortment of equine influenza virus genes.

We describe a rapid method for genetic characterisation of influenza virus genes using reverse transcription and amplification by polymerase chain reaction (RT/PCR) of all virus segments simultaneously (multiplex RT/PCR) using primers based on the conserved terminal sequences. The product has been shown to be suitable for determination of partial nucleotide sequences which can be used to search nucleotide sequence databases and rapidly map the genetic origin of each segment. We illustrate the use of the method by analysing genetic reassortment in H7N7 equine influenza viruses.

Characterisation of the influenza virus associated protein kinase and its resemblance to casein kinase II.

The protein kinase activity associated with purified influenza virus has been examined. By use of a radiolabelled photoreactive ATP analogue (3'-O-(4-benzoyl) benzoyl adenosine triphosphate) a 47 kD polypeptide has been identified that binds ATP. A comparison of the sensitivity of the kinase activity and the 47 kDa polypeptide labelling to inhibitors indicate that the 47 kDa polypeptide is likely to represent the major protein kinase activity of the virus. The virus associated protein kinase phosphorylates the synthetic peptide RREEETEEE, a peptide substrate for casein kinase II. Antiserum directed against casein kinase II revealed a positive signal in immunoblots of purified virus. We propose that the major protein kinase activity associated with purified virus preparations is host cell casein kinase II.

Sequence analysis of the equine H7 influenza virus haemagglutinin gene.

The nucleotide sequences of ten haemagglutinin genes of representative H7N7 equine influenza viruses isolated between 1956 and 1977 have been determined by primer extension sequencing. Their nucleotide and deduced amino acid sequences demonstrate a high degree of homology. These equine viruses can be divided into two distinct subgroups, the prototype-like, and a group comprising the early American isolates and the remaining equine viruses. The equine H7 haemagglutinins form a quite distinct group compared to H7 haemagglutinins isolated from other species. Each of these equine H7 haemagglutinins possess a tetrabasic amino acid cleavage site separating the HA1 and HA2 domains but, in addition, all ten contain a nine amino acid insertion prior to the tetrabasic sequence. The haemagglutinin glycoproteins of all ten viruses are capable of cleavage activation in virus infected primary chicken embryo fibroblast cells.

Inhibition of the growth of influenza viruses in vitro by 4-guanidino-2,4-dideoxy-N-acetylneuraminic acid.

The sialidase inhibitor 4-guanidino-2,4-dideoxy-2,3-dehydro-N- acetylneuraminic acid was tested for growth inhibitory effects against a panel of avian influenza A viruses encompassing all nine neuraminidase subtypes. Growth in tissue culture of viruses from each subtype was inhibited by this compound at concentrations within a range previously found effective against human N1 and N2 viruses. This compound may prove a selective agent for the treatment (and prevention) of influenza virus infections.

4-Guanidino-Neu5Ac2en fails to protect chickens from infection with highly pathogenic avian influenza virus.

The effectiveness of the novel sialidase inhibitor 4-guanidino-Neu5Ac2en, which is highly effective in mouse and ferret models of influenza virus infection (von Itzstein et al. (1993) Nature 363, 418-423), has been assessed as a prophylactic agent in the prevention of infection of chickens with highly pathogenic avian influenza viruses. At best a small delay in the onset of pyrexia and death was observed with one strain of fowl plague virus, but not with two other strains. These results demonstrate that a locally acting drug may be ineffective if virus can escape from the site of inoculation and replicate elsewhere.

Mutational changes in the hemagglutinin of equine H3 influenza viruses result in the introduction of a glycosylation site which enhances the infectivity of the viruses.

The complete amino acid sequences of the hemagglutinin (HA) glycoprotein of three equine-2 influenza viruses from tropical Africa are presented in comparison with that of a well characterized European equine-2 virus (Suffolk/89) and a consensus sequence from the database. The sequences of the tropical African viruses were deduced from the complete nucleotide sequences of their HA genes reported earlier. Mutational changes in the nucleotide sequences resulted in amino acid changes in the HA which led to the introduction of a new asparagine-linked (N-linked) glycosylation site in two viruses. This new glycosylation site enhanced the infectivity of these viruses as investigated by plaque assay, virus titration in embryonated chicken eggs and tunicamycin treatment. The role of N-linked glycosylation of influenza virus HA glycoprotein in virus infectivity, antigenicity and immunogenicity is discussed in the light of the results of our previous and present investigations.

Prolonged nasal shedding and viraemia of cytopathogenic bovine virus diarrhoea virus in experimental late-onset mucosal disease.

A calf persistently infected with bovine virus diarrhoea virus (BVDV) was super-infected with a heterologous BVDV strain, C874, which contained non-cytopathogenic and cytopathogenic viruses. High titres of cytopathogenic BVDV were recovered in the three to four weeks after the challenge. Thereafter low titres of cytopathogenic virus were recovered repeatedly from the blood and the nose, with the titres in nasal secretions increasing in the four weeks before the onset of clinical signs. Neutralising antibodies against the challenge cytopathic virus (C874cp) were first detected 21 days after the super-infection, but these antibodies failed to neutralise the persisting non-cytopathogenic and cytopathogenic viruses isolated from the animal during the course of the infection. Serum collected from 105 days after the super-infection neutralised the cytopathogenic viruses isolated on day 105 and postmortem. These data indicate that unaltered wild-type C874cp was not directly responsible for the late-onset mucosal disease.

Aptamer-based biosensors for the rapid visual detection of flu viruses.

RNA aptamers showing affinity and specificity for different strains of human influenza virus were assembled onto gold nanoparticles that subsequently formed a gold nanoshell (AuNS) around the viral envelope. These shells could be visualised by transmission electron microscopy (TEM). Changes in size and structure of the AuNS coated virus can be used to detect the viruses. We show that sedimentation with a low cost centrifuge and visual determination can detect 3 × 10(8) viral particles.

Influenza gain-of-function experiments: their role in vaccine virus recommendation and pandemic preparedness.

In recent years, controversy has arisen regarding the risks and benefits of certain types of gain-of-function (GOF) studies involving avian influenza viruses. In this article, we provide specific examples of how different types of data, including information garnered from GOF studies, have helped to shape the influenza vaccine production process-from selection of candidate vaccine viruses (CVVs) to the manufacture and stockpiling of safe, high-yield prepandemic vaccines for the global community. The article is not written to support a specific pro- or anti-GOF stance but rather to inform the scientific community about factors involved in vaccine virus selection and the preparation of prepandemic influenza vaccines and the impact that some GOF information has had on this process.

Evaluation of influenza virus antiviral susceptibility testing in Europe: results from the first external quality assessment exercise.

BACKGROUND: The first antiviral susceptibility testing external quality assessment (EQA) was held for European influenza reference laboratories during winter 2010/11. OBJECTIVES: To assess European network influenza antiviral susceptibility testing capability and provide participants with an independent performance evaluation. STUDY DESIGN: The EQA panel contained ten coded specimens of inactivated human influenza A and B viruses with reduced susceptibility to neuraminidase inhibitors (NAI), or adamantanes. Twenty-four laboratories from 19 member states of the WHO European region analysed the panel using phenotypic (determination of 50% inhibitory concentration (IC(50)) values by neuraminidase (NA) enzyme inhibition assay) and/or genotypic methods. RESULTS: All 24 laboratories returned genotypic data for A(H1N1)pdm09 influenza virus, 18 (75%) for former seasonal A(H1N1), 16 (67%) for A(H3N2) and 15 (63%) for influenza B virus, correctly identifying NAI or adamantane reduced susceptibility-associated substitutions in the NA (mean 84%; range 52-100%) or M2 (mean 85%; range 73-94%), respectively. Thirteen laboratories (54%) returned phenotypic NAI susceptibility data. Despite inter-laboratory and inter-assay IC(50) value variation, all 13 laboratories correctly identified oseltamivir reduced susceptibility/resistance in pure preparations of A(H1N1) oseltamivir-resistant viruses. However, only 11 (85%) identified oseltamivir reduced susceptibility/resistance in a mixture of A(H1N1)pdm09 oseltamivir-sensitive/-resistant viruses. Furthermore, 3 laboratories (23%) considered oseltamivir-sensitive influenza B virus reduced susceptible/resistant. CONCLUSIONS: Detection of NA-H275Y in A(H1N1) viruses was achieved by most laboratories. IC(50) values and interpretation thereof varied for a sensitive/resistant virus mixture and for influenza B virus. The results of this exercise will assist harmonisation of antiviral susceptibility testing, interpretation and reporting within the European network through targeted training.

Enhanced mechanical properties of nanocrystalline boron carbide by nanoporosity and interface phases.

Ceramics typically have very high hardness, but low toughness and plasticity. Besides intrinsic brittleness associated with rigid covalent or ionic bonds, porosity and interface phases are the foremost characteristics that lead to their failure at low stress levels in a brittle manner. Here we show that, in contrast to the conventional wisdom that these features are adverse factors in mechanical properties of ceramics, the compression strength, plasticity and toughness of nanocrystalline boron carbide can be noticeably improved by introducing nanoporosity and weak amorphous carbon at grain boundaries. Transmission electron microscopy reveals that the unusual nanosize effect arises from the deformation-induced elimination of nanoporosity mediated by grain boundary sliding with the assistance of the soft grain boundary phases. This study has important implications in developing high-performance ceramics with ultrahigh strength and enhanced plasticity and toughness.