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Influenza viruses pose great threats to public health and cause enormous economic losses every year. Previous work has revealed the viral factors associated with the virulence of influenza viruses in mammals. However, taking prior viral knowledge represented by heterogeneous categorical and discrete information into account to explore virus virulence is scarce in the existing work. How to make full use of the preceding domain knowledge in virulence study is challenging but beneficial. This paper proposes a general framework named ViPal for virulence prediction in mice that incorporates discrete prior viral mutation and reassortment information based on all eight influenza segments. The posterior regularization technique is leveraged to transform prior viral knowledge into constraint features and integrated into the machine learning models. Experimental results on influenza genomic datasets validate that our proposed framework can improve virulence prediction performance over baselines. The comparison between ViPal and other existing methods shows the computational efficiency of our framework with comparable or superior performance. Moreover, the interpretable analysis through SHAP (SHapley Additive exPlanations) identifies the scores of constraint features contributing to the prediction. We hope this framework could provide assistance for the accurate detection of influenza virulence and facilitate flu surveillance., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Current Situation: The global influenza surveillance and response system (GISRS), coordinated by the World Health Organization (WHO), is a global framework for surveillance of influenza and other respiratory viruses, data collection, laboratory capacity building, genomic data submission and archival, standardization, and calibration of reagents and vaccine strains, production of seasonal influenza vaccines and creating a facilitatory regulatory environment for the same. GAPS: WHO-designated national influenza centers (NICs) are entrusted with establishing surveillance in their respective countries. National and subnational surveillance remains weak in most parts of the world because of varying capacities of the NICs, lack of funds, poor human and veterinary surveillance mechanisms, lack of intersectoral coordination, and varying commitments of the local government., Way Forward: As influenza viruses have a wide variety of nonhuman hosts, it is critical to strengthen surveillance at local levels for timely detection of untypable or novel strains with potential to cause epidemics or pandemics. In this article, we have proposed possible strategies to strengthen and expand local capacities for respiratory virus surveillance through the designated NICs of the WHO., Competing Interests: Conflict of interest The authors have no competing interest to declare., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Influenza viruses are major human pathogens, responsible for respiratory diseases affecting millions of people worldwide, with high morbidity and significant mortality. Infections by influenza can be controlled by vaccines and antiviral drugs. However, this virus is constantly under mutations, limiting the effectiveness of these clinical antiviral strategies. It is therefore urgent to develop new ones. Influenza hemagglutinin (HA) is involved in receptor binding and promotes the pH-dependent fusion of viral and cell endocytic membranes. HA-targeted peptides may emerge as a novel antiviral option to block this viral entry step. In this study, we evaluated three HA-derived (lipo)peptides using fluorescence spectroscopy. Peptide membrane interaction assays were performed at neutral and acidic pH to better resemble the natural conditions in which influenza fusion occurs. We found that peptide affinity towards membranes decreases upon the acidification of the environment. Therefore, the released peptides would be able to bind their complementary domain and interfere with the six-helix bundle formation necessary for viral fusion, and thus for the infection of the target cell. Our results provide new insight into molecular interactions between HA-derived peptides and cell membranes, which may contribute to the development of new influenza virus inhibitors., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Next-generation sequencing is regularly used to identify viral sequences in DNA or RNA samples of infected hosts. A major step of most pipelines for virus detection is to map sequence reads against known virus genomes. Due to small differences between the sequences of related viruses, and due to several biological or technical errors, mapping underlies uncertainties. As a consequence, the resulting list of detected viruses can lack robustness. A new approach for generating artificial sequencing reads together with a strategy of resampling from the original findings is proposed that can help to assess the robustness of the originally identified list of viruses. From the original mapping result in form of a SAM file, a set of statistical distributions are derived. These are used in the resampling pipeline to generate new artificial reads which are again mapped versus the reference genomes. By summarizing the resampling procedure, the analyst receives information about whether the presence of a particular virus in the sample gains or losses evidence, and thus about the robustness of the original mapping list but also that of individual viruses in this list. To judge robustness, several indicators are derived from the resampling procedure such as the correlation between original and resampling read counts, or the statistical detection of outliers in the differences of read counts. Additionally, graphical illustrations of read count shifts via Sankey diagrams are provided. To demonstrate the use of the new approach, the resampling approach is applied to three real-world data samples, one of them with laboratory-confirmed Influenza sequences, and to artificially generated data where virus sequences have been spiked into the sequencing data of a host. By applying the resampling pipeline, several viruses drop from the original list while new viruses emerge, showing robustness of those viruses that remain in the list. The evaluation of the new approach shows that the resampling approach is helpful to analyze the viral content of a biological sample, to rate the robustness of original findings and to better show the overall distribution of findings. The method is also applicable to other virus detection pipelines based on read mapping., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Hunters are at a higher risk for exposure to zoonotic pathogens due to their close interactions with wildlife and arthropod vectors. In this study, high throughput sequencing was used to explore the viromes of two tick species, Amblyomma dissimile and Haemaphysalis juxtakochi, removed from hunted wildlife in Trinidad and Tobago. We identified sequences from 3 new viral species, from the viral families Orthomyxoviridae, Chuviridae and Tetraviridae in A. dissimile., (Copyright © 2021 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Introduction: Digital immunoassays (DIAs) and molecular point-of-care (POC) tests for influenza were recently developed. We aimed to evaluate and compare the positive rate with molecular POC tests and DIAs in detecting influenza virus A, B and respiratory syncytial virus (RSV)., Methods: A prospective observational study was conducted in 2019-2020. Nasopharyngeal swab samples were collected from adult outpatients with influenza-like illness who visited four hospitals and clinics in Japan. DIAs were performed at each facility. The clinical diagnosis was determined based on the findings of DIAs, history taking, and physical assessment. Molecular POC test and reverse transcription polymerase chain reaction (RT-PCR) were performed later., Results: A total of 182 patients were evaluated. The positive rate for influenza virus with molecular POC test was significantly higher than that with DIAs (51.6% versus 40.7%, p = 0.046). In patients who tested positive for influenza virus with only molecular POC test, the presence of influenza virus was confirmed by RT-PCR. In a comparison between the patients who were positive for influenza virus with only molecular POC test and those with both molecular POC test and DIA, the percentage of patients who sought consultation within 18 h after the onset of symptoms was significantly higher in the molecular POC test only group than in the both methods group (70.0% versus 43.2%, p = 0.044)., Conclusions: A molecular POC test could contribute to the accurate diagnosis of influenza in patients with influenza-like illness, especially those who visited a hospital immediately after the onset of symptoms., Competing Interests: Declaration of competing interest This study was funded by Roche Diagnostics K.K. The sponsor also performed RT-PCR analysis in this study. The sponsor had no control over the interpretation, writing, or publication of this work. The authors have no conflict of interest directly relevant to the content of this article., (Copyright © 2021 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Amino acid substitutions in influenza virus neuraminidase (NA) that cause resistance to neuraminidase inhibitors (NAI) generally result in virus attenuation. However, influenza viruses may acquire secondary substitutions in the NA and hemagglutinin (HA) proteins that can restore viral fitness. To assess to which extent this happens, the emergence of NAI resistance substitutions and secondary - potentially compensatory - substitutions was quantified in influenza viruses of immunocompetent individuals included in the Influenza Resistance Information Study (IRIS; NCT00884117). Known resistance substitutions were detected by mutation specific RT-PCR in viruses of 57 of 1803 (3.2%) oseltamivir-treated individuals, including 39 individuals infected with A/H1N1pdm09 [H275Y] virus and 18 with A/H3N2 [R292K] virus. A total of fifteen and ten other amino acid substitutions were acquired in HA and NA respectively, of A/H1N1pdm09, A/H3N2 and influenza B viruses upon treatment with oseltamivir but none of these was associated with resistance to oseltamivir. All cultured viruses with the known resistance substitutions H275Y or R292K showed reduced susceptibility to oseltamivir in the NA-star assay. Upon next-generation sequencing, the vast majority of NAI resistant A/H1N1pdm09 and A/H3N2 viruses had no resistance-associated secondary substitutions at high frequency. Only in two A/H1N1pdm09 [H275Y] viruses, the potentially compensatory secondary substitutions HA-D52N and NA-R152K were detected. We conclude that the emergence of secondary substitutions that may restore viral fitness upon the emergence of known influenza virus NAI resistance substitutions was a rare event in this immunocompetent population., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: To describe the prevalence, clinical features and complications of human metapneumovirus (hMPV) infections in a population of adults hospitalized with influenza-like illness (ILI)., Methods: This was a retrospective, observational, multicenter cohort study using prospectively collected data from adult patients hospitalized during influenza virus circulation, for at least 24 h, for community-acquired ILI (with symptom onset <7 days). Data were collected from five French teaching hospitals over six consecutive winters (2012-2018). Respiratory viruses were identified by multiplex reverse transcription polymerase chain reaction (RT-PCR) on nasopharyngeal specimens. hMPV + patients were compared with hMPV- patients, influenza+ and respiratory syncytial virus (RSV)+ patients using multivariate logistic regressions. Primary outcome was the prevalence of hMPV in patients hospitalized for ILI., Results: Among the 3148 patients included (1449 (46%) women, 1988 (63%) aged 65 and over; 2508 (80%) with chronic disease), at least one respiratory virus was detected in 1604 (51%, 95% confidence interval (CI) 49-53), including 100 cases of hMPV (100/3148, 3% 95% CI 3-4), of which 10 (10%) were viral co-infection. In the hMPV + patients, mean length of stay was 7 days, 62% (56/90) developed a complication, 21% (14/68) were admitted to intensive care unit and 4% (4/90) died during hospitalization. In comparison with influenza + patients, hMPV + patients were more frequently >65 years old (adjusted odds ratio (aOR) = 3.3, 95% CI 1.9-6.3) and presented more acute heart failure during hospitalization (aOR = 1.8, 95% CI 1.0-2.9). Compared with RSV + patients, hMPV + patients had less cancer (aOR = 0.4, 95% CI 0.2-0.9) and were less likely to smoke (aOR = 0.5, 95% CI 0.2-0.9) but had similar outcomes, especially high rates of respiratory and cardiovascular complications., Conclusions: Adult hMPV infections mainly affect the elderly and patients with chronic conditions and are responsible for frequent cardiac and pulmonary complications similar to those of RSV infections. At-risk populations would benefit from the development of antivirals and vaccines targeting hMPV., (Copyright © 2020 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Objective: The COVID-19 outbreak in Wuhan, Hubei, China, followed the seasonal influenza epidemic. Since some COVID-19 cases may have been misdiagnosed as seasonal influenza in January 2020, before testing capacity was adequate, it is relevant to study the proportions of influenza and COVID-19 cases among influenza-like-illness (ILI) patients and their temporal pattern., Results: This study analyzed the record of the ILI patients with a recent travel history to Wuhan who arrived in Hong Kong between 31 December 2019 and 21 January 2020. We found that the proportion of COVID-19 cases among the total ILI patients is much smaller than a study among ILI in Wuhan. This difference in proportion could be due to the difference in sampling. We argue that it is essential to consider both samples when inferring the number of COVID-19 cases from ILI patients., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: Few studies have explored the different lineages of influenza B viruses, which cause excessive fatalities. This study aimed to estimate age- and sex-specific excessive mortality associated with influenza subtypes/lineages in Shanghai, during 2010-2015., Methods: We used quasi-Poisson regression models to fit weekly numbers of deaths from various causes by adjusting long-term trend, seasonal trend, and absolute humidity as confounding factors. The mortality burden associated with influenza subtypes/lineages was estimated by age and sex., Results: The average influenza-associated excessive mortality rate in deaths coded as all-cause was 27.66 (95%CI: 22.30-33.88) per 100,000 person-years. The excessive deaths from six underlying causes in people aged ≥65 years weight heavily. Compared to influenza B(Victoria) lineage, influenza B(Yamagata) lineage had more effect on the influenza-associated disease burden, particularly in patients with respiratory and circulatory diseases. There was no statistical significance of sex in the mortality burden associated with influenza., Conclusion: Disproportionate mortality associated with influenza B virus is dominated by B(Yamagata) lineage in Shanghai, and the evidence for sex differences in the influenza-associated mortality burden for respiratory and circulatory diseases is limited. Improving the vaccination of the elderly and strengthening laboratory-based surveillance of influenza B lineages in Shanghai are needed to reduce this influenza's disease burden., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Conventional influenza vaccines are designed to stimulate neutralizing antibodies against immunodominant but highly variable hemagglutinin antigens. Inherent limitations include suboptimal protection against rapidly changing seasonal influenza viruses and a lack of protection against antigenically novel pandemic influenza. New technologies for developing influenza vaccines that induce more broadly protective and durable immunity are a growing area of research and focus on a variety of approaches, including targeting conserved antigens and stimulating cross-reactive T cell responses. This review highlights a new effort to track the development of universal influenza vaccine technologies. The Universal Influenza Vaccine Technology Landscape is intended to provide stakeholders and funders with a common source of information to monitor research progress and identify opportunities for informed investments and collaboration., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Influenza viruses have been studied for over 80 years, yet much about the basic viral lifecycle remain unknown. However, new imaging, biochemical, and sequencing techniques have revealed significant insight into many age-old questions of influenza virus biology. In this review, we will cover the role of imaging techniques to describe unique aspects of influenza virus assembly, biochemical techniques to study viral genomic organization, and next-generation sequencing to explore influenza genomic evolution. Our goal is to provide a brief overview of how emerging techniques are being used to answer basic questions about influenza viruses. This is not a comprehensive list of emerging techniques, rather ones that we feel will continue to make significant contributions to field of influenza biology., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Broadly neutralizing antibodies (bnAbs) are potential therapeutic molecules and valuable tools for studying conserved viral targets for vaccine and drug design. Interestingly, antibody responses to conserved epitopes can be highly convergent at the molecular level. Human antibodies targeting a number of viral antigens have often been found to utilize a restricted set of immunoglobulin germline genes in different individuals. Here we review recent knowledge on V H 1-69-encoded antibodies in antiviral responses to influenza virus, HCV, and HIV-1. These antibodies share common genetic and structural features, and often develop neutralizing activity against a broad spectrum of viral strains. Understanding the genetic and structural characteristics of such antibodies and the target epitopes should help advance novel strategies to elicit bnAbs through vaccination., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Human adenoviruses (HAdVs) are prevalent in pediatric and adult patients with severe acute respiratory disease (ARD). To date, there have been no widely used HAdV vaccines available. In this report, we developed a cold-adapted attenuated influenza virus, termed rg HAdV-Flu ca, carrying epitopes from HAdV hexon protein in the backbone of the ca influenza vaccine neuraminidase (NA) gene using reverse genetics. Rg HAdV-Flu ca virus exhibited a cold-adapted (ca) phenotype, and its morphological characteristics were observed using electron microscopy. Moreover, BALB/c mice were immunized intranasally (i.n.) with 10 5 , 10 6 or 10 7 TCID50 rg HAdV-Flu ca. Results showed a specific, robust antibody response against influenza and HAdV in a dose-dependent manner. More importantly, potent humoral, mucosal and cellular immune responses protected against subsequent wild-type HAdV-3 or HAdV-7 challenges, as determined by a significant decrease in viral titers and a noticeable alleviation of histopathological alterations in the lung tissue of challenged mice. These findings demonstrate that rg HAdV-Flu ca warrants attention as a potential vaccine candidate against HAdV infection., (Copyright © 2018. Published by Elsevier B.V.)

Background: Acute encephalitis/encephalopathy (AE/E) is a rare and severe complication of common childhood infections; however, a treatment strategy based on clinical and pathological evidence has not been established., Methods: The clinical data and aetiological results using a rapid and comprehensive virological detection system of 62 Japanese children diagnosed with AE/E from 2010 to 2014 were collected. We assessed clinical differences between causes and effectiveness of our multiplex PCR system to establish a pathogen-based treatment strategy for AE/E., Results: Suspected causes were detected in 84% of patients, and our multiplex PCR system contributed to diagnosing 38% of the patients. Furthermore, a negative virus PCR might be important for inferring underlying disease. Most cases were triggered by human herpes virus (HHV) 6/7 (32%) and influenza virus (24%). The causes of AE/E depended on age (p=0.00089) but not on sex (p=0.94). The median age of HHV6/7-associated AE/E was 2.3years, which is lower than the median ages of AE/E associated with other viruses. Major initial treatments were pulse steroid therapy (83.9%) and acyclovir (71%). Most of the patients in this study had good prognoses: 77% recovered without neurological sequalae., Conclusions: Our virological detection system was useful for detecting the cause of AE/E, and may also contribute to construction of pathogen-based treatment strategies for AE/E. Our data indicated the possibility that early intervention with pulse steroid therapy could be effective for treating AE/E. Further investigation for selection of antiepileptic drugs and additional therapies might be required to prevent progression of AE/E., (Copyright © 2017 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

Trypanosoma cruzi, the etiologic agent of Chagas disease, is a protozoan parasite with a life cycle that alternates between replicative and non-replicative forms, but the components and mechanisms that regulate its cell cycle are poorly described. In higher eukaryotes, cyclins are proteins that activate cyclin-dependent kinases (CDKs), by associating with them along the different stages of the cell cycle. These cyclin-CDK complexes exert their role as major modulators of the cell cycle by phosphorylating specific substrates. For the correct progression of the cell cycle, the mechanisms that regulate the activity of cyclins and their associated CDKs are diverse and must be controlled precisely. Different types of cyclins are involved in specific phases of the eukaryotic cell cycle, preferentially activating certain CDKs. In this work, we characterized TcCYC6, a putative coding sequence of T. cruzi which encodes a protein with homology to mitotic cyclins. The overexpression of this sequence, fused to a tag of nine amino acids from influenza virus hemagglutinin (TcCYC6-HA), showed to be detrimental for the proliferation of epimastigotes in axenic culture and affected the cell cycle progression. In silico analysis revealed an N-terminal segment similar to the consensus sequence of the destruction box, a hallmark for the degradation of several mitotic cyclins. We experimentally determined that the TcCYC6-HA turnover decreased in the presence of proteasome inhibitors, suggesting that TcCYC6 degradation occurs via ubiquitin-proteasome pathway. The results obtained in this study provide first evidence that TcCYC6 expression and degradation are finely regulated in T. cruzi., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

A desirable vaccine against respiratory syncytial virus (RSV) should induce neutralizing antibodies without eliciting abnormal T cell responses to avoid vaccine-enhanced pathology. In an approach to deliver RSV neutralizing epitopes without RSV-specific T cell antigens, we genetically engineered chimeric influenza virus expressing RSV F262-276 neutralizing epitopes in the globular head domain as a chimeric hemagglutinin (HA) protein. Immunization of mice with formalin-inactivated recombinant chimeric influenza/RSV F262-276 was able to induce RSV protective neutralizing antibodies and lower lung viral loads after challenge. Formalin-inactivated RSV immune mice showed high levels of pulmonary inflammatory cytokines, macrophages, IL-4-producing T cells, and extensive histopathology. However, RSV-specific T cell responses and enhancement of pulmonary histopathology were not observed after RSV infection of inactivated chimeric influenza/RSV F262-276. This study provides evidence that an inactivated vaccine platform of chimeric influenza/RSV virus can be developed into a safe RSV vaccine candidate without priming RSV-specific T cells and immunopathology., From the Clinical Editor: Respiratory syncytial virus (RSV) is a major cause of respiratory tract illness and morbidity in children. Hence, there is a need to develop an effective vaccine against this virus. In this article, the authors engineered chimeric influenza virus to express RSV neutralizing epitopes. The positive findings in in-vivo experiments provide a beginning for future clinical trials and perhaps eventual product realization., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Human adenoviruses (HAdVs) are known to cause a broad spectrum of diseases in pediatric and adult patients. As this time, there is no specific therapy for HAdV infection. This study used reverse genetics (RG) to successfully rescue a recombinant influenza virus, termed rFLU/HAdV, with the HAdV hexon protein antigenic epitope sequence inserted in the influenza non-structural (NS1) protein gene. rFLU/HAdV morphological characteristics were observed using electron microscopy. Furthermore, BALB/c mice immunized twice intranasally (i.n.) with 10(4) TCID50 or 10(5) TCID50 rFLU/HAdV showed robust humoral, mucosal, and cell-mediated immune responses in vivo. More importantly, these specific immune responses could protect against subsequent wild-type HAdV-3 (BJ809) or HAdV-7 (BJ1026) challenge, showing a significant reduction in viral load and a noticeable alleviation of histopathological changes in the challenged mouse lung in a dose-dependent manner. These findings highlighted that recombinant rFLU/HAdV warrants further investigation as a promising HAdV candidate vaccine and underscored that the immuno-protection should be confirmed in primate models., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Seasonal influenza vaccine strains are routinely updated when influenza viruses acquire mutations in exposed regions of the hemagglutinin and neuraminidase glycoproteins. Ironically, although thousands of viral isolates are sequenced each year, today's influenza surveillance community places less emphasis on viral genetic information and more emphasis on classical serological assays when choosing vaccine strains. Here, I argue that these classical serological assays are oversimplified and that they fail to detect influenza mutations that facilitate escape of particular types of human antibodies. I propose that influenza vaccine strains should be updated more frequently even when classical serological assays fail to detect significant antigenic alterations., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Vaccination remains the most effective means to prevent morbidity and mortality caused by influenza epidemics and pandemics. Live attenuated influenza vaccine (LAIV) has been proven to be effective in preventing influenza with broad cross reactivity to drifted strains. Owing to the sophisticated nature of the influenza vaccine production process, the time needed to develop high yield LAIV strains for vaccine production and product release remains a constant challenge. This review summarizes LAIV production process with highlights on the experiences gained during the past decade generating seasonal and pandemic LAIV seeds by reverse genetics strategy., (Copyright © 2014 Elsevier B.V. All rights reserved.)

BlockLogo is a web-server application for the visualization of protein and nucleotide fragments, continuous protein sequence motifs, and discontinuous sequence motifs using calculation of block entropy from multiple sequence alignments. The user input consists of a multiple sequence alignment, selection of motif positions, type of sequence, and output format definition. The output has BlockLogo along with the sequence logo, and a table of motif frequencies. We deployed BlockLogo as an online application and have demonstrated its utility through examples that show visualization of T-cell epitopes and B-cell epitopes (both continuous and discontinuous). Our additional example shows a visualization and analysis of structural motifs that determine the specificity of peptide binding to HLA-DR molecules. The BlockLogo server also employs selected experimentally validated prediction algorithms to enable on-the-fly prediction of MHC binding affinity to 15 common HLA class I and class II alleles as well as visual analysis of discontinuous epitopes from multiple sequence alignments. It enables the visualization and analysis of structural and functional motifs that are usually described as regular expressions. It provides a compact view of discontinuous motifs composed of distant positions within biological sequences. BlockLogo is available at: http://research4.dfci.harvard.edu/cvc/blocklogo/ and http://met-hilab.bu.edu/blocklogo/., (© 2013.)

Protein-protein interactions (PPIs) play a key role in many biological processes, including virus replication in the host cell. Since most of the PPIs are functionally essential, a possible strategy to inhibit virus replication is based on the disruption of viral protein complexes by peptides or small molecules that interfere with subunit interactions. In particular, an attractive target for antiviral drugs is the binding between the subunits of essential viral enzymes. This review describes the development of new antiviral compounds that inhibit herpesvirus and influenza virus replication by blocking interactions between subunit proteins of their polymerase complexes., (Copyright © 2013 Elsevier B.V. All rights reserved.)

High evolvability of influenza virus and the complex nature of its antagonistic interaction with the host immune system make it difficult to predict which strain of virus will become epidemic next and when it will emerge. To investigate the most likely time at which a new successful strain emerges every year in seasonal influenza, we use an individual-based model that takes into account the seasonality in transmission rate and host cross-immunity against a current viral strain due to previous infections with other strains. Our model deals with antigenic evolution of influenza virus that originated by point mutations at amino acid sites that constitute epitope and is driven by host immune response. Under the range of parameters by which influenza virus shows a "trunk" shape in its phylogenetic tree, as is typical in influenza A virus evolution, we find that most successful mutant strains emerge in an early part of the epidemic season, and that the time when the number of infected hosts reaches a maximum tends to be more than one season after viral emergence. This carryover of the epidemic peak timing implies that we can predict the strain that will become dominant in the epidemic in the following year., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Influenza-like illnesses (ILI) are estimated to cause millions of deaths annually. Despite this disease burden, the etiologic causes of ILI are poorly described for many geographical regions., Methods: Beginning in April 2010, we conducted an observational cohort study at five hospitals in Mexico City, enrolling subjects who met the criteria for ILI. Evaluations were conducted at enrollment and on day 28, with the collection of clinical data and a nasopharyngeal swab (or nasal aspirate in children). Swabs were tested by multiplex PCR for 15 viral pathogens and real-time PCR for influenza., Results: During the first year, 1065 subjects were enrolled in this study, 55% of whom were hospitalized; 24% of all subjects were children. One or more pathogens were detected by PCR in 64% of subjects, most commonly rhinovirus (25% of all isolates) and influenza (24% of isolates). Six percent of subjects died, and of those, 54% had no pathogen identified. Rhinovirus was the most common pathogen among those who died, although it did not have the highest case fatality rate., Conclusions: Multiple respiratory viruses beyond influenza are associated with significant morbidity and mortality among adults and children in Mexico City. Detection of these agents could be useful for the adjustment of antibiotic treatment in severe cases., (Copyright © 2013 International Society for Infectious Diseases. All rights reserved.)

Background: The hemagglutinin (HA) and neuraminidase (NA) genes encode surface glycoproteins of influenza virus. These two proteins are involved in pathogenicity and are the primary targets of the immune system. Mutations in the HA and NA genes can result in antigenic drift in an influenza viral strain. A comparative sequencing method using MALDI MS combined with base-specific cleavage has been applied for the surveillance of these viral mutations. This approach shows advantages in high throughput and efficiency than the traditional direct sequencing methods in targeted sequence analysis., Methods: Base-specific cleavage assay with RNAse A was combined with MALDI-MS for the analysis of the HA and NA genes of 2 influenza viral strains. The mass peak patterns from the spectra were compared with the in silico digest result of reference gene sequences from the database to achieve comparative sequencing and screening of novel mutations., Results: The HA and NA genes of two influenza lab strains were comparatively sequenced using the base-specific cleavage and MALDI-MS approach. Mutations could be exactly identified if more than one observation (mass peak changes) were detected in the spectrum. Mutations with only one observation could be located in a small area for further validation., Conclusions: We showed a proof of a principle that base-specific combined with MALDI-MS comparative sequencing approach can be utilized for targeted sequence analysis and potentially rapid and cost effective screening of emerging viral mutations., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The main objectives of this study were to describe the characteristics of children with influenza infection during the postpandemic outbreak, and to compare sociodemographic and clinical data between patients who required hospitalization and those managed on an outpatient basis with a matched case-control study design. This is a multicentre paediatric study in Spain that included patients aged 6 month to 18 years in whom influenza infection was confirmed by real-time reverse transcription-polymerase chain reaction between December 2010 and March 2011. Among the 143 admitted patients, the main reason for admission was respiratory failure (123/143). In 55 there was some previously known disease. The median age was lower in patients without comorbidity (1.8 years: interquartile range 1.0-3.0 versus 5.3 years: interquartile range 1.3-10.7); p <0.01). The lag time from onset of symptoms to starting antiviral treatment was correlated with the length of hospital stay (Rho Spearman = + 0.32; p 0.01). Twenty patients required admission to the paediatric intensive care units, all due to respiratory failure. Children with chest X-ray opacities in more than one quadrant more frequently required admission to intensive care. Having a neurological disease conferred the highest risk of requiring hospitalization (OR 17.18) in a multivariate analysis. This study concludes that influenza in the paediatric population requiring hospitalization during the postpandemic season affected mainly children with neurological or pulmonary comorbidities and children of parents with a lower educational level. Most of the influenza infections caused respiratory symptoms, although neurological manifestations were also observed. Early initiation of oseltamivir was associated with a shorter length of hospital stay., (© 2012 The Authors Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.)

We explore the dynamics that occur when multiple variants of a virus are released in a population, where hosts can make an immune response to only one epitope of the virus. A heterogeneous immune response was implemented by varying the probability for making a response at a locus of the virus variants. A framework based on an SIR model is presented which allows for the explicit representation of the virus variants and the immune histories of the population. From this we are able to calculate the proportion of the population infected by each variant and the number of times a host has been infected when there have been multiple epidemics. When there are discrete epidemics, we use the framework to calculate the proportion of the population infected analytically, demonstrating a method which reduces the calculations required in comparison with solving the full system of differential equations. However, when there are multiple strains present in the population at any time, we show that the full set of ODEs must be solved to fully describe the infection dynamics., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In hepatitis C virus (HCV) infection, CD8(+) T cell responses have been shown to be important in viral clearance. Examining the efficacy of CD8(+) T cell vaccines against HCV has been limited by the lack of an HCV infectious model in mice and the differences between MHC restriction in humans and mice. Using HLA-A2 transgenic HHD mice, we demonstrate that intranasally delivered Pam2Cys-based lipopeptides containing HLA-A2-restricted HCV epitopes can induce polyfunctional CD8(+) T cell responses in several organs including the liver. To examine the activity of these responses in an infectious context, we developed a recombinant influenza virus that expresses the NS5B(2594-2602) epitope from non-structural protein 5B of hepatitis C virus (PR8-HCV(NS5B)). We showed that mice inoculated with a lipopeptide containing the NS5B epitope had reduced viral loads following challenge with the PR8-HCV(NS5B) virus. This reduction was associated with the induction of NS5B(2594-2602)-specific IFN-γ and TNF-α co-producing CD8(+) T cells. The T cell receptor usage in the NS5B(2594-2602) response was found to exhibit a Vβ8.1/8.2 bias that was characterized by a narrow repertoire and a common CDR3β motif. This work has identified CD8(+) T cell functions induced by lipopeptides that are associated with viral control and demonstrate the potential of lipopeptide-based vaccines as candidates for treatment of HCV infection., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Influenza virus (IV) infection can cause severe pneumonia and death. Therapeutic actions are limited to vaccines and a few anti-viral drugs. These target viral functions thereby selecting resistant variants. During replication IV activates the Raf/MEK/ERK-cascade and the transcription factor NF-kappaB. Both result in virus supportive and anti-viral effects by promoting viral genome transport for virus assembly and by inducing expression of pro-inflammatory host factors. Apart from tissue damage caused by the virus lytic replication, an imbalanced overproduction of anti-viral cytokines can cause severe lung damage as observed in human H5-type IV infections. Recently we showed that inhibition of NF-kappaB activity reduces the virus titer in vitro and in vivo. We have now analyzed whether inhibition of these pathways, allows simultaneous reduction of virus titers and virus-induced cytokines. The results show that inhibition of either pathway indeed leads to decreased virus titers and cytokine expression. This was not only true for infected permanent cells or primary mouse alveolar epithelial cells, but also in infected mice. Hereby we demonstrate for the first time in vitro and in vivo that virus titers and pro-inflammatory cytokine expression can be modulated simultaneously. This could provide a new rationale of future therapeutic strategies to treat IV pneumonia., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Efforts to develop new antiviral chemotherapeutic approaches are focusing on compounds that target either influenza virus replication itself or host factor(s) that are critical to influenza replication. Host protease mediated influenza hemagglutinin (HA) cleavage is critical for activation of virus infectivity and as such is a chemotherapeutic target. Influenza pathogenesis involves a "vicious cycle" in which host proteases activate progeny virus which in turn amplifies replication and stimulates further protease activities which may be detrimental to the infected host. Aprotinin, a 58 amino acid polypeptide purified from bovine lung that is one of a family of host-targeted antivirals that inhibit serine proteases responsible for influenza virus activation. This drug and similar agents, such as leupeptin and camostat, suppress virus HA cleavage and limit reproduction of human and avian influenza viruses with a single arginine in the HA cleavage site. Site-directed structural modifications of aprotinin are possible to increase its intracellular targeting of cleavage of highly virulent H5 and H7 hemagglutinins possessing multi-arginine/lysine cleavage site. An additional mechanism of action for serine protease inhibitors is to target a number of host mediators of inflammation and down regulate their levels in virus-infected hosts. Aprotinin is a generic drug approved for intravenous use in humans to treat pancreatitis and limit post-operative bleeding. As an antiinfluenzal compound, aprotinin might be delivered by two routes: (i) a small-particle aerosol has been approved in Russia for local respiratory application in mild-to-moderate influenza and (ii) a proposed intravenous administration for severe influenza to provide both an antiviral effect and a decrease in systemic pathology and inflammation., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Recent accumulating evidence suggests that the human immune system possesses a variety of innate receptors that recognize, distinguish, and respond to viral infections and to vaccination. These include Toll-like receptors, C-type lectin receptors, RIG-I-like receptors, Nod-like receptors and possibly AIM2-like receptors. However, the precise mechanisms by which these receptors exert their critical roles in the induction of virus-specific adaptive immune responses have not been fully elucidated. In this review, we discuss recent advances in our understanding of the innate immune recognition of viruses and the differential connection to the adaptive immune responses induced by infection or vaccination, with a particular focus on the influenza virus., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Antiviral monitoring of influenza viruses circulating in Italy has been carried out since 2007 by the National Influenza Centre (NIC), using both phenotypic and sequence-based assays. Here, we report results of the susceptibility evaluation to neuraminidase (NA) inhibitors (NAIs, zanamivir and oseltamivir) and adamantanes of nearly 300 influenza type A and B seasonal viruses isolated in Italy during six recent seasons, together with over 30 pandemic (H1N1) 2009 virus strains. The present work is the first such study conducted in Italy, aimed to develop national data on antiviral drug profile and to establish a nationwide surveillance programme on antiviral susceptibility. Sequencing of the NA gene was undertaken either to confirm the phenotypic findings or to identify any NA change, in potentially resistant viruses (outliers), which might be associated with reduced susceptibility to NAIs. The 50% inhibitory concentration values (IC(50)s) showed slightly different sensitivities of the seasonal Italian isolates to the two NAI drugs, depending on the specific NA subtype. We found mean zanamivir IC(50)s of 0.74, 1.33 and 7 nM, and oseltamivir IC(50)s of 0.67, 2.34 and 30.1 nM for the N2, N1 and B NAs, respectively. The pandemic (H1N1) 2009 viruses showed IC(50)values overall comparable to the seasonal N1 viruses from previous years, showing mean zanamivir IC(50)s of 1.02 nM and mean oseltamivir IC(50)s of 2.82 nM. Oseltamivir resistance was found in a total of 19 seasonal N1viruses of 2007/2008 and 2008/2009, and in three pandemic (H1N1) 2009 strains. A gradual increase of resistance to adamantanes was observed among the N2 viruses isolated in recent seasons; no resistant viruses were found among the seasonal N1 strains, whereas all the pandemic (H1N1) 2009 isolates analysed were resistant to the M2 blockers., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Influenza virus is reliant on numerous host cell functions during its replication cycle. RNA interference technology, applied on a genome-wide level, has identified human host factors that are necessary for efficient virus replication and provides new insight into how influenza virus interacts with its host at the molecular level., (Copyright © 2011 Institut Pasteur. Published by Elsevier SAS. All rights reserved.)

Consumer-resource dynamics of hosts with their pathogens are modulated by complex interactions between various branches of hosts' immune systems and the imperfectly perceived pathogen. Multistrain SIR models tend to sweep competitive interaction terms between different pathogen strains into a single parameter representing cross-immunity. After reviewing several hypotheses about the generation of immune responses, we look into the consequences of assuming that hosts with identical immune repertoires respond to new pathogens identically. In particular, we vary the breadth of the typical immune response, or the average number of pathogen epitopes a host perceives, and the probability of perceiving a particular epitope. The latter quantity in our model is equivalent both to the degree of diversity in host responses at the population level and the relative immunodominance of different epitopes. We find that a sharp transition to strain coexistence occurs as host responses become narrow or skewed toward one epitope. Increasing the breadth of the immune response and the immunogenicity of different epitopes typically increases the range of cross-immunity values in which chaotic strain dynamics and competitive exclusion occur. Models attempting to predict the outcomes of strain competition should thus consider the potential diversity and specificity of hosts' responses to infection., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The accumulation of cross-immunity in the host population is an important factor driving the antigenic evolution of viruses such as influenza A. Mathematical models have shown that the strength of temporary non-specific cross-immunity and the basic reproductive number are both key determinants for evolutionary branching of the antigenic phenotype. Here we develop deterministic and stochastic versions of one such model. We examine how the time of emergence or introduction of a novel strain affects co-existence with existing strains and hence the initial establishment of a new evolutionary branch. We also clarify the roles of cross-immunity and the basic reproductive number in this process. We show that the basic reproductive number is important because it affects the frequency of infection, which influences the long term immune profile of the host population. The time at which a new strain appears relative to the epidemic peak of an existing strain is important because it determines the environment the emergent mutant experiences in terms of the short term immune profile of the host population. Strains are more likely to coexist, and hence to establish a new clade in the viral phylogeny, when there is a significant time overlap between their epidemics. It follows that the majority of antigenic drift in influenza is expected to occur in the earlier part of each transmission season and this is likely to be a key surveillance period for detecting emerging antigenic novelty.

Neuraminidase (NA) inhibitors are a class of antivirals designed to target the conserved residues of the influenza NA active site. While there are many conserved residues in the NA active site that are involved in NA inhibitor binding, only a few have been demonstrated to confer resistance. As such, little is known regarding the potential of the other conserved residues in the NA active site to cause NA inhibitor resistance. Two conserved residues (E227 and E276) of an N1 NA that have not previously been associated with resistance to NA inhibitors were investigated. Site-directed mutagenesis was used to generate three alternative amino acids at each residue. Reverse genetics was used to generate recombinant mutant viruses which were characterized for growth, NA activity and NA inhibitor sensitivity. Of the six recombinant viruses expressing NA with mutations at either E227 or E276, only the E227D and E276D viruses were able to grow without supplementary NA activity, and all mutant viruses had a significant reduction in NA activity. The E227D virus demonstrated significantly reduced sensitivity to zanamivir while the E276D virus did not demonstrate any significant changes in NA inhibitor sensitivity. Interestingly, the resistance profiles of E227D and E276D in N1 NA were significantly different from these sites that have been reported for N2 NA. This study confirmed the essential role of NA active site residues in viral fitness, and identified clear differences in the role of residues E227 and E276 in NA inhibitor resistance with N1 and N2 neuraminidases.

A classical limitation of early life immunization is the interference by maternally derived antibodies, which are known to inhibit the immune response to modified-live and killed vaccines. Several studies have convincingly shown that even minute amounts of maternally derived antibodies against equine influenza can strongly interfere with successful vaccination of foals born to immune mares. In this study we evaluated the response of foals born to vaccinated mares to immunization with a canarypox-vectored recombinant vaccine against equine influenza virus H3N8. The recombinant vaccine was able to efficiently prime foals in the presence of maternally derived immunity against influenza as was evidenced by a clear anamnestic antibody response when a secondary vaccination with the same vaccine was performed. The canarypox-vectored recombinant influenza vaccine therefore offers a unique opportunity to overcome the limitations of early life vaccination in the face of maternally derived immunity in foals.

Almost 30 years ago it was proposed that the selection for antiviral drug resistance should be used as an indicator of antiviral drug activity. In addition to discriminating between cellular toxicity and specific activity directed against a viral target, drug resistant mutants have been used to confirm the mechanism of action of antiviral drugs, to discover the functions of several viral proteins and to provide insights into viral evolution and fitness. Drug resistance has also become a standard component of both the preclinical and clinical drug development process. For HIV and increasingly for other viruses drug resistance testing has become standard-of-care in clinical practice. A few selected examples are provided to illustrate each of these points.

This study examined whether increased antigen expression resulted in enhanced antigen-specific immune responses in the context of DNA vaccines. To increase antigen expression, two copies of antigen expression cassettes were arranged in a plasmid pDX. BALB/c mice were intramuscularly immunized with various constructs that express influenza antigens and analysed for DNA-raised immunity. The plasmid pDX that expresses two copies of the antigen gene induced stronger antigen-specific immune responses than the plasmid pGA which expresses single antigen gene. To explore the in vivo transgene expression by pDX and pGA, luciferase activity was measured in the muscles transduced with luciferase expression plasmids. The pDX expressing two copies of luciferase induced the highest luciferase activity, which corresponded to the results from vaccination. We concluded that increasing the number of antigen expression cassettes in a vaccine construct improved antigen expression in the transduced tissue, which induced stronger DNA-raised immune responses.

The discovery that the juvenile cardiomyopathy known as Keshan disease likely has a dual etiology that involves both a nutritional deficiency of the essential trace mineral selenium (Se) as well as an infection with an enterovirus provided the impetus for additional studies of relationships between nutrition and viral infection. An amyocarditic strain of coxsackievirus B3, CVB3/0, converted to virulence when it was inoculated into Se-deficient mice. This conversion was accompanied by changes in the genetic structure of the virus so that its genome closely resembled that of other known virulent CVB3 strains. Similar alterations in virulence and genomic composition of CVB3/0 could be observed in mice fed normal diets but genetically deprived of the antioxidant selenoenzyme glutathione peroxidase (knockout mice). More recent research has shown that a mild strain of influenza virus, influenza A/Bangkok/1/79, also exhibits increased virulence when given to Se-deficient mice. This increased virulence is accompanied by multiple changes in the viral genome in a segment previously thought to be relatively stable. Epidemic neuropathy in Cuba has features that suggest a combined nutritional/viral etiology. Further research, both basic and applied, is needed to assess properly the possible role of malnutrition in contributing to the emergence of novel viral diseases.

A "new" influenza virus will appear at some time in the future. This virus will arise by natural processes, which we do not fully understand, or it might be created by some bioterrorist. The world's population will have no immunity to the new virus, which will spread like wild-fire, causing much misery, economic disruption and many deaths. Vaccines will take time to develop and the only means of control, at least in the early stages of the epidemic, are anti-viral drugs, of which the neuraminidase inhibitors currently seem the most effective.

CD4+ and CD8+ T cells are key components of immune response against tumors and viruses. Many techniques have been used to clone and expand these cells in vitro for purposes of immunotherapy. Here, we describe an improved method to obtain large quantities of tumor and virus-specific human CD4+ and CD8+ T-cell clones. T cells derived from peripheral blood mononuclear cells (PBMCs) of healthy donors were stimulated several times by peptide pulsed monocyte-derived mature dendritic cells (DCs) in the presence of exogenous cytokines. T cells specific for influenza or melanoma antigens were detected by IFN-gamma intracellular staining and were cloned by limiting dilution. Specific polyclonal T-cell populations were derived for all epitopes presented by mature DCs. Nine different populations were cloned and clones were raised from eight of them. Clonality was verified by HLA/peptide tetramer staining. With additional rounds of stimulation after the cloning procedure, it was possible to obtain from 10(9) to 10(12) of each clone. Furthermore, clones could be maintained in culture in the presence of IL-2 for at least 1 month without losing their antigen-specific reactivity (e.g. cytokine secretion, cytolytic activity and proliferation). Importantly, a majority of the CD8+ T-cell clones recognized endogenously processed antigens. This method is of value for the purposes of adoptive anti-virus or anti-tumor immunotherapy.

The Na,K-ATPase is an essential plasma membrane transporter of mammalian cells composed of two subunits, alpha and beta, of which there are several isoforms. We investigated the effect of a substitution, S364P, on the subcellular localization and enzymatic activity of the wild-type alpha2 and alpha2L111R,N122D (alpha2RD) subunits. The substitutions, L111R and N122D, lower the affinity of the alpha2 subunit for the inhibitor ouabain roughly one thousand-fold (E. A. Jewell and J. B. Lingrel, J. Biol. Chem. 266, 16925-16930, 1991) and were introduced into the alpha2 subunit to distinguish its enzymatic activity from that of the endogenous alpha1 subunit of COS-7 cells. The S364P substitution is located in the ATP binding site, only five residues from the aspartyl residue which is phosphorylated during the catalytic cycle of the Na,K-ATPase. This substitution dramatically decreases the amount of enzymatic activity associated with expression of the alpha2RD subunit. Despite the fact that S364P substitution does not block association of the alpha2RD subunit with the endogenous beta1 subunit, it prevents the alpha2 and alpha2RD subunits from accumulating in the plasma membrane and results in their localization in the endoplasmic reticulum.

The influenza virus genome is composed of eight negative-strand RNA segments. In cells coinfected with two or more influenza strains, the genomic RNAs reassort at random, resulting in progeny viruses (reassortants) that contain genes derived from each parent. Genetic reassortment among influenza viruses occurs naturally and plays an important role in viral epidemiology and pathogenicity. The reassortment process is also utilized for the annual production of influenza vaccines. Each year, the two gene segments that encode the major surface antigens of the current virulent, wild-type viruses are reassorted with the remaining six gene segments of a laboratory-derived vaccine or "master donor" strain. As the gene reassortment appears to be random, identifying a progeny virus with the desired gene constellation can be labor-intensive. We developed a streamlined, cost-effective method to genotype influenza viruses that combines multiplex reverse transcription-polymerase chain reaction (RT-PCR) and fluorescent single-strand conformation polymorphism (SSCP) analysis. This method utilizes oligonucleotide primers labeled with one of three fluorescent dyes to generate RT-PCR products for each gene segment in a multiplex configuration. The RT-PCR products of the reassortants, wild-type, and master donor viruses are then electrophoresed under SSCP conditions. The viral origin of each gene segment can be identified by fluorescence and mobility shift patterns of the corresponding RT-PCR products. We demonstrate the utility of this method in differentiating the genes of a master donor strain, several wild-type viruses, and vaccine reassortants.

Despite many successes, atomic force microscopy (AFM) of biological specimens at room temperature is still severely limited by at least two factors: the softness and the thermal motion of flexible multi-domain/subunit molecules. Both problems can be overcome by imaging biological structures at cryogenic temperatures. Even though the instrumentation is considerably more complex and earlier attempts were largely unsuccessful, cryo-AFM has recently been demonstrated on a number of biological specimens, using an AFM operated in liquid nitrogen vapor under ambient pressure. In this brief review, both the method of instrumentation and the latest biological applications are discussed. Not only has the cryo-AFM attained high resolution on those specimens that could not be well imaged at room temperature, but it has also produced potentially important information on several specimens. These results firmly establish the cryo-AFM as a useful and versatile structural probe in biology with its own unique capabilities.