Selection of DNA aptamers that bind to influenza A viruses with high affinity and broad subtype specificity.

Many cases of influenza are reported worldwide every year. The influenza virus often acquires new antigenicity, which is known as antigenic shift; this results in the emergence of new virus strains, for which preexisting immunity is not found in the population resulting in influenza pandemics. In the event a new strain emerges, diagnostic tools must be developed rapidly to detect the novel influenza strain. The generation of high affinity antibodies is costly and takes time; therefore, an alternative detection system, aptamer detection, provides a viable alternative to antibodies as a diagnostic tool. In this study, we developed DNA aptamers that bind to HA1 proteins of multiple influenza A virus subtypes by the SELEX procedure. To evaluate the binding properties of these aptamers using colorimetric methods, we developed a novel aptamer-based sandwich detection method employing our newly identified aptamers. This novel sandwich enzyme-linked aptamer assay successfully detected the H5N1, H1N1, and H3N2 subtypes of influenza A virus with almost equal sensitivities. These findings suggest that our aptamers are attractive candidates for use as simple and sensitive diagnostic tools that need sandwich system for detecting the influenza A virus with broad subtype specificities.

No evidence of a link between influenza vaccines and Guillain-Barre syndrome-associated antiganglioside antibodies.

BACKGROUND: Guillain-Barre syndrome (GBS) is a rare autoimmune disease characterized by acute, progressive peripheral neuropathy and is commonly associated with the presence of antiganglioside antibodies. Previously, influenza vaccination was linked with the increased incidence of GBS; however, whether antiganglioside antibodies are subsequently induced remains unresolved. METHODS: Sera from human subjects vaccinated with seasonal influenza vaccines from the 2007-2008, 2008-2009, or 1976-1977 influenza seasons were screened for the induction of immunity to influenza and the presence of antiganglioside antibodies pre- and post-vaccination. Likewise, sera from mice vaccinated with seasonal influenza vaccines (1988-1989, 2007-2008) or "swine flu" pandemic vaccines (1976, 2009) were assessed in the same manner. Viruses were also screened for cross-reacting ganglioside epitopes. RESULTS: Antiganglioside antibodies were found to recognize influenza viruses; this reactivity correlated with virus glycosylation. Antibodies to influenza viruses were detected in human and mouse sera, but the prevalence of antiganglioside antibodies was extremely low. CONCLUSIONS: Although the correlation between antiganglioside antibody cross-reactivity and glycosylation of viruses suggests the role of shared carbohydrate epitopes, no correlation was observed between hemagglutinin-inhibition titers and the induction of antiganglioside antibodies after influenza vaccination.

Novel genotyping and quantitative analysis of neuraminidase inhibitor resistance-associated mutations in influenza a viruses by single-nucleotide polymorphism analysis.

Neuraminidase (NA) inhibitors are among the first line of defense against influenza virus infection. With the increased worldwide use of the drugs, antiviral susceptibility surveillance is increasingly important for effective clinical management and for public health epidemiology. Effective monitoring requires effective resistance detection methods. We have developed and validated a novel genotyping method for rapid detection of established NA inhibitor resistance markers in influenza viruses by single nucleotide polymorphism (SNP) analysis. The multi- or monoplex SNP analysis based on single nucleotide extension assays was developed to detect NA mutations H275Y and I223R/V in pandemic H1N1 viruses, H275Y in seasonal H1N1 viruses, E119V and R292K in seasonal H3N2 viruses, and H275Y and N295S in H5N1 viruses. The SNP analysis demonstrated high sensitivity for low-content NA amplicons (0.1 to 1 ng/µl) and showed 100% accordant results against a panel of defined clinical isolates. The monoplex assays for the H275Y NA mutation allowed precise and accurate quantification of the proportions of wild-type and mutant genotypes in virus mixtures (5% to 10% discrimination), with results comparable to those of pyrosequencing. The SNP analysis revealed the lower growth fitness of an H275Y mutant compared to the wild-type pandemic H1N1 virus by quantitatively genotyping progeny viruses grown in normal human bronchial epithelial cells. This novel method offers high-throughput screening capacity, relatively low costs, and the wide availability of the necessary equipment, and thus it could provide a much-needed approach for genotypic screening of NA inhibitor resistance in influenza viruses.

Oseltamivir-resistant pandemic H1N1/2009 influenza virus possesses lower transmissibility and fitness in ferrets.

The neuraminidase (NA) inhibitor oseltamivir offers an important immediate option for the control of influenza, and its clinical use has increased substantially during the recent H1N1 pandemic. In view of the high prevalence of oseltamivir-resistant seasonal H1N1 influenza viruses in 2007-2008, there is an urgent need to characterize the transmissibility and fitness of oseltamivir-resistant H1N1/2009 viruses, although resistant variants have been isolated at a low rate. Here we studied the transmissibility of a closely matched pair of pandemic H1N1/2009 clinical isolates, one oseltamivir-sensitive and one resistant, in the ferret model. The resistant H275Y mutant was derived from a patient on oseltamivir prophylaxis and was the first oseltamivir-resistant isolate of the pandemic virus. Full genome sequencing revealed that the pair of viruses differed only at NA amino acid position 275. We found that the oseltamivir-resistant H1N1/2009 virus was not transmitted efficiently in ferrets via respiratory droplets (0/2), while it retained efficient transmission via direct contact (2/2). The sensitive H1N1/2009 virus was efficiently transmitted via both routes (2/2 and 1/2, respectively). The wild-type H1N1/2009 and the resistant mutant appeared to cause a similar disease course in ferrets without apparent attenuation of clinical signs. We compared viral fitness within the host by co-infecting a ferret with oseltamivir-sensitive and -resistant H1N1/2009 viruses and found that the resistant virus showed less growth capability (fitness). The NA of the resistant virus showed reduced substrate-binding affinity and catalytic activity in vitro and delayed initial growth in MDCK and MDCK-SIAT1 cells. These findings may in part explain its less efficient transmission. The fact that the oseltamivir-resistant H1N1/2009 virus retained efficient transmission through direct contact underlines the necessity of continuous monitoring of drug resistance and characterization of possible evolving viral proteins during the pandemic.

Drugs in development for influenza.

The emergence and global spread of the 2009 pandemic H1N1 influenza virus reminds us that we are limited in the strategies available to control influenza infection. Vaccines are the best option for the prophylaxis and control of a pandemic; however, the lag time between virus identification and vaccine distribution exceeds 6 months and concerns regarding vaccine safety are a growing issue leading to vaccination refusal. In the short-term, antiviral therapy is vital to control the spread of influenza. However, we are currently limited to four licensed anti-influenza drugs: the neuraminidase inhibitors oseltamivir and zanamivir, and the M2 ion-channel inhibitors amantadine and rimantadine. The value of neuraminidase inhibitors was clearly established during the initial phases of the 2009 pandemic when vaccines were not available, i.e. stockpiles of antivirals are valuable. Unfortunately, as drug-resistant variants continue to emerge naturally and through selective pressure applied by use of antiviral drugs, the efficacy of these drugs declines. Because we cannot predict the strain of influenza virus that will cause the next epidemic or pandemic, it is important that we develop novel anti-influenza drugs with broad reactivity against all strains and subtypes, and consider moving to multiple drug therapy in the future. In this article we review the experimental data on investigational antiviral agents undergoing clinical trials (parenteral zanamivir and peramivir, long-acting neuraminidase inhibitors and the polymerase inhibitor favipiravir [T-705]) and experimental antiviral agents that target either the virus (the haemagglutinin inhibitor cyanovirin-N and thiazolides) or the host (fusion protein inhibitors [DAS181], cyclo-oxygenase-2 inhibitors and peroxisome proliferator-activated receptor agonists).

Emergence of H5N1 avian influenza viruses with reduced sensitivity to neuraminidase inhibitors and novel reassortants in Lao People's Democratic Republic.

Pandemic influenza viruses can emerge through continuous evolution and the acquisition of specific mutations or through reassortment. This study assessed the pandemic potential of H5N1 viruses isolated from poultry outbreaks occurring from July 2006 to September 2008 in the Lao People's Democratic Republic (PDR). We analyzed 29 viruses isolated from chickens and ducks and two from fatal human cases in 2007. Prior to 2008, all H5N1 isolates in Lao PDR were from clade 2.3.4; however, clade 2.3.2 was introduced in September 2008. Of greatest concern was the circulation of three isolates that showed reduced sensitivity to the neuraminidase (NA) inhibitor oseltamivir in an enzyme inhibition assay, each with different NA mutations - V116A, I222L and K150N, and a previously unreported S246N mutation. In addition, six isolates had an S31N mutation in the M2 protein, which conferred resistance to amantadine not previously reported in clade 2.3.4 viruses. Two H5N1 reassortants were isolated whose polymerase genes, PB1 and PB2, were homologous to those of Eurasian viruses giving rise to a novel H5N1 genotype, genotype P. All H5N1 viruses retained avian-like receptor specificity, but four had altered affinities for alpha2,3-linked sialic acid. This study shows that, in a genetically similar population of H5N1 viruses in Lao PDR, mutants emerged with natural resistance to antivirals and altered affinities for alpha2,3-linked sialic acids, together with reassortants with polymerase genes homologous to Eurasian viruses. These changes may contribute to the emergence of a pandemic influenza strain and are critical in devising surveillance strategies.

Field assessment of an H5N1 inactivated vaccine in chickens and ducks in Lao PDR.

Despite the extensive use of poultry vaccines to control the spread of H5N1 influenza in poultry, H5N1 outbreaks continue to occur in domestic birds. Our objective was to determine the duration of the neutralizing antibody response under field conditions after vaccination with a laboratory-tested inactivated reverse genetics-derived H5N3 vaccine. H5N3 hemagglutination inhibition (HI) and virus neutralization (VN) antibodies were observed 40 weeks after vaccination of chickens with two doses and vaccination of ducks with one dose. Cross-clade antibodies to an H5N1 virus (A/chicken/Laos/A0464/07) antigenically distinct from the vaccine strain were detected in ducks after a single vaccination and were sustained for 28 weeks (for 40 weeks when a boost vaccination was given). Our results indicate that this inactivated H5N3 vaccine can produce long-lasting antibodies to homologous and heterologous viruses under field conditions.

TNF/iNOS-producing dendritic cells are the necessary evil of lethal influenza virus infection.

Respiratory infection with highly pathogenic influenza A viruses is characterized by the exuberant production of cytokines and chemokines and the enhanced recruitment of innate inflammatory cells. Here, we show that challenging mice with virulent influenza A viruses, including currently circulating H5N1 strains, causes the increased selective accumulation of a particular dendritic cell subset, the tipDCs, in the pneumonic airways. These tipDCs are required for the further proliferation of influenza-specific CD8(+) T cells in the infected lung, because blocking their recruitment in CCR2(-/-) mice decreases the numbers of CD8(+) effectors and ultimately compromises virus clearance. However, diminution rather than total elimination of tipDC trafficking by treatment with the peroxisome proliferator-activated receptor-gamma agonist pioglitazone moderates the potentially lethal consequences of excessive tipDC recruitment without abrogating CD8(+) T cell expansion or compromising virus control. Targeting the tipDCs in this way thus offers possibilities for therapeutic intervention in the face of a catastrophic pandemic.

Changes in H5N1 influenza virus hemagglutinin receptor binding domain affect systemic spread.

The HA of influenza virus is a receptor-binding and fusion protein that is required to initiate infection. The HA receptor-binding domain determines the species of sialyl receptors recognized by influenza viruses. Here, we demonstrate that changes in the HA receptor-binding domain alter the ability of the H5N1 virus to spread systemically in mice. The A/Vietnam/1203/04 (VN1203) and A/Hong Kong/213/03 (HK213) viruses are consistently lethal to domestic chickens but differ in their pathogenicity to mammals. Insertion of the VN1203 HA and neuraminidase (NA) genes into recombinant HK213 virus expanded its tissue tropism and increased its lethality in mice; conversely, insertion of HK213 HA and NA genes into recombinant VN1203 virus decreased its systemic spread and lethality. The VN1203 and HK213 HAs differ by 10 aa, and HK213 HA has shown greater binding affinity for synthetic alpha2,6-linked sialyl receptor. Introduction of an S227N change and removal of N-linked glycosylation at residue 158 increased the alpha2,6-binding affinity of VN1203 HA. Recombinant VN1203 virus carrying the S227N change alone or with the residue-158 glycosylation site removed showed reduced lethality and systemic spread in mice but not in domestic chickens. Wild-type VN1203 virus exhibited the greatest efficiency in systemic spread after intramuscular inoculation and in infection of mouse bone marrow-derived dendritic cells and conventional pulmonary dendritic cells. These results show that VN1203 HA glycoprotein confers pathogenicity by facilitating systemic spread in mice; they also suggest that a minor change in receptor binding domain may modulate the virulence of H5N1 viruses.

Viral parkinsonism.

Parkinson's disease is a debilitating neurological disorder that affects 1-2% of the adult population over 55 years of age. For the vast majority of cases, the etiology of this disorder is unknown, although it is generally accepted that there is a genetic susceptibility to any number of environmental agents. One such agent may be viruses. It has been shown that numerous viruses can enter the nervous system, i.e. they are neurotropic, and induce a number of encephalopathies. One of the secondary consequences of these encephalopathies can be parkinsonism, that is both transient as well as permanent. One of the most highlighted and controversial cases of viral parkinsonism is that which followed the 1918 influenza outbreak and the subsequent induction of von Economo's encephalopathy. In this review, we discuss the neurological sequelae of infection by influenza virus as well as that of other viruses known to induce parkinsonism including Coxsackie, Japanese encephalitis B, St. Louis, West Nile and HIV viruses.

Intramuscularly administered neuraminidase inhibitor peramivir is effective against lethal H5N1 influenza virus in mice.

The replication efficiency and multi-organ dissemination of some influenza A (H5N1) viruses requires a rapid re-evaluation of the available antiviral strategies. We assessed five regimens of the neuraminidase (NA) inhibitor peramivir in mice inoculated with H5N1 virus. The regimens differed by: (1) frequency of administration on first day (once vs twice); (2) duration of administration (1 day vs 8 days); (3) route of administration (intramuscular [IM] injection alone or followed by oral administration). In all regimens, BALB/c mice were administered 30 mg/kg peramivir IM 1 h after lethal challenge with 5 MLD(50) of A/Vietnam/1203/04 (H5N1) influenza virus. When given only on the day of inoculation, a single IM injection produced a 33% survival rate, which increased to 55% with two injections. Eight-day regimens significantly increased survival; two IM injections followed by seven daily IM injections was the most effective regimen (100% survival; inhibition of replication in lungs and brain). When this 8-day regimen began at 24h after inoculation, 78% of mice survived; 56% survived when treatment began at 48 hours. Anti-HA antibody titer differed with the peramivir regimen and corresponded to the severity of disease. Overall, our results demonstrate that IM administration of peramivir is effective in promoting the survival of mice infected with systemically replicating H5N1 virus.

Oseltamivir prophylactic regimens prevent H5N1 influenza morbidity and mortality in a ferret model.

BACKGROUND: Current oseltamivir prophylactic regimens may not be as effective against highly pathogenic H5N1 influenza viruses as they are against less pathogenic strains. An optimal regimen is urgently needed. METHODS: Ferrets were given the neuraminidase inhibitor oseltamivir orally for 10 days (5 or 10 mg/kg once daily or 2.5 or 5 mg/kg twice daily). Prophylaxis was initiated 1 day before infection, and oseltamivir was given 4 h before the ferrets were inoculated with a lethal dose of A/Vietnam/1203/04 (H5N1) influenza virus. RESULTS: At a dose of 5 mg/kg once daily, oseltamivir prevented death but not clinical signs of infection in ferrets; severe pathology was observed in the lungs, brain, and liver. At 10 mg/kg once daily, oseltamivir reduced clinical symptoms and systemic virus replication, but pathology was observed in the internal organs. The best results were obtained at a dose of 2.5 or 5 mg/kg given twice daily. Both regimens resulted in 100% survival and the absence of clinical symptoms, systemic virus spread, and organ pathology. Serum antibody titers were comparable across regimens and were sufficient to protect against rechallenge. CONCLUSIONS: An increased dose of oseltamivir or twice-daily administration effectively protects ferrets against morbidity and mortality caused by H5N1 infection and does not interfere with the development of protective antibodies against subsequent H5N1 infection.

Efficacy of oseltamivir therapy in ferrets inoculated with different clades of H5N1 influenza virus.

Highly pathogenic H5N1 influenza viruses have infected an increasing number of humans in Asia, with high mortality rates and the emergence of multiple distinguishable clades. It is not known whether antiviral drugs that are effective against contemporary human influenza viruses will be effective against systemically replicating viruses, such as these pathogens. Therefore, we evaluated the use of the neuraminidase (NA) inhibitor oseltamivir for early postexposure prophylaxis and for treatment in ferrets exposed to representatives of two clades of H5N1 virus with markedly different pathogenicities in ferrets. Ferrets were protected from lethal infection with the A/Vietnam/1203/04 (H5N1) virus by oseltamivir (5 mg/kg of body weight/day) given 4 h after virus inoculation, but higher daily doses (25 mg/kg) were required for treatment when it was initiated 24 h after virus inoculation. For the treatment of ferrets inoculated with the less pathogenic A/Turkey/15/06 (H5N1) virus, 10 mg/kg/day of oseltamivir was sufficient to reduce the lethargy of the animals, significantly inhibit inflammation in the upper respiratory tract, and block virus spread to the internal organs. Importantly, all ferrets that survived the initial infection were rechallenged with homologous virus after 21 days and were completely protected from infection. Direct sequencing of the NA or HA1 gene segments in viruses isolated from ferret after treatment showed no amino acid substitutions known to cause drug resistance in conserved residues. Thus, early oseltamivir treatment is crucial for protection against highly pathogenic H5N1 viruses and the higher dose may be needed for the treatment of more virulent viruses.

H5N1 influenza viruses in Lao People's Democratic Republic.

A prospective surveillance program for influenza viruses was established in Lao People's Democratic Republic (PDR) in July of 2005. We report isolation of H5N1 virus genetically distinct from H5N1 circulating in 2004, which indicates reintroduction of H5N1 into Lao PDR after its disappearance (i.e., no virologic or serologic evidence) for 2 years.

Epididymal stone formation and decreased sperm production in roosters vaccinated with a killed strain of avian infectious bronchitis virus.

Our objective was to determine if vaccination with killed avian infectious bronchitis virus (AIBV) causes epididymal calcium stones in the rooster as is seen following vaccination with live attenuated AIBV. Specific-pathogen-free roosters were divided into three groups: nonvaccinated (NONVAC), live attenuated AIBV-vaccinated (LVAC), and killed AIBV-vaccinated (KVAC) groups. Roosters were vaccinated at 2, 6, 10, and 14 wk of age and the epididymal region was observed at 27 wk of age. Epididymal stones were present in 13% of NONVAC, 50% of KVAC, and 64% of LVAC roosters. Histologically, immune cells were seen in the interstitium of efferent ductules containing stones. We conclude that use of a killed vaccine does not reduce the incidence of epididymal stones.

Avian infectious bronchitis virus: a possible cause of reduced fertility in the rooster.

The formation of epididymal stones in the rooster epididymis is a widespread problem that has detrimental effects on sperm production and fertility. The cause of epididymal stones is unknown, but an infectious agent, the avian infectious bronchitis virus (AIBV), has been implicated. The goal of this study was to determine if administering the live attenuated AIBV vaccine to male chicks increases the incidence of stones in the epididymal region of the adult rooster. Specific pathogen free (SPF) Leghorn roosters were divided into two groups: a vaccine-free group (n = 7) and a group vaccinated with AIBV (n = 12). The vaccine was administered orally at 2, 4, 10, and 14 wk of age. Blood was drawn weekly to monitor antibodies to AIBV. At 26 wk of age, blood was obtained to determine testosterone concentrations, and reproductive tracts were removed to analyze daily sperm production and to detect epididymal stones. Nine of 12 vaccinated roosters developed stones, whereas those not given the vaccine did not develop stones. Serum testosterone concentrations were significantly (P < 0.05) reduced in vaccinated roosters with epididymal stones (3.6 +/- 0.30 ng/ml) when compared with nonvaccinated roosters that did not have epididymal stones (7.0 +/- 1.63 ng/ml). Testis weight was significantly (P < 0.05) reduced in vaccinated roosters with epididymal stones (12.1 +/- 0.76 g), as compared with nonvaccinated roosters without epididymal stones (15.2 +/- 0.81 g). Daily sperm production was significantly (P < 0.05) decreased in vaccinated roosters with epididymal stones (5.03 +/- 0.31 x 10(8) sperm/testis/day) when compared with nonvaccinated roosters without epididymal stones (7.43 +/- 0.52 x 10(8) sperm/testis/day). Comparing daily sperm production on a per gram basis, vaccinated roosters with epididymal stones had 4.38 +/- 0.14 x 10(7) sperm/g of testis, which was significantly (P < 0.05) smaller than nonvaccinated roosters without epididymal stones, which had 5.17 +/- 0.17 x 10(7) sperm/g of testis. We conclude that the use of a live attenuated AIBV vaccine increases the incidence of epididymal stones in roosters, resulting in decreased sperm production and decreased serum testosterone concentrations.