Microscope studies of symptomless growth of Botrytis cinerea in Lactuca sativa and Arabidopsis thaliana.

The grey mould pathogen Botrytis cinerea forms systemic associations in some hosts, spreading into plant organs produced a considerable time after initial infection. These infections may have no macroscopic symptoms during much of the hosts' lifetime and are at least partially within the host tissue. The aim of the studies reported here was to locate and visualize these infections at a cellular level in Lactuca sativa (lettuce) and Arabidopsis thaliana. Symptomless but infected plants were produced by dry spore inoculation of plants growing in conditions previously shown to result in fungal spread from the initial inoculation site to newly developing plant organs. Tissue taken from inoculated plants was examined using confocal laser scanning microscopy. Two B. cinerea isolates were used: B05.10 and its GFP-labelled derivative Bcgfp1-3. Spore germination on leaf surfaces was followed by development of subcuticular inclusions and plant cell damage in single infected epidermal cells and sometimes a few nearby cells. Sparsely branched long hyphae arose and spread from the inclusions, mostly on the outer surface of the epidermal layer but occasionally below the cuticle or epidermal cells, where further inclusions formed. This was consistent with the pattern in time of recovery of B. cinerea from surface-sterilized leaf tissue. In the late symptomless phase, mycelium arising from internal fungal inclusions formed mycelial networks on the surface of leaves. Symptomless exterior mycelium grew on the roots in A. thaliana.

Cross-resistance between myclobutanil and tebuconazole and the genetic basis of tebuconazole resistance in Venturia inaequalis.

BACKGROUND: Myclobutanil is one of the most widely used demethylation inhibitor (DMI) fungicides for the management of apple scab, caused by Venturia inaequalis. Strains of V. inaequalis resistant to myclobutanil have been reported across the world. Tebuconazole, another DMI fungicide, has been proposed as an alternative to myclobutanil, and the extent of cross-resistance with myclobutanil therefore needs to be evaluated. The sensitivity to tebuconazole and myclobutanil of a total of 40 isolates was determined. Half the isolates came from an isolated orchard which had never been sprayed with fungicides and half from orchards sprayed regularly with myclobutanil, but still with disease control problems. The progeny of a tebuconazole resistant (R) × sensitive (S) V. inaequalis cross were analyzed in order to improve understanding of the genetic control of tebuconazole sensitivity. RESULTS: There is cross-resistance between myclobutanil and tebuconazole (r = 0.91; P < 0.001). Sensitivity to tebuconazole of the progeny of a R × S cross varied quantitatively in a pattern which implied at least two gene loci differing between the parental strains. In addition, the asymmetric distribution of the sensitivity in the progeny implied possible epistatic effects. CONCLUSION: Resistance to myclobutanil and tebuconazole is strongly correlated. At least two genes are involved in the control of tebuconazole resistance in V. inaequalis.

The effect of competition on the control of invading plant pathogens.

New invading pathogen strains must compete with endemic pathogen strains to emerge and spread. As disease control measures are often non-specific, that is, they do not distinguish between strains, applying control not only affects the invading pathogen strain but the endemic as well. We hypothesize that the control of the invasive strain could be compromised due to the non-specific nature of the control.A spatially explicit model, describing the East African cassava mosaic virus-Uganda strain (EACMV-UG) outbreak, is used to evaluate methods of controlling both disease incidence and spread of invading pathogen strains in pathosystems with and without an endemic pathogen strain present.We find that while many newly introduced or intensified control measures (such as resistant cultivars or roguing) decrease the expected incidence, they have the unintended consequence of increasing, or at least not reducing, the speed with which the invasive pathogen spreads geographically. We identify the controls that cause this effect and methods in which these controls may be applied to prevent it.We found that the spatial spread of the invading strain is chiefly governed by the incidence at the wave front. Control can therefore be applied, or intensified, once the wave front has passed without increasing the pathogen's rate of spread.When trade of planting material occurs, it is possible that the planting material is already infected. The only forms of control in this study that reduces the speed of geographic spread, regardless of the presence of an endemic strain, are those that reduce the amount of trade and the distance over which trade takes place. Synthesis and applications. The best control strategy depends on the presence of competing endemic strains. Applying or intensifying the control can slow the rate of spread when absent but increase it if present. Imposing trade restrictions before the epidemic has reached a given area and intensifying other control methods only when the wave front has passed is the most effective way of both slowing down spread and controlling incidence when a competing endemic strain is present and is the safest approach when its presence is unknown.

Lessons from the reestablishment of Public Health Laboratory activities in Puerto Rico after Hurricane Maria.

Public Health Laboratories (PHLs) in Puerto Rico did not escape the devastation caused by Hurricane Maria. We implemented a quality management system (QMS) approach to systematically reestablish laboratory testing, after evaluating structural and functional damage. PHLs were inoperable immediately after the storm. Our QMS-based approach began in October 2017, ended in May 2018, and resulted in the reestablishment of 92% of baseline laboratory testing capacity. Here, we share lessons learned from the historic recovery of the largest United States' jurisdiction to lose its PHL capacity, and provide broadly applicable tools for other jurisdictions to enhance preparedness for public health emergencies.

Asymptomatic Host Plant Infection by the Widespread Pathogen Botrytis cinerea Alters the Life Histories, Behaviors, and Interactions of an Aphid and Its Natural Enemies.

Plant pathogens can profoundly affect host plant quality as perceived by their insect herbivores, with potentially far-reaching implications for the ecology and structure of insect communities. Changes in host plants may have direct effects on the life-histories of their insect herbivores, which can then influence their value as prey to their natural enemies. While there have been many studies that have explored the effects of infection when plants show symptoms of disease, little is understood about how unexpressed infection may affect interactions at higher trophic levels. We examined how systemic, asymptomatic, and seed-borne infection by the ubiquitous plant pathogen Botrytis cinerea, infecting two varieties of the lettuce Lactuca sativa, affected aphids (the green peach aphid, Myzus persicae) and two widely used biocontrol agents (the parasitoid Aphidius colemani and the ladybird predator Adalia bipunctata). Lettuce varieties differed in host plant quality. Asymptomatic infection reduced chlorophyll content and dry weight of host plants, irrespective of plant variety. Aphids reared on asymptomatic plants were smaller, had reduced off-plant survival time and were less fecund than aphids reared on uninfected plants. Parasitoids showed reduced attack rates on asymptomatically infected plants, and wasps emerging from hosts reared on such plants were smaller and showed reduced starvation resistance. When given a choice in an olfactometer, aphids preferentially chose uninfected plants of one variety (Tom Thumb) but showed no preference with the second (Little Gem) variety. Parasitoids preferentially chose aphids on uninfected plants, irrespective of host plant variety, but ladybirds did not show any such preference. These results suggest that the reduced quality of plants asymptomatically infected by Botrytis cinerea negatively affects the life history of aphids and their parasitoids, and alters the behaviors of aphids and parasitoids, but not of ladybirds. Fungal pathogens are ubiquitous in nature, and this work shows that even when host plants are yet to show symptoms, pathogens can affect interactions between insect herbivores and their natural enemies. This is likely to have important implications for the success of biological control programs.

Dose-dependent selection drives lineage replacement during the experimental evolution of SDHI fungicide resistance in Zymoseptoria tritici.

Fungicide resistance is a constant threat to agricultural production worldwide. Molecular mechanisms of fungicide resistance have been studied extensively in the wheat pathogen Zymoseptoria tritici. However, less is known about the evolutionary processes driving resistance development. In vitro evolutionary studies give the opportunity to investigate this. Here, we examine the adaptation of Z. tritici to fluxapyroxad, a succinate dehydrogenase (Sdh) inhibitor. Replicate populations of Z. tritici derived from the sensitive isolate IPO323 were exposed to increasing concentrations of fluxapyroxad with or without UV mutagenesis. After ten increases in fungicide concentration, sensitivity had decreased dramatically, with replicate populations showing similar phenotypic trajectories. Sequencing the Sdh subunit B, C, and D encoding genes identified seven mutations associated with resistance to fluxapyroxad. Mutation frequency over time was measured with a pyrosequencing assay, revealing sequential lineage replacement in the UV-mutagenized populations but not in the untreated populations. Repeating selection from set time-points with different fungicide concentrations revealed that haplotype replacement of Sdh variants was driven by dose-dependent selection as fungicide concentration changed, and was not mutation-limited. These findings suggest that fungicide field applications may select for highly insensitive Sdh variants with higher resistance factors if the fungicide concentration is increased to achieve a better disease control. However, in the absence or presence of lower fungicide concentrations, the spread of these strains might be restricted if the underlying Sdh mutations carry fitness penalties.

High-Fat Feeding Protects Mice From Ventilator-Induced Lung Injury, Via Neutrophil-Independent Mechanisms.

OBJECTIVE: Obesity has a complex impact on acute respiratory distress syndrome patients, being associated with increased likelihood of developing the syndrome but reduced likelihood of dying. We propose that such observations are potentially explained by a model in which obesity influences the iatrogenic injury that occurs subsequent to intensive care admission. This study therefore investigated whether fat feeding protected mice from ventilator-induced lung injury. DESIGN: In vivo study. SETTING: University research laboratory. SUBJECTS: Wild-type C57Bl/6 mice or tumor necrosis factor receptor 2 knockout mice, either fed a high-fat diet for 12-14 weeks, or age-matched lean controls. INTERVENTIONS: Anesthetized mice were ventilated with injurious high tidal volume ventilation for periods up to 180 minutes. MEASUREMENTS AND MAIN RESULTS: Fat-fed mice showed clear attenuation of ventilator-induced lung injury in terms of respiratory mechanics, blood gases, and pulmonary edema. Leukocyte recruitment and activation within the lungs were not significantly attenuated nor were a host of circulating or intra-alveolar inflammatory cytokines. However, intra-alveolar matrix metalloproteinase activity and levels of the matrix metalloproteinase cleavage product soluble receptor for advanced glycation end products were significantly attenuated in fat-fed mice. This was associated with reduced stretch-induced CD147 expression on lung epithelial cells. CONCLUSIONS: Consumption of a high-fat diet protects mice from ventilator-induced lung injury in a manner independent of neutrophil recruitment, which we postulate instead arises through blunted up-regulation of CD147 expression and subsequent activation of intra-alveolar matrix metalloproteinases. These findings may open avenues for therapeutic manipulation in acute respiratory distress syndrome and could have implications for understanding the pathogenesis of lung disease in obese patients.

Analysis of Cryptic, Systemic Botrytis Infections in Symptomless Hosts.

Botrytis species are generally considered to be aggressive, necrotrophic plant pathogens. By contrast to this general perception, however, Botrytis species could frequently be isolated from the interior of multiple tissues in apparently healthy hosts of many species. Infection frequencies reached 50% of samples or more, but were commonly less, and cryptic infections were rare or absent in some plant species. Prevalence varied substantially from year to year and from tissue to tissue, but some host species routinely had high prevalence. The same genotype was found to occur throughout a host, representing mycelial spread. Botrytis cinerea and Botrytis pseudocinerea are the species that most commonly occur as cryptic infections, but phylogenetically distant isolates of Botrytis were also detected, one of which does not correspond to previously described species. Sporulation and visible damage occurred only when infected tissues were stressed, or became mature or senescent. There was no evidence of cryptic infection having a deleterious effect on growth of the host, and prevalence was probably greater in plants grown in high light conditions. Isolates from cryptic infections were often capable of causing disease (to varying extents) when spore suspensions were inoculated onto their own host as well as on distinct host species, arguing against co-adaptation between cryptic isolates and their hosts. These data collectively suggest that several Botrytis species, including the most notorious pathogenic species, exist frequently in cryptic form to an extent that has thus far largely been neglected, and do not need to cause disease on healthy hosts in order to complete their life-cycles.

Detection of Zymoseptoria tritici SDHI-insensitive field isolates carrying the SdhC-H152R and SdhD-R47W substitutions.

BACKGROUND: Succinate dehydrogenase inhibitor (SDHI) fungicides are important in the management of Zymoseptoria tritici in wheat. New active ingredients from this group of fungicides have been introduced recently and are widely used. Because the fungicides act at a single enzyme site, resistance development in Z. tritici is classified as medium-to-high risk. RESULTS: Isolates from Irish experimental plots in 2015 were tested against the SDHI penthiopyrad during routine monitoring. The median of the population was approximately 2 times less sensitive than the median of the baseline population. Two of the 93 isolates were much less sensitive to penthiopyrad than the least sensitive of the baseline isolates. These isolates were also insensitive to most commercially available SDHIs. Analysis of the succinate dehydrogenase coding genes confirmed the presence of the substitutions SdhC-H152R and SdhD-R47W in the very insensitive isolates. CONCLUSION: This is the first report showing that the SdhC-H152R mutation detected in laboratory mutagenesis studies also exists in the field. The function and relevance of this mutation, combined with SdhD-R47W, still needs to be determined. © 2016 Society of Chemical Industry.

The effect of succinate dehydrogenase inhibitor/azole mixtures on selection of Zymoseptoria tritici isolates with reduced sensitivity.

BACKGROUND: Combining fungicides with different modes of action is regarded as one of the most effective means of slowing the selection of resistance. Field trials were used to study the effects of such mixtures on selection for Zymoseptoria tritici with reduced sensitivity to the succinate dehydrogenase inhibitors (SDHIs) and azole fungicides. The SDHI isopyrazam and the azole epoxiconazole were applied individually as solo products, and together in a preformulated mixture. All fungicide treatments were included at both full and half the recommended doses. RESULTS: Compared with using epoxiconazole alone, mixing epoxiconazole with isopyrazam led to an increase in epoxiconazole-sensitive isolates. In contrast, all treatments containing isopyrazam reduced the sensitivity of Z. tritici to isopyrazam compared with those without. Reducing doses to half the recommended rate had no effect on sensitivity of isolates to either active ingredient. In a subgroup of isolates least sensitive to isopyrazam, non-synonymous mutations were found in the SdhC and SdhD subunits, but their presence was unrelated to sensitivity. CONCLUSION: Mixing an azole and SDHI was clearly beneficial for the azole, but not for the SDHI component. This dynamic might change if strains conferring reduced sensitivity to the SDHIs were to arise. © 2015 Society of Chemical Industry.

Transient Microgeographic Clines during B Chromosome Invasion.

The near-neutral model of B chromosome evolution predicts that the invasion of a new population should last some tens of generations, but the details on how it proceeds in real populations are mostly unknown. Trying to fill this gap, we analyze here a natural population of the grasshopper Eyprepocnemis plorans at three time points during the last 35 years. Our results show that B chromosome frequency increased significantly during this period and that a cline observed in 1992 had disappeared in 2012 once B chromosome frequency reached an upper limit at all sites sampled. This indicates that, during B chromosome invasion, transient clines for B chromosome frequency are formed at the invasion front on a microgeographic scale. Computer simulation experiments showed that the pattern of change observed for genotypic frequencies is consistent with the existence of B chromosome drive through females and selection against individuals with a high number of B chromosomes.

Paralog re-emergence: a novel, historically contingent mechanism in the evolution of antimicrobial resistance.

Evolution of resistance to drugs and pesticides poses a serious threat to human health and agricultural production. CYP51 encodes the target site of azole fungicides, widely used clinically and in agriculture. Azole resistance can evolve due to point mutations or overexpression of CYP51, and previous studies have shown that fungicide-resistant alleles have arisen by de novo mutation. Paralogs CYP51A and CYP51B are found in filamentous ascomycetes, but CYP51A has been lost from multiple lineages. Here, we show that in the barley pathogen Rhynchosporium commune, re-emergence of CYP51A constitutes a novel mechanism for the evolution of resistance to azoles. Pyrosequencing analysis of historical barley leaf samples from a unique long-term experiment from 1892 to 2008 indicates that the majority of the R. commune population lacked CYP51A until 1985, after which the frequency of CYP51A rapidly increased. Functional analysis demonstrates that CYP51A retains the same substrate as CYP51B, but with different transcriptional regulation. Phylogenetic analyses show that the origin of CYP51A far predates azole use, and newly sequenced Rhynchosporium genomes show CYP51A persisting in the R. commune lineage rather than being regained by horizontal gene transfer; therefore, CYP51A re-emergence provides an example of adaptation to novel compounds by selection from standing genetic variation.

Botrytis species: relentless necrotrophic thugs or endophytes gone rogue?

Plant pathology has a long-standing tradition of classifying microbes as pathogens, endophytes or saprophytes. Lifestyles of pathogens are categorized as biotrophic, necrotrophic or hemibiotrophic. Botrytis species are considered by many to be archetypal examples of necrotrophic fungi, with B. cinerea being the most extensively studied species because of its broad host range and economic impact. In this review, we discuss recent work which illustrates that B. cinerea is capable of colonizing plants internally, presumably as an endophyte, without causing any disease or stress symptoms. The extent of the facultative endophytic behaviour of B. cinerea and its relevance in the ecology and disease epidemiology may be vastly underestimated. Moreover, we discuss the recent discovery of a novel Botrytis species, B. deweyae, which normally grows as an endophyte in ornamental daylilies (Hemerocallis), but displays facultative pathogenic behaviour, and is increasingly causing economic damage. We propose that the emergence of endophytes 'gone rogue' as novel diseases may be related to increased inbreeding of hybrid lines and reduced genetic diversity. These observations lead us to argue that the sometimes inflexible classification of pathogenic microbes by their lifestyles requires serious reconsideration. There is much more variety to the interactions of Botrytis with its hosts than the eye (or the plant pathologist) can see, and this may be true for other microbes interacting with plants.

Alterations in the predicted regulatory and coding regions of the sterol 14α-demethylase gene (CYP51) confer decreased azole sensitivity in the oilseed rape pathogen Pyrenopeziza brassicae.

The incidence and severity of light leaf spot epidemics caused by the ascomycete fungus Pyrenopeziza brassicae on UK oilseed rape crops are increasing. The disease is currently controlled by a combination of host resistance, cultural practices and fungicide applications. We report decreases in sensitivity of modern UK P. brassicae isolates to the azole (imidazole and triazole) class of fungicides. By cloning and sequencing the P. brassicae CYP51 (PbCYP51) gene, encoding the azole target sterol 14α-demethylase, we identified two non-synonymous mutations encoding substitutions G460S and S508T associated with reduced azole sensitivity. We confirmed the impact of the encoded PbCYP51 changes on azole sensitivity and protein activity by heterologous expression in a Saccharomyces cerevisiae mutant YUG37:erg11 carrying a controllable promoter of native CYP51 expression. In addition, we identified insertions in the predicted regulatory regions of PbCYP51 in isolates with reduced azole sensitivity. The presence of these insertions was associated with enhanced transcription of PbCYP51 in response to subinhibitory concentrations of the azole fungicide tebuconazole. Genetic analysis of in vitro crosses of sensitive and resistant isolates confirmed the impact of PbCYP51 alterations in coding and regulatory sequences on a reduced sensitivity phenotype, as well as identifying a second major gene at another locus contributing to resistance in some isolates. The least sensitive field isolates carry combinations of upstream insertions and non-synonymous mutations, suggesting that PbCYP51 evolution is ongoing and the progressive decline in azole sensitivity of UK P. brassicae populations will continue. The implications for the future control of light leaf spot are discussed.

Detection and molecular characterisation of Pyrenopeziza brassicae isolates resistant to methyl benzimidazole carbamates.

BACKGROUND: Methyl benzimidazole carbamate (MBC) fungicides are used to control the oilseed rape pathogen Pyrenopeziza brassicae. Resistance to MBCs has been reported in P. brassicae, but the molecular mechanism(s) associated with reductions in sensitivity have not been verified in this species. Elucidation of the genetic changes responsible for resistance, hypothesised to be target-site mutations in ß-tubulin, will enable resistance diagnostics and thereby inform resistance management strategies. RESULTS: P. brassicae isolates were classified as sensitive, moderately resistant or resistant to MBCs. Crossing P. brassicae isolates of different MBC sensitivities indicated that resistance was conferred by a single gene. The MBC-target encoding gene ß-tubulin was cloned and sequenced. Reduced MBC sensitivity of field isolates correlated with ß-tubulin amino acid substitutions L240F and E198A. The highest level of MBC resistance was measured for isolates carrying E198A. Negative cross-resistance between MBCs and the fungicides diethofencarb and zoxamide was only measured in E198A isolates. PCR-RFLP was used to screen isolates for the presence of L240F and E198A. The substitutions E198G and F200Y were also detected in DNA samples from P. brassicae populations after cloning and sequencing of PCR products. The frequencies of L240F and E198A in different P. brassicae populations were quantified by pyrosequencing. There were no differences in the frequencies of these alleles between P. brassicae populations sampled from different locations or after fungicide treatment regimes. CONCLUSIONS: The molecular mechanisms affecting sensitivity to MBCs in P. brassicae have been identified. Pyrosequencing assays are a powerful tool for quantifying fungicide-resistant alleles in pathogen populations.

Identification of cellular proteins required for replication of human immunodeficiency virus type 1.

Cellular proteins are essential for human immunodeficiency virus type 1 (HIV-1) replication and may serve as viable new targets for treating infection. Using gene trap insertional mutagenesis, a high-throughput approach based on random inactivation of cellular genes, candidate genes were found that limit virus replication when mutated. Disrupted genes (N=87) conferring resistance to lytic infection with several viruses were queried for an affect on HIV-1 replication by utilizing small interfering RNA (siRNA) screens in TZM-bl cells. Several genes regulating diverse pathways were found to be required for HIV-1 replication, including DHX8, DNAJA1, GTF2E1, GTF2E2, HAP1, KALRN, UBA3, UBE2E3, and VMP1. Candidate genes were independently tested in primary human macrophages, toxicity assays, and/or Tat-dependent ß-galactosidase reporter assays. Bioinformatics analyses indicated that several host factors present in this study participate in canonical pathways and functional processes implicated in prior genome-wide studies. However, the genes presented in this study did not share identity with those found previously. Novel antiviral targets identified in this study should open new avenues for mechanistic investigation.

Inhibition of influenza A virus replication by antagonism of a PI3K-AKT-mTOR pathway member identified by gene-trap insertional mutagenesis.

BACKGROUND: Host genes serving potential roles in virus replication may be exploited as novel antiviral targets. METHODS: Small interfering RNA (siRNA)-mediated knockdown of host gene expression was used to validate candidate genes in screens against six unrelated viruses, most importantly influenza. A mouse model of influenza A virus infection was used to evaluate the efficacy of a candidate FDA-approved drug identified in the screening effort. RESULTS: Several genes in the PI3K-AKT-mTOR pathway were found to support broad-spectrum viral replication in vitro by RNA interference. This led to the discovery that everolimus, an mTOR inhibitor, showed in vitro antiviral activity against cowpox, dengue type 2, influenza A, rhino- and respiratory syncytial viruses. In a lethal mouse infection model of influenza A (H1N1 and H5N1) virus infection, everolimus treatment (1 mg/kg/day) significantly delayed death but could not prevent mortality. Fourteen days of treatment was more beneficial in delaying the time to death than treatment for seven days. Pathological findings in everolimus-treated mice showed reduced lung haemorrhage and lung weights in response to infection. CONCLUSIONS: These results provide proof of concept that cellular targets can be identified by gene knockout methods, and highlight the importance of the PI3K-AKT-mTOR pathway in supporting viral infections.

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.

Validation of a method for preparing influenza H5N1 simulated samples.

Avian influenza virus type A subtype H5N1 and potentially other novel influenza A viruses continue to pose a concern with mutation into a form easily transmitted between humans. The ability to rapidly detect and characterize influenza viruses, and distinguish seasonal and novel influenza A viruses such as H5N1, remains important to minimize morbidity and mortality in humans. As with other rare and emerging viral pathogens, clinical specimens from persons with H5N1 infections are extremely rare. Consequently, development of standardized methods and accepted criteria are necessary for both ensuring the validity of available diagnostic methods and for assessing the potential of new diagnostic tests that can detect and differentiate H5N1 and other novel influenza A viruses. Additionally, genotypic and antigenic evolution of H5N1 poses a challenge with maintaining updated reference virus strains. In this report, a method for preparing simulated samples using defined procedures and carefully selected H5N1 virus strains is described, and the reliability for using these samples in an evaluation protocol with a laboratory test for differentiating H5N1 virus from other influenza A viruses is evaluated.

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.