Novel Genotypes of H9N2 Influenza A Viruses Isolated from Poultry in Pakistan Containing NS Genes Similar to Highly Pathogenic H7N3 and H5N1 Viruses

The impact of avian influenza caused by H9N2 viruses in Pakistan is now significantly more severe than in previous years. Since all gene segments contribute towards the virulence of avian influenza virus, it was imperative to investigate the molecular features and genetic relationships of H9N2 viruses prevalent in this region. Analysis of the gene sequences of all eight RNA segments from 12 viruses isolated between 2005 and 2008 was undertaken. The hemagglutinin (HA) sequences of all isolates were closely related to H9N2 viruses isolated from Iran between 2004 and 2007 and contained leucine instead of glutamine at position 226 in the receptor binding pocket, a recognised marker for the recognition of sialic acids linked α2–6 to galactose. The neuraminidase (NA) of two isolates contained a unique five residue deletion in the stalk (from residues 80 to 84), a possible indication of greater adaptation of these viruses to the chicken host. The HA, NA, nucleoprotein (NP), and matrix (M) genes showed close identity with H9N2 viruses isolated during 1999 in Pakistan and clustered in the A/Quail/Hong Kong/G1/97 virus lineage. In contrast, the polymerase genes clustered with H9N2 viruses from India, Iran and Dubai. The NS gene segment showed greater genetic diversity and shared a high level of similarity with NS genes from either H5 or H7 subtypes rather than with established H9N2 Eurasian lineages. These results indicate that during recent years the H9N2 viruses have undergone extensive genetic reassortment which has led to the generation of H9N2 viruses of novel genotypes in the Indian sub-continent. The novel genotypes of H9N2 viruses may play a role in the increased problems observed by H9N2 to poultry and reinforce the continued need to monitor H9N2 infections for their zoonotic potential.     

Cell Culture-Selected Substitutions in Influenza A(H3N2) Neuraminidase Affect Drug Susceptibility Assessment

Assessment of drug susceptibility has become an integral part of influenza virus surveillance. In this study, we describe the drug resistance profile of influenza A(H3N2) virus, A/Mississippi/05/2011, collected from a patient treated with oseltamivir and detected via surveillance. An MDCK cell-grown isolate of this virus exhibited highly reduced inhibition by the neuraminidase (NA) inhibitors (NAIs) oseltamivir (8,005-fold), zanamivir (813-fold), peramivir (116-fold), and laninamivir (257-fold) in the NA inhibition assay. Sequence analysis of its NA gene revealed a known oseltamivir-resistance marker, the glutamic acid-to-valine substitution at position 119 (E119V), and an additional change, threonine to isoleucine at position 148 (T148I). Unlike E119V, T148I was not detected in the clinical sample but acquired during viral propagation in MDCK cells. Using recombinant proteins, T148I by itself was shown to cause only a 6-fold increase in the zanamivir 50% inhibitory concentration (IC₅₀) and had no effect on inhibition by other drugs. The T148I substitution reduced NA activity by 50%, most likely by affecting the positioning of the 150 loop at the NA catalytic site. Using pyrosequencing, changes at T148 were detected in 35 (23%) of 150 MDCK cell-grown A(H3N2) viruses tested, which was lower than the frequency of changes at D151 (85%), an NA residue previously implicated in cell selection. We demonstrate that culturing of the A(H3N2) viruses (n = 11) at a low multiplicity of infection delayed the emergence of the NA variants with changes at position 148 and/or 151, especially when conducted in MDCK-SIAT1 cells. Our findings highlight the current challenges in monitoring susceptibility of influenza A(H3N2) viruses to the NAI class of antiviral drugs.     

Evolution of Oseltamivir Resistance Mutations in Influenza A(H1N1) and A(H3N2) Viruses during Selection in Experimentally Infected Mice

The evolution of oseltamivir resistance mutations during selection through serial passages in animals is still poorly described. Herein, we assessed the evolution of neuraminidase (NA) and hemagglutinin (HA) genes of influenza A/WSN/33 (H1N1) and A/Victoria/3/75 (H3N2) viruses recovered from the lungs of experimentally infected BALB/c mice receiving suboptimal doses (0.05 and 1 mg/kg of body weight/day) of oseltamivir over two generations. The traditional phenotypic and genotypic methods as well as deep-sequencing analysis were used to characterize the potential selection of mutations and population dynamics of oseltamivir-resistant variants. No oseltamivir-resistant NA or HA changes were detected in the recovered A/WSN/33 viruses. However, we observed a positive selection of the I222T NA substitution in the recovered A/Victoria/3/75 viruses, with a frequency increasing over time and with an oseltamivir concentration from 4% in the initial pretherapy inoculum up to 28% after two lung passages. Although the presence of mixed I222T viral populations in mouse lungs only led to a minimal increase in oseltamivir 50% enzyme-inhibitory concentrations (IC₅₀s) (by a mean of 5.7-fold) compared to that of the baseline virus, the expressed recombinant A/Victoria/3/75 I222T NA protein displayed a 16-fold increase in the oseltamivir IC₅₀ level compared to that of the recombinant wild type (WT). In conclusion, the combination of serial in vivo passages under neuraminidase inhibitor (NAI) pressure and temporal deep-sequencing analysis enabled, for the first time, the identification and selection of the oseltamivir-resistant I222T NA mutation in an influenza H3N2 virus. Additional in vivo selection experiments with other antivirals and drug combinations might provide important information on the evolution of antiviral resistance in influenza viruses.     

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 μM (0.5 μg/ml) (50% effective concentrations [EC₅₀s] of 0.19 to 22.48 μM and 0.03 to 3.53 μg/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 μM (0.5 μg/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.In the United States alone, seasonal influenza is responsible annually for infecting between 5 and 20% of the American population, resulting in more than 200,000 hospitalizations and 36,000 deaths (8). Globally, seasonal influenza causes between 250,000 and 500,000 deaths every year (60). Influenza is not only a disease of great medical importance but also of economic importance. Despite available vaccines, a recent study predicted that in the United States influenza results in direct medical costs of the order of $10.4 billion each year, with the total economic burden for the United States being projected at $87.1 billion each year (44). It is widely accepted that vaccination remains the most effective approach for the prevention of viral infections (48). Although there is a safe and effective annual trivalent influenza vaccine, a large proportion of the global population does not receive the yearly influenza vaccine. This can be due to a variety of reasons, including the lack of access to adequate health care, unavailability of vaccine supply, allergies, and adverse reactions. During the 2009 pandemic (pdm), in addition to the vaccination and epidemiological control measures being exerted by health care officials, antivirals targeting influenza offer an essential tool in treating infected patients, in addition to protecting those at high risk of infection, such as the young, elderly, and health care workers.Currently, there are two classes of anti-influenza drugs licensed in the United States for use in the treatment and management of influenza infections in humans: M2 ion channel blockers (also known as adamantanes) and neuraminidase (NA) inhibitors (NAIs) (30). Influenza antivirals are highly effective in the treatment of influenza infections if used promptly following the onset of symptoms or following exposure (45, 46). Both the M2 blockers amantadine and rimantadine are taken by the patient orally (45). However, of the two available NAIs, only oseltamivir is available as an oral formulation (zanamivir has to be inhaled [14, 53]), although other routes of administration have been investigated (31). The use of the M2 blockers amantadine and rimantadine is limited due to the rapid emergence of transmissible drug-resistant mutant viruses and the fact that they offer protection only against influenza A virus infections (32). The high prevalence of adamantane resistance in seasonal A(H3N2) viruses and oseltamivir resistance in seasonal A(H1N1) viruses is reflected in the CDC recommendations for the use of influenza antivirals (6).The majority of adamantane-resistant A(H3N2) and A(H1N1) viruses circulating globally in recent years share the same mutation, S31N, in the M2 protein (20), although other resistance-conferring mutations have been detected also (including A30T, L26F, and V27A) (20, 49). The globally spread oseltamivir-resistant seasonal A(H1N1) viruses share the same mutation, H275Y (H274Y in N2 subtype amino acid numbering), in the drug-targeted enzyme neuraminidase, although other mutations are known to cause reduced susceptibility in vitro (19, 47, 50).Seasonal A(H1N1) viruses resistant to both the adamantanes and the NAI oseltamivir have previously been reported, without an apparent link to treatment (12, 50). Currently, zanamivir is the only drug effective against both adamantane-resistant and/or oseltamivir-resistant influenza viruses, but due to the fact that it has to be inhaled, it is less suitable for use with several high-risk groups, including the severely ill (41), infants (33), and the elderly (22). Furthermore, zanamivir may decrease pulmonary function, so it is not recommended for the treatment of infections in individuals with chronic underlying lung and heart disease conditions (23).Since 1997, there have been several outbreaks of highly pathogenic avian influenza A(H5N1) infections in poultry, with a substantial number of infections occurring in humans (1). The overall case fatality of A(H5N1) infections in humans is over 60% and, unlike seasonal influenza, is most deadly in the young and healthy (ages 10 to 19 years) (59). Oseltamivir is the medication of choice for treating individuals infected with A(H5N1) (17). However, resistance in A(H5N1) viruses has been detected following the treatment of patients with oseltamivir (18, 38). In addition, naturally occurring reduced susceptibility to oseltamivir (35, 40) and possibly to zanamivir (29) has been documented for circulating A(H5N1) viruses, including novel mutations in the NA (29, 35). Adamantane resistance is widely spread among A(H5N1) viruses that carry mutations at amino acid residues 26, 27, and 31 in the M2 protein (13, 35) and among swine viruses circulating in Eurasia (27).In April 2009, a novel reassortant A(H1N1) virus was first identified as circulating in humans in both Mexico and the United States (7, 9). Since April, the virus has continued to transmit among humans, and on 11 June 2009 the World Health Organization classified the outbreak as the first influenza pandemic of the 21st century (58). The 2009 A(H1N1) pandemic viruses consist of a unique combination of gene segments, including those of the North American (triple reassortants) and Eurasian swine lineages (27, 54). The 2009 A(H1N1) pandemic viruses are resistant to the adamantanes and sensitive to the NAIs (3, 16). Yet, concerns exist about the possibility of acquisition of resistance to the NAI oseltamivir, since the majority of A(H1N1) viruses which have been circulating predominantly worldwide during the 2008-2009 influenza season are oseltamivir resistant due to the resistance-conferring H275Y mutation in the NA. Such an acquisition of resistance by the 2009 A(H1N1) pandemic viruses would be a major setback and would further limit the already sparse therapeutic options (15, 57). There have been laboratory-confirmed cases of oseltamivir-resistant 2009 A(H1N1) pandemic viruses (each carrying the H275Y resistance-conferring mutation in the NA) in the United States (5).Collectively, these recent findings emphasize not only the need for new effective antivirals to control and treat influenza infections but also the need to identify new molecular targets (47).One such compound which is currently being investigated and undergoing clinical trials for the treatment of influenza infections is favipiravir (T-705), a pyrazine derivative (2, 26, 31). Favipiravir targets the RNA-dependent RNA polymerase (RdRp), a component of influenza virus different from that of currently licensed influenza antivirals (24, 25). It was shown that favipiravir can inhibit the viral replication of influenza type A, B, and C viruses (24, 25, 55). Favipiravir reduces influenza virus replication by selectively inhibiting the viral RdRp, since it does not affect the synthesis of host cellular DNA and RNA (25). Favipiravir has also shown great potential to act as a broad-spectrum antiviral against many RNA viruses, as reviewed by Furuta and coworkers (26).The purpose of this study was to evaluate the ability, in vitro, of favipiravir to inhibit the viral replication of contemporary influenza viruses as well as viruses with pandemic potential, including viruses resistant to the currently available and licensed anti-influenza drugs. In this report we demonstrate that favipiravir is a potent inhibitor of seasonal influenza A and B virus replication, including that of drug-resistant and drug-sensitive viruses. In addition, favipiravir was shown to effectively inhibit influenza A viruses of other antigenic subtypes, including A(H2N2), viruses of avian origin [A(H4N2), A(H7N2), and A(H5N1)], and viruses of swine origin [A(H1N1) and A(H1N2)], as well as the 2009 A(H1N1) pandemic viruses.     

Application of a Seven-Target Pyrosequencing Assay To Improve the Detection of Neuraminidase Inhibitor-Resistant Influenza A(H3N2) Viruses

National U.S. influenza antiviral surveillance incorporates data generated by neuraminidase (NA) inhibition (NI) testing of isolates supplemented with NA sequence analysis and pyrosequencing analysis of clinical specimens. A lack of established correlates for clinically relevant resistance to NA inhibitors (NAIs) hinders interpretation of NI assay data. Nonetheless, A(H3N2) viruses are commonly monitored for moderately or highly reduced inhibition in the NI assay and/or for the presence of NA markers E119V, R292K, and N294S. In 2012 to 2013, three drug-resistant A(H3N2) viruses were detected by NI assay among isolates (n = 1,424); all showed highly reduced inhibition by oseltamivir and had E119V. In addition, one R292K variant was detected among clinical samples (n = 1,024) by a 3-target pyrosequencing assay. Overall, the frequency of NAI resistance was low (0.16% [4 of 2,448]). To screen for additional NA markers previously identified in viruses from NAI-treated patients, the pyrosequencing assay was modified to include Q136K, I222V, and deletions encompassing residues 245 to 248 (del245-248) and residues 247 to 250 (del247-250). The 7-target pyrosequencing assay detected NA variants carrying E119V, Q136, and del245-248 in an isolate from an oseltamivir-treated patient. Next, this assay was applied to clinical specimens collected from hospitalized patients and submitted for NI testing but failed cell culture propagation. Of the 27 clinical specimens tested, 4 (15%) contained NA changes: R292K (n = 2), E119V (n = 1), and del247-250 (n = 1). Recombinant NAs with del247-250 or del245-248 conferred highly reduced inhibition by oseltamivir, reduced inhibition by zanamivir, and normal inhibition by peramivir and laninamivir. Our results demonstrated the benefits of the 7-target pyrosequencing assay in conducting A(H3N2) antiviral surveillance and testing for clinical care.     

In Vitro Generation of Neuraminidase Inhibitor Resistance in A(H5N1) Influenza Viruses

To identify mutations that can arise in highly pathogenic A(H5N1) viruses under neuraminidase inhibitor selective pressure, two antigenically different strains were serially passaged with increasing levels of either oseltamivir or zanamivir. Under oseltamivir pressure, both A(H5N1) viruses developed a H274Y neuraminidase mutation, although in one strain the mutation occurred in combination with an I222M neuraminidase mutation. The H274Y neuraminidase mutation reduced oseltamivir susceptibility significantly (900- to 2,500-fold compared to the wild type). However the dual H274Y/I222M neuraminidase mutation had an even greater impact on resistance, with oseltamivir susceptibility reduced significantly further (8,000-fold compared to the wild type). A similar affect on oseltamivir susceptibility was observed when the dual H274Y/I222M mutations were introduced, by reverse genetics, into a recombinant seasonal human A(H1N1) virus and also when an alternative I222 substitution (I222V) was generated in combination with H274Y in A(H5N1) and A(H1N1) viruses. These viruses remained fully susceptible to zanamivir but demonstrated reduced susceptibility to peramivir. Following passage of the A(H5N1) viruses in the presence of zanamivir, the strains developed a D198G neuraminidase mutation, which reduced susceptibility to both zanamivir and oseltamivir, and also an E119G neuraminidase mutation, which demonstrated significantly reduced zanamivir susceptibility (1,400-fold compared to the wild type). Mutations in hemagglutinin residues implicated in receptor binding were also detected in many of the resistant strains. This study identified the mutations that can arise in A(H5N1) under either oseltamivir or zanamivir selective pressure and the potential for dual neuraminidase mutations to result in dramatically reduced drug susceptibility.Large-scale outbreaks of highly pathogenic A(H5N1) avian influenza affecting poultry have occurred throughout many parts of Asia, North Africa, and the Middle East since 2003 (1). The virus, which now appears to be enzootic in many regions, has on occasion caused zoonotic infections in humans (1). Humans who acquire the infection develop severe pneumonia that can progress to acute respiratory distress syndrome with high risk of mortality. For the 6-year period 2003 to 2008, 395 confirmed A(H5N1) virus human infections were reported, and 250 were fatal (a case fatality rate of 63%) (http://www.who.int/csr/disease/avian_influenza/en/index.html). Human-to-human transmission of A(H5N1) virus appears to be rare and has been associated only with very close unprotected contact with severely ill patients (30). Of concern is the potential for the A(H5N1) virus to become easily transmissible between humans, which, because of the lack of prior immunity to this strain in humans, might result in a global influenza pandemic. Based on these theoretical concerns and the experiences of large-scale morbidity and mortality from previous influenza pandemics, many countries have prepared plans to address or mitigate such an occurrence, including the stockpiling of inactivated A(H5N1) influenza vaccines, as well as anti-influenza drugs. Because multiple vaccine doses may be necessary to achieve protection and some time would be required to generate a vaccine with an antigenically matched strain (1), antiviral drugs could play a critical role in the treatment or prophylaxis of influenza, particularly during the early stages of a pandemic. The oral neuraminidase (NA) inhibitor oseltamivir (Tamiflu) has been the most widely used anti-influenza drug for the treatment of A(H5N1) virus -infected patients and has been stockpiled for potential broad use. Results from uncontrolled clinical trials suggest that the use of oseltamivir may increase the survival rate of patients with A(H5N1) virus infection, particularly if administered early in the course of illness (1). However, oseltamivir-resistant A(H5N1) virus variants with an H274Y NA mutation have been isolated from treated patients and may be associated with clinical deterioration and fatal outcomes (9). Viruses with the H274Y NA mutations are susceptible to the NA inhibitor zanamivir, which has led to the inclusion of inhaled zanamivir, together with oseltamivir, in pandemic drug stockpiles. The volume of drug that might be used in the event of a pandemic would be significantly greater than has ever been used previously for treatment of seasonal influenza. There is concern that this may lead to a high frequency of drug resistance. While previous studies have identified a number of NA inhibitor resistance mutations that have arisen in seasonal influenza viruses under drug pressure, little is known about which NA inhibitor resistance mutations might arise in highly pathogenic A(H5N1) viruses. To investigate this question, two A(H5N1) strains from different phylogenetic clades were subjected to serial passage in Madin-Darby canine kidney (MDCK) cells in the presence of increasing levels of either oseltamivir or zanamivir, and the resultant viruses were analyzed functionally and genetically.     

Characterization of a Large Cluster of Influenza A(H1N1)pdm09 Viruses Cross-Resistant to Oseltamivir and Peramivir during the 2013-2014 Influenza Season in Japan

Between September 2013 and July 2014, 2,482 influenza 2009 pandemic A(H1N1) [A(H1N1)pdm09] viruses were screened in Japan for the H275Y substitution in their neuraminidase (NA) protein, which confers cross-resistance to oseltamivir and peramivir. We found that a large cluster of the H275Y mutant virus was present prior to the main influenza season in Sapporo/Hokkaido, with the detection rate for this mutant virus reaching 29% in this area. Phylogenetic analysis suggested the clonal expansion of a single mutant virus in Sapporo/Hokkaido. To understand the reason for this large cluster, we examined the in vitro and in vivo properties of the mutant virus. We found that it grew well in cell culture, with growth comparable to that of the wild-type virus. The cluster virus also replicated well in the upper respiratory tract of ferrets and was transmitted efficiently between ferrets by way of respiratory droplets. Almost all recently circulating A(H1N1)pdm09 viruses, including the cluster virus, possessed two substitutions in NA, V241I and N369K, which are known to increase replication and transmission fitness. A structural analysis of NA predicted that a third substitution (N386K) in the NA of the cluster virus destabilized the mutant NA structure in the presence of the V241I and N369K substitutions. Our results suggest that the cluster virus retained viral fitness to spread among humans and, accordingly, caused the large cluster in Sapporo/Hokkaido. However, the mutant NA structure was less stable than that of the wild-type virus. Therefore, once the wild-type virus began to circulate in the community, the mutant virus could not compete and faded out.     

Characterization of Drug-Resistant Influenza Virus A(H1N1) and A(H3N2) Variants Selected In Vitro with Laninamivir

Neuraminidase inhibitors (NAIs) play a major role for managing influenza virus infections. The widespread oseltamivir resistance among 2007-2008 seasonal A(H1N1) viruses and community outbreaks of oseltamivir-resistant A(H1N1)pdm09 strains highlights the need for additional anti-influenza virus agents. Laninamivir is a novel long-lasting NAI that has demonstrated in vitro activity against influenza A and B viruses, and its prodrug (laninamivir octanoate) is in phase II clinical trials in the United States and other countries. Currently, little information is available on the mechanisms of resistance to laninamivir. In this study, we first performed neuraminidase (NA) inhibition assays to determine the activity of laninamivir against a set of influenza A viruses containing NA mutations conferring resistance to one or many other NAIs. We also generated drug-resistant A(H1N1) and A(H3N2) viruses under in vitro laninamivir pressure. Laninamivir demonstrated a profile of susceptibility that was similar to that of zanamivir. More specifically, it retained activity against oseltamivir-resistant H275Y and N295S A(H1N1) variants and the E119V A(H3N2) variant. In vitro, laninamivir pressure selected the E119A NA substitution in the A/Solomon Islands/3/2006 A(H1N1) background, whereas E119K and G147E NA changes along with a K133E hemagglutinin (HA) substitution were selected in the A/Quebec/144147/2009 A(H1N1)pdm09 strain. In the A/Brisbane/10/2007 A(H3N2) background, a large NA deletion accompanied by S138A/P194L HA substitutions was selected. This H3N2 variant had altered receptor-binding properties and was highly resistant to laninamivir in plaque reduction assays. Overall, we confirmed the similarity between zanamivir and laninamivir susceptibility profiles and demonstrated that both NA and HA changes can contribute to laninamivir resistance in vitro.     

危重型甲型H1N1流感患儿二例的护理

目的探讨危重型H1N1甲型流感患儿的护理措施。方法对2例危重型甲型H1N1流感患儿实行全方位护理,特别是加强消毒隔离及医护人员的自我防护、呼吸功能障碍的护理和监护、药物使用和循环功能的监护等。结果 2例患儿的住院时间分别为14d和18d,均痊愈出院。结论危重型甲型H1N1流感患儿病情发展迅速,易发生并发症,全方位、高质量的护理措施对患儿的成功救治和防止疾病传播具有重要意义。     

甲型H1N1流感的预防

<正>甲型H1N1流感(猪流感)是甲型(A型)流感病毒引起的猪或人的一种急性、人畜共患呼吸道传染性疾病。2009年3月,墨西哥和美国等国家先后发生人感染新型猪流感病毒疫情。甲型H1N1流感可以人传染人,其传染途径与季     

Susceptibility of Highly Pathogenic H5N1 Influenza Viruses to the Neuraminidase Inhibitor Oseltamivir Differs In Vitro and in a Mouse Model

While the neuraminidase (NA) inhibitor oseltamivir is currently our first line of defense against a pandemic threat, there is little information about whether in vitro testing can predict the in vivo effectiveness of antiviral treatment. Using a panel of five H5N1 influenza viruses (H5 clades 1 and 2), we determined that four viruses were susceptible to the drug in vitro (mean 50% inhibitory concentration [IC₅₀], 0.1 to 4.9 nM), and A/Turkey/65-1242/06 virus was slightly less susceptible (mean IC₅₀, 10.8 nM). Two avian viruses showed significantly greater NA enzymatic activity (V_max) than the human viruses, and the five viruses varied in their affinity for the NA substrate MUNANA (K_m, 64 to 300 μM) and for oseltamivir carboxylate (K_i, 0.1 to 7.9 nM). The protection of mice provided by a standard oseltamivir regimen (20 mg/kg/day for 5 days) also varied among the viruses used. We observed (i) complete protection against the less virulent A/chicken/Jogjakarta/BBVET/IX/04 virus; (ii) moderate protection (60 to 80% survival) against three viruses, two of which are neurotropic; and (iii) no protection against A/Turkey/65-1242/06 virus, which induced high pulmonary expression of proinflammatory mediators (interleukin-1α [IL-1α], IL-6, alpha interferon, and monocyte chemotactic protein 1) and contained a minor subpopulation of drug-resistant clones (I117V and E119A NA mutations). We found no correlation between in vitro susceptibility and in vivo protection (Spearman rank correlation coefficient ρ = −0.1; P > 0.05). Therefore, the in vivo efficacy of oseltamivir against highly pathogenic H5N1 influenza viruses cannot be reliably predicted by susceptibility testing, and more prognostic ways to evaluate anti-influenza compounds must be developed. Multiple viral and host factors modulate the effectiveness of NA inhibitor regimens against such viruses and new, more consistently effective treatment options, including combination therapies, are needed.Highly pathogenic avian H5N1 influenza viruses have spread intercontinentally and evolved into 10 phylogenetically distinct hemagglutinin (HA) clades; the most diverse, clade 2, comprises five subclades (33). Large outbreaks among poultry continue in far-ranging geographical areas, although human infections remain rare (411 confirmed cases since May 2003) (34). However, the pandemic potential of H5N1 influenza viruses should not be underestimated, and preparedness requires that appropriate prophylactic and therapeutic antiviral regimens be established. Importantly, human H5N1 infection differs markedly from human seasonal influenza (35). Viral pneumonia is considered a primary cause of death from H5N1 infection, but disseminated disease and multiorgan failure with renal and cardiac dysfunction, Reye''s syndrome, and hemorrhage often occur (1, 4, 38). Infectious virus and viral RNA have been isolated from the upper and lower respiratory tract, brain, intestines, feces, blood, cerebrospinal fluid, and even from the placentas and fetuses of pregnant women (9, 35).Antiviral drugs can play an important role in the initial response to pandemic influenza. One of the two classes of anti-influenza drugs, M2-ion channel blockers (amantadine and rimantadine), has limited usefulness, because clade 1 H5N1 viruses are frequently resistant (3, 22), although representatives of clade 2 are susceptible to adamantanes (15, 26). Most H5N1 isolates are susceptible in vitro to the second class of drugs, neuraminidase (NA) inhibitors (oseltamivir and zanamivir) (12). Natural genetic variations in NA were reported to affect the susceptibility of H5N1 viruses to oseltamivir in vitro (23), and some clade 2 viruses were found to be 15 to 30 times less susceptible to oseltamivir than clade 1 viruses, based on their 50% inhibitory concentrations (IC₅₀s) (18). Reduced susceptibility may be caused by NA antigenic mutation(s) and by the emergence of specific NA mutations under drug selection pressure (18, 23). NA mutations at positions 274 (H→Y) and 294 (N→S) are considered markers of the oseltamivir-resistant H5N1 phenotype (6, 17).The NA enzyme inhibition assay measures the decrease in functional NA activity in the presence of the drug. This assay is considered the most reliable in vitro method of quantifying the susceptibility of seasonal influenza viruses to NA inhibitors, and it is well correlated with their susceptibility in animal models (29). However, it is unknown whether in vitro data can accurately predict the effectiveness of antiviral drugs against H5N1 viruses in vivo, since viral and host factors that modulate disease manifestations are incompletely understood (20). Experimental animal models are a logical approach to estimating drug effectiveness in vivo against lethal influenza virus infection. Studies in mice showed that more prolonged oseltamivir treatment is required to inhibit residual replication of a highly virulent representative of clade 1, A/Vietnam/1203/04 (H5N1) virus, than to inhibit a less virulent 1997 isolate (36). In a ferret model, the best antiviral effect against H5N1 virus was achieved by increasing the dose of oseltamivir and initiating treatment early (8). These observations show that the optimal dose and duration of an anti-H5N1 regimen may depend on virus virulence, although other viral factors can play a role. Some characteristics, such as the ability to spread systemically, tissue tropism (including neurotropism), virus fitness, the characteristics of individual virus proteins, and a preference for binding to α2,3- or α2,6-linked sialic acid receptors, clearly differ among H5N1 viruses and may affect the protection offered by antiviral therapy. It is also unknown whether the hypercytokinemia reported in human cases of H5N1 infection (7) represents an appropriate immune response or immune dysregulation that may alter the outcome of drug therapy.In the present study, we compared the in vitro NA inhibitor susceptibility and NA protein properties (enzymatic activity, affinity for substrate, and affinity for NA inhibitors) of five highly pathogenic H5N1 influenza viruses with the efficacy of oseltamivir treatment in a mouse model. Viruses of clade 1 and of the more diverse clade 2 were represented. Virus replication in the lungs and brain and production of proinflammatory cytokines were assessed, and virus clones were sequenced to identify minor subpopulations of variants and determine their effect on antiviral treatment. Here we demonstrate that the in vivo efficacy in mice of NA inhibitors against highly pathogenic H5N1 influenza viruses cannot be reliably predicted by susceptibility testing in vitro.     

甲型H1N1流感病毒相关发作性睡病

2009甲型(H1N1 pdm09)流感大流行后,几个单价大流行性流感疫苗通过快速程序被许可使用。发作性睡病作为疫苗的副作用被发现,主要是在接受用欧洲的灭活纯化协议生产的AS03佐剂A(H1N1)流感疫苗的人群。感染野生甲型(H1N1)大流行流感病毒后没有接种疫苗的人群,发作性睡病的发病也在增加,这提示此种病毒抗原的作用在发作性睡病疾病发展中具有一定作用。本文进行了发作性睡病背景简介,概述了流感疫苗制剂的不同类型,探讨了自身免疫性疾病和自然感染之间的关系。     

Multiple Influenza A (H3N2) Mutations Conferring Resistance to Neuraminidase Inhibitors in a Bone Marrow Transplant Recipient

Immunocompromised patients are predisposed to infections caused by influenza virus. Influenza virus may produce considerable morbidity, including protracted illness and prolonged viral shedding in these patients, thus prompting higher doses and prolonged courses of antiviral therapy. This approach may promote the emergence of resistant strains. Characterization of neuraminidase (NA) inhibitor (NAI)-resistant strains of influenza A virus is essential for documenting causes of resistance. In this study, using quantitative real-time PCR along with conventional Sanger sequencing, we identified an NAI-resistant strain of influenza A (H3N2) virus in an immunocompromised patient. In-depth analysis by deep gene sequencing revealed that various known markers of antiviral resistance, including transient R292K and Q136K substitutions and a sustained E119K (N2 numbering) substitution in the NA protein emerged during prolonged antiviral therapy. In addition, a combination of a 4-amino-acid deletion at residues 245 to 248 (Δ245-248) accompanied by the E119V substitution occurred, causing resistance to or reduced inhibition by NAIs (oseltamivir, zanamivir, and peramivir). Resistant variants within a pool of viral quasispecies arose during combined antiviral treatment. More research is needed to understand the interplay of drug resistance mutations, viral fitness, and transmission.     

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.In the spring of 2009, an antigenically novel influenza A virus (H1N1) was detected in North America (7). The rapid widespread transmission of the virus resulted in the declaration of an influenza pandemic by the World Health Organization (WHO) (42). The 2009 pandemic influenza A (H1N1) virus (H1N1pdm) was determined to be a reassortant with a combination of gene segments that had not been previously described (12, 21). Phylogenetic analysis of the full genome sequences revealed that in the late 1990s, reassortment between seasonal influenza A virus (H3N2), classical swine influenza virus, and North American avian influenza viruses led to the appearance of triple-reassortant H3N2 and H1N2 swine influenza viruses that have since circulated in pigs in North America (40). The pandemic virus was a result of further reassortment between a triple-reassortant swine influenza virus and a Eurasian avian influenza virus-like swine influenza virus, resulting in the acquisition of two gene segments, coding for the M protein and neuraminidase (NA), from the Eurasian avian influenza virus-like swine influenza virus lineage. Recent genome sequence analysis performed with pandemic viruses collected in different regions found variants with characteristic amino acid changes, including 2 amino acid changes in the NA (21, 29). The reports identified three NA variants among the H1N1pdm viruses: one variant group has V106 and N248 (referred to as the A/California/04/2009 group); the second variant, named the A/Osaka/164/2009 group, is characterized by I106 and N248; and the third NA variant group contains I106 and D248, such as the A/New York/18/2009 strain.Currently circulating triple-reassortant swine influenza viruses in the United States do not contain any known markers of adamantane resistance (L26F, V27A, A30V, A30T, S31N, and G34E) (10, 25), whereas the Eurasian avian-like influenza viruses as well as the pandemic virus contain the adamantane resistance-conferring change S31N in the M2 protein. Currently, two classes of antiviral drugs are approved for use by the FDA for the control of influenza virus infections: adamantanes (M2 blockers) and neuraminidase inhibitors (NAIs). Resistance to adamantanes makes the NAIs oseltamivir and zanamivir the only pharmaceutical options available for use for the control of infections caused by the pandemic virus. Monitoring of resistance to NAIs is mainly based on the NA inhibition assay (23, 39, 41), which allows the detection of resistance conferred by known and novel mutations. However, the NA inhibition assay requires virus isolation and propagation, and the detection of resistance by the NA inhibition assay requires confirmation by sequencing of the NA gene segment to identify the markers of resistance and their presence in the original clinical material.Prior to the 2007-2008 influenza season, the frequency of resistance to NAIs had been very low (<0.5%) among field isolates (28, 35, 36). During the 2007-2008 influenza season, seasonal H1N1 viruses resistant to oseltamivir emerged and spread globally (3, 17, 31, 39), and by April of 2009, the majority of the H1N1 viruses were resistant to oseltamivir but sensitive to zanamivir. Of note, nearly all of the 2009 pandemic H1N1 viruses were sensitive to NAIs (8); only sporadic cases of oseltamivir-resistant viruses with the H275Y mutation in the NA gene segment were reported to the WHO, and they were mainly detected following antiviral drug treatment (5, 6, 42). The H275Y mutation is equivalent to the H274Y mutation in the N2 subtype amino acid numbering. Throughout the text, amino acids are described with the N1 numbering, and the corresponding N2 amino acid numbering is shown in parentheses, when it differs from the N1 numbering. Recent reports on the emergence of oseltamivir resistance highlight the need for close monitoring of the susceptibility of the pandemic H1N1 virus to the available drugs (5, 6, 42). Such information is needed to make informed decisions on measures aimed at managing pandemic virus infections.The molecular markers of NAI resistance are type and subtype specific and are also drug specific (1, 23). The H275Y (H274Y) change is the most commonly reported mutation conferring resistance to oseltamivir in the N1 subtype of NA. This change has been reported not only in seasonal H1N1 viruses but also in highly pathogenic H5N1 viruses (13, 22, 23, 31, 33). The H275Y (H274Y) mutation is also known to reduce susceptibility to the investigational NAI peramivir (23). The amino acid replacement N295S (N294S) in N1 has also been shown to reduce susceptibility to oseltamivir and zanamivir (33, 43). In addition, recent studies have demonstrated that mutations in other residues located in and around the NA active site can alter the susceptibilities of viruses to NAIs. For instance, changes at residues V116, I117, E119, Q136, D199 (D198), and I223 (I222) were associated with reduced susceptibility to NAIs in both seasonal and H5N1 viruses (26-28, 30, 32, 39). Moreover, crystal structure studies with the NAs of H1N1 and H5N1 viruses (9, 37) suggested that mutations at amino acids Q136, K150, and D151 (37) may affect susceptibility to oseltamivir and zanamivir, presumably by interfering with the binding of the drug to the NA. Changes at these residues were reported to reduce the susceptibilities to NAIs of viruses with the N1 enzyme (34; CDC, Atlanta, GA, unpublished data).It is important to develop the tools necessary for the rapid detection of NA markers known or suspected of affecting susceptibility to NAIs. Pyrosequencing has previously been shown to provide a rapid and high-throughput method for the detection of molecular markers of drug resistance in seasonal as well as highly pathogenic avian influenza viruses (4, 8, 15, 16, 19, 30, 31, 38).Here we report on the design and validation of pyrosequencing assays for the detection of signature markers in the M2 and NA gene segments of the pandemic H1N1 viruses.     

Assessment of Pandemic and Seasonal Influenza A (H1N1) Virus Susceptibility to Neuraminidase Inhibitors in Three Enzyme Activity Inhibition Assays

The neuraminidase inhibitors (NAIs) zanamivir and oseltamivir are currently the only antiviral drugs effective for the treatment and prophylaxis of 2009 pandemic influenza A (H1N1) virus infections. The proven potential of these viruses to acquire NAI resistance during treatment emphasizes the need to assess their NAI susceptibility. The 50% inhibitory concentrations (IC₅₀s) are known to vary depending on the neuraminidase inhibition (NI) test used; however, few side-by-side comparisons of different NI assays have been done. In the present study, a panel of 11 isolates representing 2009 seasonal and pandemic influenza H1N1 viruses, including oseltamivir-resistant H275Y variants, were tested in three functional NI assays: chemiluminescent (CL), fluorescent (FL), and colorimetric (CM). The sensitivities of the viruses to zanamivir, oseltamivir, and three investigational NAIs (peramivir, R-125489, and A-315675) were assessed. All isolates with the exception of H275Y variants were sensitive to all five NAIs by all three NI assays. The H275Y variants showed substantially elevated IC₅₀s against oseltamivir and peramivir. The three NI assays generally yielded consistent results; thus, the choice of NI assay does not appear to affect conclusions based on drug susceptibility surveillance. Each assay, however, offers certain advantages compared to the others: the CL assay required less virus volume and the FL assay provided the greatest difference in the IC₅₀s between the wild type and the variants, whereas the IC₅₀s obtained from the CM assay may be the most predictive of the drug concentrations needed to inhibit enzyme activity in humans. It would be desirable to develop an NI assay which combines the advantages of all three currently available assays but which lacks their shortcomings.For the treatment and chemoprophylaxis of infections caused by influenza A viruses, the U.S. Food and Drug Administration (FDA) has approved four drugs: amantadine and rimantadine as well as zanamivir and oseltamivir. These drugs belong to two classes, adamantanes (i.e., M2 ion-channel blockers) and neuraminidase (NA) inhibitors (NAIs), respectively. In recent years, the effectiveness of M2 blockers has been greatly compromised, which limits their usefulness in clinical practice. This is largely due to the rapid emergence and widespread circulation of adamantane-resistant influenza viruses (1, 5, 6, 7, 14, 17). More recently, the emergence and worldwide spread of seasonal H1N1 viruses resistant to oseltamivir, currently the most widely used drug against influenza infections, became a considerable public health concern (15, 21, 25, 32). Monitoring the NAI resistance of influenza viruses is an ongoing public health issue since the emergence in 2009 of pandemic viruses that are resistant to M2 blockers.Cell culture-based assays are typically not used for assessment of virus sensitivity to NAIs because of the unpredictable effect of hemagglutinin (HA) receptor binding (2, 34). Instead, drug susceptibility can be monitored by functional (biochemical) NA inhibition (NI) assays, and subsequent genotypic methods are generally required to identify the molecular marker(s) of resistance in the NA. The principle underlying the functional methods relies on the enzymatic nature of the NA, a viral surface glycoprotein and antigen. NA acts by cleaving the terminal neuraminic acid (also called sialic acid) from receptors recognized by influenza viral HA, thus facilitating the release of progeny virions from infected cells and preventing self-aggregation (29). Structurally, NAIs mimic the natural substrate, neuraminic acid, and produce tight interactions, with conserved residues of the NA active site competing with neuraminic acid for binding (11, 23). Preincubation of virus with NAIs leads to the inhibition of enzyme activity, which is detected after the addition of enzyme substrate. Most NI assays commonly used for virus surveillance utilize as substrates small synthetic conjugates that produce either a luminescent or a fluorescent signal upon cleavage by the NA enzyme. The chemiluminescent (CL) assay uses the 1,2-dioxetane derivative of neuraminic acid substrate in the influenza neuraminidase inhibitor resistance detection (NA-Star) kit (8), while the fluorescent (FL) assay employs 2′-O-(4-methylumbelliferyl)-N-acetylneuraminic acid substrate (MUNANA) (30). The results of the NI assays are expressed as the 50% inhibitory concentration (IC₅₀), which represents the NAI concentration that inhibits 50% of the enzyme activity of the virus. As the NA activity of clinical specimens is usually insufficient for determining the IC₅₀ due to a low viral content, NI assays, using either the substrate provided with the NA-Star kit or the MUNANA substrate, require virus propagation in cell cultures or embryonated chicken eggs. It is noteworthy that IC₅₀s are specific to the virus type/subtype and to the individual NAI tested (8, 19, 20, 24, 32, 37). The IC₅₀s obtained can be used for assessment of virus susceptibility to NAIs, including detection of resistant viruses, as well as for comparing the potencies of antiviral drugs belonging to the NAI class. Although both the CL and FL assays allow reliable detection of NAI resistance, the more recently developed CL assay was reported to be about 70 times more sensitive in detecting NA activity and has a greater linear range than the FL assay (8). The CL assay was also selected for use in the global drug susceptibility surveillance program by the Neuraminidase Inhibitor Susceptibility Network (NISN) (37, 39) and by other surveillance laboratories (28, 32). It should also be noted that IC₅₀s may vary even for the same virus when the NI assay is done using the NA-Star substrate (CL assay) and the MUNANA substrate (FL assay), according to reports on seasonal viruses (37). Whether one of the two assays, the CL or FL assay, more reliably predicts the level of resistance and the drug concentration required for the NA activity inhibition in vivo are key points of interest and remain to be elucidated.A third assay, the colorimetric (CM) assay, which utilizes fetuin as the substrate of the NA, is typically used to determine the titer of anti-NA antibodies because small substrates do not effectively compete with antibodies (3, 31). This assay is not widely used for antiviral susceptibility testing. Unlike the NA-Star and MUNANA synthetic substrates, fetuin is a large, natural, and soluble bovine glycoprotein that contains abundant neuraminic acids at the ends of its oligosaccharide moiety (which include the presence of two residues of α2,3-linked sialic acid and one residue of α2,6-linked sialic acid) (4, 33) and has been used as a substrate in NA-catalyzed reactions (3). Given that NAIs compete with the enzyme substrate for binding to the active site, the structure of the substrate can potentially influence the outcome of the competition and, as a result, the IC₅₀. In this respect, fetuin may represent a better natural substrate for the enzyme-neuraminic acid attached via an α2,3 or α2,6 linkage to oligosaccharide chains on the cell surface. Furthermore, since the cleavage of each neuraminic acid is chemically converted, the CM assay can be a quantifiable method from which the resulting IC₅₀s would correlate more closely to the NA activity of the virus tested. Despite these apparent advantages to the use of fetuin, the CM method relies on chemical reactions that are time-consuming, cumbersome, and impractical for high-throughput use. In addition, the assay requires concentrated virus stocks for testing. Thus, fetuin is still considered an undefined substrate that does not confer sufficient sensitivity or specificity for use in routine NAI susceptibility assays (34). The potential usefulness of a large substrate such as fetuin for assessment of the NAI susceptibilities of novel H1N1 viruses or novel inhibitors remains largely unexplored.Resistance to NAIs is not defined as clearly as that to adamantanes. In NI assays, a drug-resistant virus should have IC₅₀s consistently greater than the threshold value that is determined for each viral type/subtype and drug tested (27, 32, 37). Since the 2007-2008 influenza season, about a decade after the introduction of NAIs into clinical use, an NA framework mutation, H275Y (H274Y in N2 numbering), was consistently and most commonly detected in oseltamivir-resistant H1N1 viruses isolated worldwide (15, 21, 25, 32). Although the H275Y substitution represents the most-defined oseltamivir resistance marker of influenza viruses carrying the NA of the N1 subtype (35), novel NAI resistance-associated mutations—determined by elevated IC₅₀s in NI assays—continue to be revealed (21, 22, 32). Importantly, oseltamivir-resistant viruses from the ongoing H1N1 pandemic have been detected and reported around the world (9, 10, 26, 38). Seasonal and 2009 pandemic H1N1 viruses have the same phylogenetically distant NA gene ancestors (16), which necessitates the comprehensive assessment of the drug susceptibilities of the new pandemic viruses. Therefore, it is necessary to evaluate existing NI assays in order to better understand which assay may be the most sensitive for the detection of NAI resistance and/or the most predictive of virus susceptibility to NAIs in vivo.In the present study, we assessed the susceptibilities of a panel of seasonal and pandemic H1N1 influenza viruses, including virus variants bearing the established oseltamivir resistance mutation, H275Y in the NA, against five NAIs: two FDA-approved NAIs, zanamivir and oseltamivir, and three investigational NAIs, peramivir, R-125489 (the bioactive metabolite of the prodrug CS-8958 [laninamivir]), and A-315675 (a bioactive form of the prodrug A-322278). In order to better characterize and assess the consistency of IC₅₀s and levels of susceptibility, these viruses were tested in the widely used CL and FL assays, as well as with the CM method.     

Influenza A Subtyping: Seasonal H1N1, H3N2, and the Appearance of Novel H1N1

Influenza virus subtyping has emerged as a critical tool in the diagnosis of influenza. Antiviral resistance is present in the majority of seasonal H1N1 influenza A infections, with association of viral strain type and antiviral resistance. Influenza A virus subtypes can be reliably distinguished by examining conserved sequences in the matrix protein gene. We describe our experience with an assay for influenza A subtyping based on matrix gene sequences. Viral RNA was prepared from nasopharyngeal swab samples, and real-time RT-PCR detection of influenza A and B was performed using a laboratory developed analyte-specific reagent-based assay that targets a conserved region of the influenza A matrix protein gene. FluA-positive samples were analyzed using a second RT-PCR assay targeting the matrix protein gene to distinguish seasonal influenza subtypes based on differential melting of fluorescence resonance energy transfer probes. The novel H1N1 influenza strain responsible for the 2009 pandemic showed a melting profile distinct from that of seasonal H1N1 or H3N2 and compatible with the predicted melting temperature based on the published novel H1N1 matrix gene sequence. Validation by comparison with the Centers for Disease Control and Prevention real-time RT-PCR for swine influenza A (novel H1N1) test showed this assay to be both rapid and reliable (>99% sensitive and specific) in the identification of the novel H1N1 influenza A virus strain.The 2009 novel influenza A/H1N1 viral pandemic has presented challenges for hospital laboratories and health care systems seeking to rapidly diagnose, treat, and limit the spread of this virus. As is the case for routine diagnosis of seasonal influenza infections, molecular amplification assays offer the potential for the sensitivity and speed needed to manage an influenza outbreak. However, standardized RT-PCR assays specific for this strain of influenza were not initially available, leaving many laboratories to diagnose this infection through indirect means.Our laboratory has used PCR for rapid detection of influenza A and B for several years, and more recently had implemented a rapid RT-PCR/melt-curve assay designed to differentiate seasonal influenza A subtypes H1N1 and H3N2.¹ This approach was initially developed for viral subtyping to guide clinicians on the appropriate antiviral therapy. Antiviral resistance has risen during recent years, with the majority of seasonal H1N1 strains no longer being sensitive to oseltamivir (Tamiflu), and seasonal H3N2 strains being largely resistant to adamantanes.² Rapid determination of influenza A subtype is essential for determining optimal therapy and for prudent use of antiviral agents. Consequently, this RT-PCR assay has become part of our influenza testing algorithm.The design of the RT-PCR assay exploits minor variations in a relatively conserved sequence within the matrix protein gene. Not surprisingly, the novel H1N1 strain of influenza that appeared in the spring of 2009 had a distinct melting temperature consistent with the published matrix gene sequence and the sequence of the fluorescence resonance energy transfer probes used in this assay designed to differentiate seasonal influenza A subtypes H1N1 and H3N2. As part of our influenza testing algorithm, this assay allowed definitive diagnosis of the 2009 influenza H1N1 from nasopharyngeal swabs within hours after arrival in the clinical laboratory.     

Strain Types and Antimicrobial Resistance Patterns of Clostridium difficile Isolates from the United States, 2011 to 2013

We determined the PCR ribotypes and antimicrobial susceptibility patterns of 508 toxigenic Clostridium difficile isolates collected between 2011 and 2013 from 32 U.S. hospitals. Of the 29 PCR ribotypes identified, the 027 strain type was the most common (28.1%), although the rates varied by geographic region. Ribotype 014/020 isolates appear to be emerging. Clindamycin and moxifloxacin resistances (36.8% and 35.8%, respectively) were the most frequent resistance phenotypes observed. Reduced susceptibility to vancomycin was observed in 39.1% of 027 isolates.