Epidemiology of Epidemic Ebola Virus Disease in Conakry and Surrounding Prefectures, Guinea, 2014-2015.

In 2014, Ebola virus disease (EVD) in West Africa was first reported during March in 3 southeastern prefectures in Guinea; from there, the disease rapidly spread across West Africa. We describe the epidemiology of EVD cases reported in Guinea's capital, Conakry, and 4 surrounding prefectures (Coyah, Dubreka, Forecariah, and Kindia), encompassing a full year of the epidemic. A total of 1,355 EVD cases, representing ≈40% of cases reported in Guinea, originated from these areas. Overall, Forecariah had the highest cumulative incidence (4× higher than that in Conakry). Case-fatality percentage ranged from 40% in Conakry to 60% in Kindia. Cumulative incidence was slightly higher among male than female residents, although incidences by prefecture and commune differed by sex. Over the course of the year, Conakry and neighboring prefectures became the EVD epicenter in Guinea.

Strengthening HIV surveillance in the antiretroviral therapy era: rationale and design of a longitudinal study to monitor HIV prevalence and incidence in the uMgungundlovu District, KwaZulu-Natal, South Africa.

BACKGROUND: South Africa has over 6,000,000 HIV infected individuals and the province of KwaZulu-Natal (KZN) is the most severely affected. As public health initiatives to better control the HIV epidemic are implemented, timely, detailed and robust surveillance data are needed to monitor, evaluate and inform the programmatic interventions and policies over time. We describe the rationale and design of the HIV Incidence Provincial Surveillance System (HIPSS) to monitor HIV prevalence and incidence. METHODS/DESIGN: The household-based survey will include a sample of men and women from two sub-districts of the uMgungundlovu municipality (Vulindlela and the Greater Edendale) of KZN, South Africa. The study is designed as two sequential cross-sectional surveys of 10,000 randomly selected individuals aged 15-49 years to be conducted one year apart. From the cross sectional surveys, two sequential cohorts of HIV negative individuals aged 15-35 years will be followed-up one year later to measure the primary outcome of HIV incidence. Secondary outcomes include the laboratory measurements for pulmonary tuberculosis, sexually transmitted infections and evaluating tests for estimating population-level HIV incidence. Antiretroviral therapy (ART) access, HIV-1 RNA viral load, and CD4 cell counts in HIV positive individuals will assess the effectiveness of the HIV treatment cascade. Household and individual-level socio-demographic characteristics, exposure to HIV programmatic interventions and risk behaviours will be assessed as predictors of HIV incidence. The incidence rate ratio of the two cohorts will be calculated to quantify the change in HIV incidence between consecutive samples. In anticipation of better availability of population-level HIV prevention and treatment programmes leading to decreases in HIV incidence, the sample size provides 84% power to detect a reduction of 30% in the HIV incidence rate between surveys. DISCUSSION: The results from HIPSS will provide critical data regarding HIV prevalence and incidence in this community and will establish whether HIV prevention and treatment efforts in a "real world", non-trial setting have an impact on HIV incidence at a population level. Importantly, the study design and methods will inform future methods for HIV surveillance.

Dual resistance to adamantanes and oseltamivir among seasonal influenza A(H1N1) viruses: 2008-2010.

Two distinct genetic clades of seasonal influenza A(H1N1) viruses have cocirculated in the recent seasons: clade 2B oseltamivir-resistant and adamantane-susceptible viruses, and clade 2C viruses that are resistant to adamantanes and susceptible to oseltamivir. We tested seasonal influenza A(H1N1) viruses collected in 2008-2010 from the United States and globally for resistance to antivirals approved by the Food and Drug Administration. We report 28 viruses with both adamantane and oseltamivir (dual) resistance from 5 countries belonging to 4 distinct genotypes. Because of limited options for antiviral treatment, emergence of dual-resistant influenza viruses poses a public health concern, and their circulation needs to be closely monitored.

Cluster of oseltamivir-resistant 2009 pandemic influenza A (H1N1) virus infections on a hospital ward among immunocompromised patients--North Carolina, 2009.

BACKGROUND: Oseltamivir resistance among 2009 pandemic influenza A (H1N1) viruses (pH1N1) is rare. We investigated a cluster of oseltamivir-resistant pH1N1 infections in a hospital ward. METHODS: We reviewed patient records and infection control measures and interviewed health care personnel (HCP) and visitors. Oseltamivir-resistant pH1N1 infections were found with real-time reverse-transcription polymerase chain reaction and pyrosequencing for the H275Y neuraminidase (NA) mutation. We compared hemagglutinin (HA) sequences from clinical samples from the outbreak with those of other surveillance viruses. RESULTS: During the period 6-11 October 2009, 4 immunocompromised patients within a hematology-oncology ward exhibited symptoms of pH1N1 infection. The likely index patient became febrile 8 days after completing a course of oseltamivir; isolation was instituted 9 days after symptom onset. Three other case patients developed symptoms 1, 3, and 5 days after the index patient. Three case patients were located in adjacent rooms. HA and NA sequences from case patients were identical. Twelve HCP and 6 visitors reported influenza symptoms during the study period. No other pH1N1 isolates from the hospital or from throughout the state carried the H275Y mutation. CONCLUSIONS: Geographic proximity, temporal clustering, presence of H275Y mutation, and viral sequence homology confirmed nosocomial transmission of oseltamivir-resistant pH1N1. Diagnostic vigilance and prompt isolation may prevent nosocomial transmission of influenza.

Detection of molecular markers of drug resistance in 2009 pandemic influenza A (H1N1) viruses by pyrosequencing.

The M2 blockers amantadine and rimantadine and the neuraminidase (NA) inhibitors (NAIs) oseltamivir and zanamivir are approved by the FDA for use for the control of influenza A virus infections. The 2009 pandemic influenza A (H1N1) viruses (H1N1pdm) are reassortants that acquired M and NA gene segments from a Eurasian adamantane-resistant swine influenza virus. NAI resistance in the H1N1pdm viruses has been rare, and its occurrence is mainly limited to oseltamivir-exposed patients. The pyrosequencing assay has been proven to be a useful tool in surveillance for drug resistance in seasonal influenza A viruses. We provide a protocol which allows the detection of adamantane resistance markers as well as the I43T change, which is unique to the H1N1pdm M2 protein. The protocol also allows the detection of changes at residues V116, I117, E119, Q136, K150, D151, D199, I223, H275, and N295 in the NA, known to alter NAI drug susceptibility. We report on the detection of the first cases of the oseltamivir resistance-conferring mutation H275Y and the I223V change in viruses from the United States using the approach described in this study. Moreover, the assay permits the quick identification of the major NA group (V106/N248, I106/D248, or I106/N248) to which a pandemic virus belongs. Pyrosequencing is well suited for the detection of drug resistance markers and signature mutations in the M and NA gene segments of the pandemic H1N1 influenza viruses.

Detection of E119V and E119I mutations in influenza A (H3N2) viruses isolated from an immunocompromised patient: challenges in diagnosis of oseltamivir resistance.

The clinical use of the neuraminidase inhibitor (NAI) oseltamivir is associated with the emergence of drug resistance resulting from subtype-specific neuraminidase (NA) mutations. The influenza A/Texas/12/2007 (H3N2) virus isolated from an oseltamivir-treated immunocompromised patient exhibited reduced susceptibility to oseltamivir in the chemiluminescent neuraminidase inhibition (NI) assay (approximately 60-fold increase in its 50% inhibitory concentration [IC(50)] compared to that for a control virus). When further propagated in cell culture, the isolate maintained reduced susceptibility to oseltamivir in both chemiluminescent and fluorescent NI assays (approximately 50- and 350-fold increases in IC(50), respectively). Sequencing analysis of the isolate revealed a mix of nucleotides coding for amino acids at position 119 of the NA [E119(V/I)]. Plaque purification of the isolate yielded E119V and E119I variants, both exhibiting reduced susceptibility to oseltamivir. The E119I variant also showed decreased susceptibility to zanamivir and the investigational NAIs peramivir and A-315675. The emergence of E119V variants in oseltamivir-treated patients has been previously reported; however, the E119I mutation detected here is a novel one which reduces susceptibility to several NAIs. Both mutations were not detected in unpropagated original clinical specimens using either conventional sequencing or pyrosequencing, suggesting that these variants were present in very low proportions (<10%) in clinical specimens and gained dominance after virus propagation in MDCK cells. All virus isolates recovered from the patient were resistant to adamantanes. Our findings highlight the potential for emergence and persistence of multidrug-resistant influenza viruses in oseltamivir-treated immunocompromised subjects and also highlight challenges for drug resistance diagnosis due to the genetic instability of the virus population upon propagation in cell culture.

In vitro antiviral activity of favipiravir (T-705) against drug-resistant influenza and 2009 A(H1N1) viruses.

Favipiravir (T-705) has previously been shown to have a potent antiviral effect against influenza virus and some other RNA viruses in both cell culture and in animal models. Currently, favipiravir is undergoing clinical evaluation for the treatment of influenza A and B virus infections. In this study, favipiravir was evaluated in vitro for its ability to inhibit the replication of a representative panel of seasonal influenza viruses, the 2009 A(H1N1) strains, and animal viruses with pandemic (pdm) potential (swine triple reassortants, H2N2, H4N2, avian H7N2, and avian H5N1), including viruses which are resistant to the currently licensed anti-influenza drugs. All viruses were tested in a plaque reduction assay with MDCK cells, and a subset was also tested in both yield reduction and focus inhibition (FI) assays. For the majority of viruses tested, favipiravir significantly inhibited plaque formation at 3.2 muM (0.5 microg/ml) (50% effective concentrations [EC(50)s] of 0.19 to 22.48 muM and 0.03 to 3.53 microg/ml), and for all viruses, with the exception of a single dually resistant 2009 A(H1N1) virus, complete inhibition of plaque formation was seen at 3.2 muM (0.5 microg/ml). Due to the 2009 pandemic and increased drug resistance in circulating seasonal influenza viruses, there is an urgent need for new drugs which target influenza. This study demonstrates that favipiravir inhibits in vitro replication of a wide range of influenza viruses, including those resistant to currently available drugs.

Detection of molecular markers of antiviral resistance in influenza A (H5N1) viruses using a pyrosequencing method.

Resistance of influenza viruses to antiviral drugs can emerge following medication or may result from natural variation. Two classes of anti-influenza virus drugs targeting either the M2 protein (amantadine and rimantadine) or neuraminidase (NA; oseltamivir and zanamivir) are currently licensed. These drugs are expected to be important in controlling the early stages of a potential pandemic. In the present study, we describe how a pyrosequencing method can be used to rapidly detect established molecular markers of resistance to M2 blockers and NA inhibitors in influenza A (H5N1) viruses. The residues L26, V27, A30, S31, and G34 in the M2 protein were targeted for pyrosequencing. The NA residues for pyrosequencing analysis included the established markers of drug resistance (H274 and N294), as well as residues of less certain relevance (V116, I117, Q136, K150, and I222). A single pair of pyro-reverse transcription (RT)-PCR primers was designed to allow amplification of an approximately 600-nucleotide-long amplicon of the NA genes of H5N1 viruses from various clades/subclades associated with infections in humans. The sensitivity of the assay was demonstrated by the successful pyrosequencing of RNA extracted from samples of serially diluted (10(-5) to 10(-7)) virus stocks with initial concentrations ranging from 10(5) to 10(8) PFU/ml. The markers of resistance were detected in samples with threshold cycle values ranging from 32 to 37, as determined by real-time RT-PCR. The pyrosequencing approach may provide a valuable tool for rapid detection of markers of drug resistance in H5N1 viruses and facilitate the elucidation of the role of such changes in natural and acquired drug resistance.

Surveillance for neuraminidase inhibitor resistance among human influenza A and B viruses circulating worldwide from 2004 to 2008.

The surveillance of seasonal influenza virus susceptibility to neuraminidase (NA) inhibitors was conducted using an NA inhibition assay. The 50% inhibitory concentration values (IC(50)s) of 4,570 viruses collected globally from October 2004 to March 2008 were determined. Based on mean IC(50)s, A(H3N2) viruses (0.44 nM) were more sensitive to oseltamivir than A(H1N1) viruses (0.91 nM). The opposite trend was observed with zanamivir: 1.06 nM for A(H1N1) and 2.54 nM for A(H3N2). Influenza B viruses exhibited the least susceptibility to oseltamivir (3.42 nM) and to zanamivir (3.87 nM). To identify potentially resistant viruses (outliers), a threshold of a mean IC(50) value + 3 standard deviations was defined for type/subtype and drug. Sequence analysis of outliers was performed to identify NA changes that might be associated with reduced susceptibility. Molecular markers of oseltamivir resistance were found in six A(H1N1) viruses (H274Y) and one A(H3N2) virus (E119V) collected between 2004 and 2007. Some outliers contained previously reported mutations (e.g., I222T in the B viruses), while other mutations [e.g., R371K and H274Y in B viruses and H274N in A(H3N2) viruses) were novel. The R371K B virus outlier exhibited high levels of resistance to both inhibitors (>100 nM). A substantial variance at residue D151 was observed among A(H3N2) zanamivir-resistant outliers. The clinical relevance of newly identified NA mutations is unknown. A rise in the incidence of oseltamivir resistance in A(H1N1) viruses carrying the H274Y mutation was detected in the United States and in other countries in the ongoing 2007 to 2008 season. As of March 2008, the frequency of resistance among A(H1N1) viruses in the United States was 8.6% (50/579 isolates). The recent increase in oseltamivir resistance among A(H1N1) viruses isolated from untreated patients raises public health concerns and necessitates close monitoring of resistance to NA inhibitors.

Monitoring seasonal influenza A evolution: rapid 2009 pandemic H1N1 surveillance with an reverse transcription-polymerase chain reaction/electro-spray ionization mass spectrometry assay.

BACKGROUND: The emergence of the pandemic H1N1 influenza strain in 2009 reinforced the need for improved influenza surveillance efforts. A previously described influenza typing assay that utilizes RT-PCR coupled to electro-spray ionization mass spectrometry (ESI-MS) played an early role in the discovery of the pandemic H1N1 influenza strain, and has potential application for monitoring viral genetic diversity in ongoing influenza surveillance efforts. OBJECTIVES: To determine the analytical sensitivity of RT-PCR/ESI-MS influenza typing assay for identifying the pandemic H1N1 strain and describe its ability to assess viral genetic diversity. STUDY DESIGN: Two sets of pandemic H1N1 samples, 190 collected between April and June of 2009, and 69 collected between October 2009 and January 2010, were processed by the RT-PCR/ESI-MS influenza typing assay, and the spectral results were compared to reference laboratory results and historical sequencing data from the Nucleotide Database of the National Center for Biotechnology Information (NCBI). RESULTS: Strain typing concordance with reference standard testing was 100% in both sample sets, and the assay demonstrated a significant increase in influenza genetic diversity, from 10.5% non-wildtype genotypes in early samples to 69.9% in late samples (P<0.001). An NCBI search demonstrated a similar increase, from 13.4% to 45.2% (P<0.001). CONCLUSIONS: This comparison of early versus late influenza samples analyzed by RT-PCR/ESI-MS demonstrates the influenza typing assay's ability as a universal influenza detection platform to provide high-fidelity pH1N1 strain identification over time, despite increasing genetic diversity in the circulating virus. The genotyping data can also be leveraged for high-throughput influenza surveillance.

RT-PCR/electrospray ionization mass spectrometry approach in detection and characterization of influenza viruses.

Reverse-transcription PCR (RT-PCR) coupled with electrospray ionization mass spectrometry (ESI-MS) is a high-throughput nucleic acid-based technology that relies on the accurate measurement of the molecular weight of PCR amplicons that can be used to deduce the base counts (number of As, Gs, Cs and Ts) of DNA. These amplicons represent highly variable regions with information-rich sequences, which are flanked by broad-range primers designed based on highly conserved loci. This technology was first introduced in 2005 for microbial identification and subtyping, and was later applied to influenza virus detection and identification. The influenza RT-PCR/ESI-MS assay allows analysis of approximately 300 samples per 24 h, and aids in the characterization of influenza viruses based on their 'core' gene signatures. Notably, this assay was used to identify one of the first cases of the 2009 H1N1 pandemic viruses. One of the main advantages of the RT-PCR/ESI-MS technology is its universality and adaptability for pathogen characterization. Efforts are being made to customize the currently used influenza surveillance assay for use in the diagnosis of the H1N1 pandemic virus. In this article, we provide a summary of known applications of the RT-PCR/ESI-MS assay in the field of influenza.

Detection of antiviral resistance and genetic lineage markers in influenza B virus neuraminidase using pyrosequencing.

We report here the design of a pyrosequencing approach for the detection of molecular markers of resistance to the neuraminidase inhibitors zanamivir and oseltamivir in influenza viruses of type B. Primers were designed to analyze the sequences at eight amino acid positions E119, R152, D198, I222, S250, H274, R371, and G402 (universal A/N2 numbering) in the neuraminidase (NA) which have been previously found to be associated with resistance or reduced susceptibility to oseltamivir and/or zanamivir in the NA inhibition assay. In addition, the designed primers could be utilized to the distinguish between the NAs of influenza B viruses from the two major lineages (Victoria and Yamagata) that have co-circulated globally in recent years, thus providing a valuable tool for virus strain surveillance.

Comprehensive assessment of 2009 pandemic influenza A (H1N1) virus drug susceptibility in vitro.

BACKGROUND: Antiviral drugs are an important option for managing infections caused by influenza viruses. This study assessed the drug susceptibility of 2009 pandemic influenza A (H1N1) viruses collected globally between April 2009 and January 2010. METHODS: Virus isolates were tested for adamantane susceptibility, using pyrosequencing to detect the S31N marker of adamantane resistance in the M2 protein and biological assays to assess viral replication in cell culture. To assess neuraminidase (NA) inhibitor (NAI) susceptibility, virus isolates were tested in chemiluminescent NA inhibition assays and by pyrosequencing to detect the H275Y (H274Y in N2 numbering) marker of oseltamivir resistance in the NA. RESULTS: With the exception of three, all viruses that were tested for adamantane susceptibility (n=3,362) were resistant to this class of drugs. All viruses tested for NAI susceptibility (n=3,359) were sensitive to two US Food and Drug Administration-approved NAIs, oseltamivir (mean ±sd 50% inhibitory concentration [IC(50)] 0.25 ±0.12 nM) and zanamivir (mean IC(50) 0.29 ±0.09 nM), except 23 (0.7%), which were resistant to oseltamivir, but sensitive to zanamivir. Oseltamivir-resistant viruses had the H275Y mutation in their NA and were detected in patients exposed to the drug through prophylaxis or treatment. NA activity of all viruses was inhibited by the NAIs peramivir, laninamivir (R-125489) and A-315675, except for H275Y variants, which exhibited approximately 100-fold reduction in peramivir susceptibility. CONCLUSIONS: This report provides data regarding antiviral susceptibility of 2009 pandemic influenza A (H1N1) surveillance viruses, the majority of which were resistant to adamantanes and sensitive to NAIs. These findings provide information essential for antiviral resistance monitoring and development of novel diagnostic tests for detecting influenza antiviral resistance.

Host cell selection of influenza neuraminidase variants: implications for drug resistance monitoring in A(H1N1) viruses.

The neuraminidase inhibitors (NAIs), oseltamivir and zanamivir, are essential for treatment and prevention of influenza A and B infections. Oseltamivir resistance among influenza A (H1N1) viruses rapidly emerged and spread globally during the 2007-2008 and 2008-2009 influenza seasons. Approximately 20% and 90% of viruses tested for NAI susceptibility at CDC during these seasons, respectively, were resistant to oseltamivir (IC(50) approximately 100-3000 time>those of sensitive viruses), based on the chemiluminescent NA inhibition assay. Pyrosequencing analysis confirmed H274Y mutation (H275Y in N1 numbering) in the neuraminidase (NA) gene of oseltamivir-resistant viruses. Full NA sequence analysis of a subset of oseltamivir-resistant and sensitive virus isolates from both seasons (n=725) showed that 53 (7.3%) had mutations at residue D151 (D-->E/G/N), while 9 (1.2%) had mutations at Q136 (Q-->K) and 2 (0.3%) had mutations at both residues. Viruses with very high IC(50) for oseltamivir and peramivir, and elevated IC(50) for zanamivir, had H274Y in addition to mutations at D151 and/or Q136, residues which can potentially confer NAI resistance based on recent N1 NA crystal structure data. Mutations at D151 without H274Y, did not elevate IC(50) for any tested NAI, however, Q136K alone significantly reduced susceptibility to zanamivir (36-fold), peramivir (80-fold) and A-315675 (114-fold) but not oseltamivir. Mutations at D151 and Q136 were present only in MDCK grown viruses but not in matching original clinical specimens (n=33) which were available for testing, suggesting that these variants were the result of cell culture selection or they were present in very low proportions. Our findings provide evidence that propagation of influenza virus outside its natural host may lead to selection of virus variants with mutations in the NA that affect sensitivity to NAIs and thus poses implications for drug resistance monitoring and diagnostics.

Genomic signature-based identification of influenza A viruses using RT-PCR/electro-spray ionization mass spectrometry (ESI-MS) technology.

BACKGROUND: The emergence and rapid spread of the 2009 H1N1 pandemic influenza A virus (H1N1pdm) in humans highlights the importance of enhancing the capability of existing influenza surveillance systems with tools for rapid identification of emerging and re-emerging viruses. One of the new approaches is the RT-PCR electrospray ionization mass spectrometry (RT-PCR/ESI-MS) technology, which is based on analysis of base composition (BC) of RT-PCR amplicons from influenza "core" genes. Combination of the BC signatures represents a "genomic print" of an influenza A virus. METHODOLOGY/PRINCIPAL FINDINGS: Here, 757 samples collected between 2006 and 2009 were tested, including 302 seasonal H1N1, 171 H3N2, 7 swine triple reassortants, and 277 H1N1pdm viruses. Of the 277 H1N1pdm samples, 209 were clinical specimens (throat, nasal and nasopharyngeal swabs, nasal washes, blood and sputum). BC signatures for the clinical specimen from one of the first cases of the 2009 pandemic, A/California/04/2009, confirmed it as an unusual, previously unrecognized influenza A virus, with "core" genes related to viruses of avian, human and swine origins. Subsequent analysis of additional 276 H1N1pdm samples revealed that they shared the genomic print of A/California/04/2009, which differed from those of North American swine triple reassortant viruses, seasonal H1N1 and H3N2 and other viruses tested. Moreover, this assay allowed distinction between "core" genes of co-circulating groups of seasonal H1N1, such as clades 2B, 2C, and their reassortants with dual antiviral resistance to adamantanes and oseltamivir. CONCLUSIONS/SIGNIFICANCE: The RT-PCR/ESI-MS assay is a broad range influenza identification tool that can be used directly on clinical specimens for rapid and accurate detection of influenza virus genes. The assay differentiates the H1N1pdm from seasonal and other nonhuman hosts viruses. Although not a diagnostic tool, this assay demonstrates its usefulness and robustness in influenza virus surveillance and detection of novel and unusual viruses with previously unseen genomic prints.

Influenza genome analysis using pyrosequencing method: current applications for a moving target.

Pyrosequencing is a high-throughput non-gel-based DNA sequencing method that was introduced in the late 1990s. It employs a DNA sequencing-by-synthesis approach based on real-time measurement of pyrophosphate released from incorporation of dNTPs. A cascade of enzymatic reactions proportionally converts the pyrophosphate to a light signal recorded in a form of peaks, known as pyrograms. Routinely, a 45-60-nucleotide sequence is obtained per reaction. Recent improvements introduced in the assay chemistry have extended the read to approximately 100 nucleotides. Since its advent, pyrosequencing has been applied in the fields of microbiology, molecular biology and pharmacogenomics. The pyrosequencing approach was first applied to analysis of influenza genome in 2005, when it played a critical role in the timely detection of an unprecedented rise in resistance to the adamantane class of anti-influenza drugs. More recently, pyrosequencing was successfully applied for monitoring the emergence and spread of influenza A (H1N1) virus resistance to oseltamivir, a newer anti-influenza drug. The present report summarizes known applications of the pyrosequencing approach for influenza genome analysis with an emphasis on drug-resistance detection.

Pyrosequencing as a tool to detect molecular markers of resistance to neuraminidase inhibitors in seasonal influenza A viruses.

Pyrosequencing has been successfully used to monitor resistance in influenza A viruses to the first class of anti-influenza drugs, M2 blockers (adamantanes). In contrast to M2 blockers, resistance to neuraminidase (NA) inhibitors (NAIs) is subtype- and drug-specific. Here, we designed a pyrosequencing assay for detection of the most commonly reported mutations associated with resistance to NAIs, a newer class of anti-influenza drugs. These common mutations occur at residues: H274 (N1), E119 (N2), R292 (N2), and N294 (N2) in seasonal influenza A viruses. Additionally, we designed primers to detect substitutions at D151 in NAs of N1 and N2 subtypes. This assay allows detection of mutations associated with resistance not only in grown viruses but also in clinical specimens, thus reducing the time needed for testing and providing an advantage for disease outbreak investigation and management. The pyrosequencing approach also allows the detection of mixed populations of virus variants at positions of interest. Analysis of viruses in the original clinical specimens reduces the potential for introducing genetic variance in the virus population due to selection by cell culture. Our results showed that, in at least one instance, a D151E change seen in N1NA after virus propagation in cell culture was not detected in the original clinical specimen. Although the pyrosequencing assay allows high throughput screening for established genetic markers of antiviral resistance, it is not a replacement for the NA inhibition assays due to insufficient knowledge of the molecular mechanisms of the NAI-resistance.

Oseltamivir resistance mutation N294S in human influenza A(H5N1) virus in Egypt.

In December 2006, three human specimens were received that were suspected positive for influenza A(H5N1). The specimens were tested using real time PCR. And the presence of A(H5N1) virus was confirmed in 2 patients (16F and 26M), The NA sequence from A(H5N1) positive specimens collected before and after antiviral therapy revealed a mutation (N294S) (N295S according to N1 numbering), previously associated with resistance to oseltamivir. When tested with NA inhibition assays, the two N294S viruses from Egypt exhibited from 57 to 138-fold reduction in susceptibility to oseltamivir, depending on the assay. To our knowledge, this is the first time oseltamivir resistance has been detected in A(H5N1) infecting a human prior to treatment.

Surveillance of resistance to adamantanes among influenza A(H3N2) and A(H1N1) viruses isolated worldwide.

Our previous reports demonstrated an alarming increase in resistance to adamantanes among influenza A(H3N2) viruses isolated in 2001-2005. To continue monitoring drug resistance, we conducted a comprehensive analysis of influenza A(H3N2) and A(H1N1) viruses isolated globally in 2005-2006. The results obtained by pyrosequencing indicate that 96.4% (n=761) of A(H3N2) viruses circulating in the United States were adamantane resistant. Drug resistance has reached 100% among isolates from some Asian countries. Analysis of correlation between the appearance of drug resistance and the evolutionary pathway of the hemagglutinin (HA) gene suggests at least 2 separate introductions of resistance into circulating populations that gave rise to identifiable subclades. It also indicates that resistant A(H3N2) viruses may have emerged in Asia in late 2001. Among A(H1N1) viruses isolated worldwide, resistance reached 15.5% in 2005-2006; in the United States alone, it was 4.0%. Phylogenetic analysis of the HA and M genes indicates that the acquisition of resistance in A(H1N1) viruses can be linked to a specific genetic group and was not a result of reassortment between A(H3N2) and A(H1N1) viruses. The results of the study highlight the necessity of close monitoring of resistance to existing antivirals as wells as the need for new therapeutics.

Crimean-Congo hemorrhagic fever virus genomics and global diversity.

Crimean-Congo hemorrhagic fever (CCHF) is a severe illness with high case fatality that occurs in Africa, Europe, the Middle East, and Asia. The complete genomes of 13 geographically and temporally diverse virus strains were determined, and CCHF viruses were found to be highly variable with 20 and 8%, 31 and 27%, and 22 and 10% nucleotide and deduced amino acid differences detected among virus S (nucleocapsid), M (glycoprotein), and L (polymerase) genome segments, respectively. Distinct geographic lineages exist, but with multiple exceptions indicative of long-distance virus movement. Discrepancies among the virus S, M, and L phylogenetic tree topologies document multiple RNA segment reassortment events. An analysis of individual segment datasets suggests genetic recombination also occurs. For an arthropod-borne virus, the genomic plasticity of CCHF virus is surprisingly high.