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The COVID-19 pandemic and subsequent implementation of nonpharmaceutical interventions (e.g., cessation of global travel, mask use, physical distancing, and staying home) reduced transmission of some viral respiratory pathogens (1). In the United States, influenza activity decreased in March 2020, was historically low through the summer of 2020 (2), and remained low during October 2020-May 2021 (

The COVID-19 pandemic and subsequent implementation of nonpharmaceutical interventions (e.g., cessation of global travel, mask use, physical distancing, and staying home) reduced transmission of some viral respiratory pathogens (1). In the United States, influenza activity decreased in March 2020, was historically low through the summer of 2020 (2), and remained low during October 2020-May 2021 (

Background Global influenza surveillance in humans and animals is a critical component of pandemic preparedness. The FluChip‐8G Insight assay was developed to subtype both seasonal and potentially pandemic influenza viruses in a single assay with a same day result. FluChip‐8G Insight uses whole gene segment RT‐PCR‐based amplification to provide robustness against genetic drift and subsequent microarray detection with artificial neural network‐based data interpretation. Objectives The objective of this study was to verify and validate the performance of the FluChip‐8G Insight assay for the detection and positive identification of human and animal origin non‐seasonal influenza A specimens. Methods We evaluated the ability of the FluChip‐8G Insight technology to type and HA and NA subtype a sample set consisting of 297 results from 180 unique non‐seasonal influenza A strains (49 unique subtypes). Results FluChip‐8G Insight demonstrated a positive percent agreement ≥93% for 5 targeted HA and 5 targeted NA subtypes except for H9 (88%), and negative percent agreement exceeding 95% for all targeted subtypes. Conclusions The FluChip‐8G Insight neural network‐based algorithm used for virus identification performed well over a data set of 297 naïve sample results, and can be easily updated to improve performance on emerging strains without changing the underlying assay chemistry.

Background The FluChip-8G Influenza A+B Assay is a multiplexed influenza RT-PCR and microarray-based assay with same day turnaround time, developed to subtype seasonal A viruses (H1N1pdm2009 and H3N2), distinguish B viruses as Yamagata or Victoria lineage, and is the only FDA cleared assay capable of positive identification of a wide variety of A subtypes as “non-seasonal” A viruses from human nasal specimens. Objective To evaluate clinical performance of the FluChip-8G Influenza A+B Assay for detection of seasonal influenza viruses in nasal and nasopharyngeal swab specimens, and to evaluate performance for detection of non-seasonal influenza viruses using contrived samples. Study Design For seasonal viruses, a multisite study of the FluChip-8G Influenza A+B Assay using prospectively and retrospectively collected nasal and nasopharyngeal swabs was performed using the FDA-cleared CDC Human Flu Dx Panel as the comparator assay. For non-seasonal viruses, testing was performed at a single site using contrived samples from 100 unique non-seasonal strains representing 41 subtypes. Results Sensitivity (95% CI) and specificity (95% CI) for each target group, respectively, from results of 1689 clinical specimens were: seasonal H1N1pdm2009: 96.4% (87.9–99.0), 99.3% (98.8–99.6), seasonal H3N2: 91.8% (87.7–94.7), 99.7% (99.2–99.9), Influenza B Victoria: 100% (94.0–100.0), 99.9% (99.6–100.0), and Influenza B Yamagata: 95.6% (89.2–98.3), 99.9% (99.6–100.0). The sensitivity and specificity from contrived influenza A non-seasonal viruses was determined to be 99.0% (94.6–99.8) and 100% (96.7–100.0). Conclusion The FluChip-8G Influenza A+B Assay has robust sensitivity and specificity for detecting and identifying all target virus groups, including non-seasonal influenza A, with same day results.

Background The evolution of influenza A viruses results in birth cohorts that have different initial influenza virus exposures. Historically, A/H3 predominant seasons have been associated with more severe influenza-associated disease; however, since the 2009 pandemic, there are suggestions that some birth cohorts experience more severe illness in A/H1 predominant seasons. Methods United States influenza virologic, hospitalization, and mortality surveillance data during 2000–2017 were analyzed for cohorts born between 1918 and 1989 that likely had different initial influenza virus exposures based on viruses circulating during early childhood. Relative risk/rate during H3 compared with H1 predominant seasons during prepandemic versus pandemic and later periods were calculated for each cohort. Results During the prepandemic period, all cohorts had more influenza-associated disease during H3 predominant seasons than H1 predominant seasons. During the pandemic and later period, 4 cohorts had higher hospitalization and mortality rates during H1 predominant seasons than H3 predominant seasons. Conclusions Birth cohort differences in risk of influenza-associated disease by influenza A virus subtype can be seen in US influenza surveillance data and differ between prepandemic and pandemic and later periods. As the population ages, the amount of influenza-associated disease may be greater in future H1 predominant seasons than H3 predominant seasons.

Background The effectiveness of influenza vaccine during 2015-2016 was reduced in some age groups as compared to that in previous 2009 pandemic influenza A(H1N1) virus (A[H1N1]pdm09 virus)-predominant seasons. We hypothesized that the age at first exposure to specific influenza A(H1N1) viruses could influence vaccine effectiveness (VE). Methods We estimated the effectiveness of influenza vaccine against polymerase chain reaction-confirmed influenza A(H1N1)pdm09-associated medically attended illness from the 2010-2011 season through the 2015-2016 season, according to patient birth cohort using data from the Influenza Vaccine Effectiveness Network. Birth cohorts were defined a priori on the basis of likely immunologic priming with groups of influenza A(H1N1) viruses that circulated during 1918-2015. VE was calculated as 100 × [1 - adjusted odds ratio] from logistic regression models comparing the odds of vaccination among influenza virus-positive versus influenza test-negative patients. Results A total of 2115 A(H1N1)pdm09 virus-positive and 14 696 influenza virus-negative patients aged ≥6 months were included. VE was 61% (95% confidence interval [CI], 56%-66%) against A(H1N1)pdm09-associated illness during the 2010-2011 through 2013-2014 seasons, compared with 47% (95% CI, 36%-56%) during 2015-2016. During 2015-2016, A(H1N1)pdm09-specific VE was 22% (95% CI, -7%-43%) among adults born during 1958-1979 versus 61% (95% CI, 54%-66%) for all other birth cohorts combined. Conclusion Findings suggest an association between reduced VE against influenza A(H1N1)pdm09-related illness during 2015-2016 and early exposure to specific influenza A(H1N1) viruses.

Influenza databases now contain over 100,000 worldwide sequence records for strains influenza A(H3N2) and A(H1N1). Although these data facilitate global research efforts and vaccine development practices, they also represent a stumbling block for researchers because of their confusing and heterogeneous annotation. Unclear passaging annotations are particularly concerning given the recent work highlighting the presence and risk of false adaptation signals introduced by cell passaging of viral isolates. With this in mind, we aim to provide a concise outline of why viruses are passaged, a clear overview of passaging annotation nomenclature currently in use, and suggestions for a standardized nomenclature going forward. Our hope is that this summary will empower researchers and clinicians alike to more easily understand a virus sample’s passage history when analyzing influenza sequences.

Currently the National Center of Biotechnology Information (NCBI) assigns individual taxonomy identifiers to each distinct influenza virus isolate submitted to GenBank. To support this practice, individual flu isolates must be manually added to the NCBI taxonomy database and unique taxonomy identifiers generated. This added layer of manual processing is unique to influenza virus and prevents automatization of the flu sequence submission process. Here we outline a new NCBI policy that normalizes influenza virus taxonomy processing but maintains features supported by the previous approach. This change will reduce the amount of manual handling necessary for flu submissions and pave the way for increased automation of the submissions process. While this automation may disrupt some historic practices, it will better align influenza virus data processing with other viruses and ultimately lower the submission burden on data providers.

The seasonality of influenza in the tropics complicates vaccination timing. We investigated influenza seasonality in northern India and found influenza positivity peaked in Srinagar (34.09°N) in January-March but peaked in New Delhi (28.66°N) in July-September. Srinagar should consider influenza vaccination in October-November, but New Delhi should vaccinate in May-June.

Highlights • Multiplex RT-PCR microarray assay for influenza typing and subtyping was evaluated. • Limits of detection were ∼200 genome copies/reaction, depending on strain. • Broad analytical reactivity over seasonal/non-seasonal strains was demonstrated. • No cross-reactivity with non-influenza organisms was observed. • Three-site inter-laboratory reproducibility was 98.5%., Background Influenza causes a significant annual disease burden, with characterization of the infecting virus important in clinical and public health settings. Rapid immunoassays are fast but insensitive, whereas real-time RT-PCR is sensitive but susceptible to genetic mutations and often requires multiple serial assays. The FluChip-8G Influenza A+B Assay provides type and subtype/lineage identification of influenza A and B, including non-seasonal A viruses, in a single microarray-based assay with same day turnaround time. Objective To evaluate key analytical performance characteristics of the FluChip-8G Influenza A+B Assay. Study design Analytical sensitivity, cross-reactivity, and multi-site reproducibility were evaluated. Results The limit of detection (LOD) for the FluChip-8G influenza A+B Assay ranged from 5.8 × 102–1.5 × 105 genome copies/mL, with most samples ∼2 × 103 genome copies/mL (∼160 genome copies/reaction). Fifty two (52) additional strains were correctly identified near the LOD, demonstrating robust reactivity. Two variant viruses (H1N1v and H3N2v) resulted in dual identification as both “non-seasonal influenza A” and A/H1N1pdm09. No reproducible cross-reactivity was observed for the 34 organisms tested, however, challenges with internal control inhibition due to crude growth matrix were observed. Lastly, samples tested near the LOD showed high reproducibility (97.0% (95% CI 94.7–98.7)) regardless of operator, site, reagent lot, or testing day. Conclusion The FluChip-8G Influenza A+B Assay is an effective new method for detecting and identifying both seasonal and non-seasonal influenza viruses, as revealed by good sensitivity and robust reactivity to 52 unique strains of influenza virus. In addition, the lack of cross-reactivity to non-influenza pathogens and high lab-to-lab reproducibility highlight the analytical performance of the assay as an alternative to real-time RT-PCR and sequencing-based assays. Clinical validation of the technology in a multi-site clinical study is the subject of a separate investigation.

Please cite this paper as: Heil et al. (2010) MChip, a low density microarray, differentiates among seasonal human H1N1, North American swine H1N1, and the 2009 pandemic H1N1. Influenza and Other Respiratory Viruses 4(6), 411–416. Background The MChip uses data from the hybridization of amplified viral RNA to 15 distinct oligonucleotides that target the influenza A matrix (M) gene segment. An artificial neural network (ANN) automates the interpretation of subtle differences in fluorescence intensity patterns from the microarray. The complete process from clinical specimen to identification including amplification of viral RNA can be completed in

Background Adamantanes resistance in H3N2 viruses has been increasing since 2000, and in 2005–2006 reached nearly 100% in most countries, with the circulation of the N-lineage. In 2006–2007, however, a significant decrease in resistance was observed in many regions. Objectives To explore potential links between adamantane resistance and the A(H3N2) viruses that circulated between 2006 and 2008. Methods A total of 1451 Influenza A (H3N2) viruses collected globally in 2001–2008 were screened for the presence of adamantane resistance markers. A subset of 100 viruses representing the broad genetic and geographic spectrum of these viruses was selected for complete genome sequencing and phylogenetic analyses. Results Full genome sequence analysis of 2006–2007 viruses revealed co-circulation of four distinct genotypes, designated A–D. Phylogenetic analyses demonstrated reassortment between viruses from the N-lineage and other viruses that had circulated in prior seasons, including those bearing an adamantane sensitive marker. Genotype D viruses became dominant in late 2006–2007 and continued to be the main H3N2 genotype in 2007–2008. Viruses of this genotype retained all N-lineage genome segments except PB2 and NP, which were acquired through reassortment. Conclusions The decrease in adamantane resistance at that time was due to transient co-circulation of genotypes that emerged through reassortment. Our findings emphasize the importance of complete genome sequencing in understanding the complex nature of the relationship between influenza virus evolution and antiviral resistance. The recent emergence of the pandemic multi-reassortant H1N1 virus underscores the importance of whole genome sequence monitoring for rapid detection of such unusual and novel strains.

The first known cases of human infection with highly pathogenic avian influenza (HPAI) H5N1 viruses in Vietnam occurred in late 2003. However, HPAI H5N1 and low-pathogenic avian influenza (LPAI) H5N2 and H9N3 viruses were isolated from domestic waterfowl during live-bird market (LBM) surveillance in Vietnam in 2001 and 2003. To understand the possible role of these early viruses in the genesis of H5N1 strains infecting people, we performed sequencing and molecular characterization. Phylogenetic analysis revealed that the hemagglutinin (HA) genes of two geese HPAI H5N1 strains belonged to clade 3, and their surface glycoprotein and replication complex genes were most closely related (98.5-99.7% homologous) to A/duck/Guangxi/22/01 (H5N1) virus, detected contemporarily in southern China, whilst the M and NS genes were derived from an A/duck/Hong Kong/2986.1/00 (H5N1)-like virus. The H5 HA gene of the duck HPAI H5N1 strain belonged to clade 5 and acquired a gene constellation from A/quail/Shantou/3846/02 (H5N1), A/teal/China/2978.1/02 (H5N1) and A/partridge/Shantou/2286/03 (H5N1)-like viruses. The phylogenetic analysis further indicated that all eight gene segments of goose and duck HPAI H5N1 and LPAI H5N2 viruses were distinct from those of H5N1 clade-1 viruses known to have caused fatal human infections in Vietnam since late 2003. The duck H9N3 isolates derived genes from aquatic-bird influenza viruses, and their H9 HA belonged to the Korean lineage. The PB2 gene of A/duck/Vietnam/340/01 (H9N3) virus had lysine at position 627. Based on the molecular characterization of specific amino acid residues in the surface and relevant internal protein-coding genes, the Vietnamese H5N1 and H9N3 virus isolates indicated specificity to avian cell surface receptor and susceptibility for currently licensed anti-influenza A virus chemotherapeutics. Our findings suggest that the H5N1 and H5N2 viruses that circulated among geese and ducks in LBMs in Hanoi, Vietnam, during 2001 and 2003 were not the immediate ancestors of the clade-1 viruses associated with fatal human infections in Vietnam. The clade-1 HPAI H5N1 viruses were independently introduced into Vietnam.

Correspondence: Dr Ruben O. Donis, Molecular Virology and Vaccines Branch, Influenza Division, NCIRD, Centers for Disease Control & Prevention, 1600 Clifton Road, NE, MS-G16, Atlanta, GA 30333, USA. Email: rvd6@cdc.gov Dr Gavin Smith, State Key Laboratory of Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Hong Kong SAR, China. E-mail: gjsmith@hku.hk *WHO ⁄OIE ⁄ FAO H5N1 Evolution Working Group members and collaborators can be found in the Appendix.

Generation of Swine Flu As the newly emerged influenza virus starts its journey to infect the world's human population, the genetic secrets of the 2009 outbreak of swine influenza A(H1N1) are being revealed. In extensive phylogenetic analyses, Garten et al. (p. 197 , published online 22 May) confirm that of the eight elements of the virus, the basic components encoded by the hemagglutinin, nucleoprotein, and nonstructural genes originated in birds and transferred to pigs in 1918. Subsequently, these formed a triple reassortant with the RNA polymerase PB1 that transferred from birds in 1968 to humans and then to pigs in 1998, coupled with RNA polymerases PA and PB2 that transferred from birds to pigs in 1998. The neuraminidase and matrix protein genes that complete the virus came from birds and entered pigs in 1979. The analysis offers insights into drug susceptibility and virulence, as well as raising the possibility of hitherto unknown factors determining host specificity. A significant question is, what is the potential for the H1 component of the current seasonal flu vaccine to act as a booster? Apart from the need for ongoing sequencing to monitor for the emergence of new reassortants, future pig populations need to be closely monitored for emerging influenza viruses.

Two genetically distinct lineages of H1N1 influenza A viruses, circulated worldwide before 1994, were antigenically indistinguishable. In 1994, viruses emerged in China, including A/Beijing/262/95, with profound antigenic differences from the contemporary circulating H1N1 strains. Haemagglutinin sequence comparisons of either a predecessor virus, A/Hebei/52/94, or one representative of the cocirculating A/Bayern/7/95-like clade, A/Shenzhen/227/95, revealed a deletion of K at position 134 (H3 numbering) in the antigenic variants. The K134 deletion conferred a selective advantage to the Chinese deletion lineage, such that it eventually gave rise to currently circulating H1 viruses. Using reverse genetics to generate viruses with either an insertion or deletion of aa 134, we have confirmed that the K134 deletion, rather than a constellation of sublineage specific amino acid changes, was sufficient for the antigenic difference observed in the Chinese deletion lineage, and reinsertion of K134 revealed the requirement of a compatible neuraminidase surface glycoprotein for viral growth.

Virulence factors of influenza A (H5N1) viruses collected in 1997 from mammalian hosts were examined using a BALB/c mouse model. Fifteen amino acid (aa) residues in four influenza virus genes which correlated with high- and low-pathogenic phenotypes in mice were identified by analyzing sequence alignments. In addition to these specific residues, the effects of aa residue 627 of the PB2 gene, and the hemagglutinin (HA) and neuraminidase (NA) genes were also investigated using a reverse genetics system established with representative viruses of low (A/Hong Kong/486/97) and high (A/Hong Kong/483/97) pathogenicity for mice. None of 15 aa residues alone had any effect on virulence. The HA and NA genes had a synergistic effect on virulence and the absence of a glycosylation site at aa154 in the HA gene also increased virulence of virus. Multiple genes are involved in virulence of Hong Kong H5N1 influenza A viruses for mice with the presence of lysine at aa627 in the PB2 gene exhibiting a significantly larger effect than the HA and NA genes.

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

The design and characterization of a low-density microarray for subtyping influenza A is presented. The microarray consisted of 15 distinct oligonucleotides designed to target only the matrix gene segment of influenza A. An artificial neural network was utilized to automate microarray image interpretation. The neural network was trained to recognize fluorescence image patterns for 68 known influenza viruses and subsequently used to identify 53 unknowns in a blind study that included 39 human patient samples and 14 negative control samples. The assay exhibited a clinical sensitivity of 95% and clinical specificity of 92%.

DNA microarrays have proven to be powerful tools for gene expression analyses and are becoming increasingly attractive for diagnostic applications, e.g., for virus identification and subtyping. The selection of appropriate sequences for use on a microarray poses a challenge, particularly for highly mutable organisms such as influenza viruses, human immunodeficiency viruses, and hepatitis C viruses. The goal of this work was to develop an efficient method for mining large databases in order to identify regions of conservation in the influenza virus genome. From these regions of conservation, capture and label sequences capable of discriminating between different viral types and subtypes were selected. The salient features of the method were the use of phylogenetic trees for data reduction and the selection of a relatively small number of capture and label sequences capable of identifying a broad spectrum of influenza viruses. A detailed experimental evaluation of the selected sequences is described in a companion paper. The software is freely available under the General Public License at http://www.colorado.edu/chemistry/RGHP/software/ .

Between 1979 and 1989, families enrolled in the Houston Family Study were prospectively monitored for influenza virus infections. Reinfection with the H3N2 subtype occurred in a number of family members, and 6 pairs of isolates (interval between collection of first and second isolate, 2-5 years) were available for molecular analysis. Changes in the hemagglutinin genes of pairs of viruses isolated from the same individuals were examined to determine the molecular basis for reinfection. The findings of this study indicate that reinfection of an individual by viruses of the same subtype may occur within a relatively short period of time when the paired strains have genetically distinct hemagglutinin genes in which amino acid changes are present in the defined antigenic sites.

Background: The hemagglutinins (HA) of human influenza viruses adapt during growth in chicken eggs. Amino acid replacements in HAs of egg-grown viruses generally appear on the terminal branches of phylogenetic trees. Our method for predicting human influenza HA evolution, which tracks replacements at positively selected codons across trees, is more accurate with terminal mutations excluded. Methods: We extracted the sequence data that contain the majority of egg-adapted replacements from our original data set, and replicated our prediction tests analyzing both internal and terminal mutations. We contrasted these results with those obtained analyzing (1) only internal mutations and (2) analyzing all mutations in our full data set. Results: Excluding egg-adapted replacements did not increase the accuracy of our predictions when other terminal mutations remained in the analysis. Deleting all terminal mutations produced the best prediction of success of viral lineages. Conclusions: Amino acid replacements on terminal branches interfered with our ability to identify which codons were under positive selection. Therefore, we recommend limiting such analyses to the internal branches of phylogenetic trees. The effects of terminal mutations may be caused by the criteria used for selecting viruses for HA sequencing.

Background: Analysis of the hemagglutinin (HA) and neuraminidase (NA) genes of influenza A and B viruses revealed worldwide circulation of multiple genetic lineages within the same type/subtype. Genetic reassortment between distinct genetic lineages was examined in this study. Methods: Nucleotide sequencing, polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) were used. Phylogenetic studies were used to analyze sequence data. Results: Influenza A (H3N2) . The NA genes of H3N2 viruses were found to have evolved as three distinct lineages represented by A/Sydney/5/97, A/Panama/2007/99, and A/Moscow/10/99, respectively. Similar genetic groups were also found among the HA genes of these viruses. PCR/RFLP data of strains isolated between June 1999 and June 2000 showed that 122 of 141 viruses examined (87%) had their NA genes closely related to that of A/Moscow/10/99 virus; 14 of 141 viruses (10%) had A/Panama/2007/99-like NA genes, and only 5 strains (3%) had A/Sydney/5/97-like NA genes. Viruses bearing A/Panama/2007/99-like HA, and either A/Moscow/10/99-like, or A/Sydney/5/97-like NA were detected, indicating that genetic reassortments of the NA genes among different lineages of H3N2 viruses had occurred. Influenza A (H1N1) . Analyses showed that the NA and HA genes of the current strains each evolved as two genetically distinct lineages, represented by A/Bayern/7/95 and A/Beijing/262/95, respectively. The NA genes of the majority of circulating H1N1 viruses were closely related to that of the current vaccine strain A/New Caledonia/20/99 virus (A/Beijing/262/95 lineage). Influenza B . The NA genes of influenza B viruses, similar to their HA genes, were found to have diverged into two genetically and antigenically distinguishable lineages represented by B/Yamagata/16/88 (recently B/Yamanashi/166/98) and B/Victoria/2/87 (recently by B/Shandong/7/97) strains. Reassortants bearing the B/Yamagata-like HA and B/Victoria-like NA were isolated from several countries. Conclusion: Monitoring both HA and NA genes/antigens is important for appropriate vaccine strain selection.

In this paper we determine the extent to which host-mediated mutations and a known sampling bias affect evolutionary studies of human influenza A. Previous phylogenetic reconstruction of influenza A (H3N2) evolution using the hemagglutinin gene revealed an excess of nonsilent substitutions assigned to the terminal branches of the tree. We investigate two hypotheses to explain this observation. The first hypothesis is that the excess reflects mutations that were either not present or were at low frequency in the viral sample isolated from its human host, and that these mutations increased in frequency during passage of the virus in embryonated eggs. A set of 22 codons known to undergo such “host-mediated” mutations showed a significant excess of mutations assigned to branches attaching sequences from egg-cultured (as opposed to cell-cultured) isolates to the tree. Our second hypothesis is that the remaining excess results from sampling bias. Influenza surveillance is purposefully biased toward sequencing antigenically dissimilar strains in an effort to identify new variants that may signal the need to update the vaccine. This bias produces an excess of mutations assigned to terminal branches simply because an isolate with no close relatives is by definition attached to the tree by a relatively long branch. Simulations show that the magnitude of excess mutations we observed in the hemagglutinin tree is consistent with expectations based on our sampling protocol. Sampling bias does not affect inferences about evolution drawn from phylogenetic analyses. However, if possible, the excess caused by host-mediated mutations should be removed from studies of the evolution of influenza viruses as they replicate in their human hosts.

Zoonotic infections with swine influenza A viruses are reported sporadically. Triple reassortant swine influenza viruses have been isolated from pigs in the United States since 1998. We report a human case of upper respiratory illness associated with swine influenza A (H1N1) triple reassortant virus infection that occurred during 2005 following exposure to freshly killed pigs.

The anti-inflammatory cytokines interleukin-10 (IL-10) and interleukin-13 (IL-13) were shown to reduce hyperalgesia in some models such as rats exposed to UV rays. In addition, IL-10 was also shown to reduce hyperalgesia in high dose of Leishmania major-induced inflammation in BALB/c mice accompanied by a significant decrease in the levels of interleukin-1β (IL-1β) in the paws of infected mice, while no effect on the levels of IL-6 was observed. In this study, we injected BALB/c mice with a high dose of L. major and treated them with IL-13 (15 ng/animal) for twelve days (excluding the weekends) and hyperalgesia was assessed using thermal pain tests. Furthermore, the levels of IL-1β and IL-6 were also assessed at different post-infection days. Our results show that IL-6 and more importantly IL-1β don't play a direct role in the L. major-induced hyperalgesia and that IL-13 induces this hyperalgesia through the down-regulation of IL-1β and another proinflammatory cytokine (most probably TNF-α). Furthermore, our data show that IL-13 leads to the upregulation of the level IL-6 which initially seems to have no direct role in the induced hyperalgesia. Therefore, we suggest that the L. major-induced hyperalgesia is mainly mediated by the cytokine cascade leading to the production of sympathetic amines.

Highly pathogenic avian influenza (HPAI) H5N1 viruses have caused dramatic economic losses to the poultry industry of Vietnam and continue to pose a serious threat to public health. As of June 2008, Vietnam had reported nearly one third of worldwide laboratory confirmed human H5N1 infections. To better understand the emergence, spread and evolution of H5N1 in Vietnam we studied over 300 H5N1 avian influenza viruses isolated from Vietnam since their first detection in 2001. Our phylogenetic analyses indicated that six genetically distinct H5N1 viruses were introduced into Vietnam during the past seven years. The H5N1 lineage that evolved following the introduction in 2003 of the A/duck/Hong Kong/821/2002-like viruses, with clade 1 hemagglutinin (HA), continued to predominate in southern Vietnam as of May 2007. A virus with a clade 2.3.4 HA newly introduced into northern Vietnam in 2007, reassorted with pre-existing clade 1 viruses, resulting in the emergence of novel genotypes with neuraminidase (NA) and/or internal gene segments from clade 1 viruses. A total of nine distinct genotypes have been present in Vietnam since 2001, including five that were circulating in 2007. At least four of these genotypes appear to have originated in Vietnam and represent novel H5N1 viruses not reported elsewhere. Geographic and temporal analyses of H5N1 infection dynamics in poultry suggest that the majority of viruses containing new genes were first detected in northern Vietnam and subsequently spread to southern Vietnam after reassorting with pre-existing local viruses in northern Vietnam. Although the routes of entry and spread of H5N1 in Vietnam remain speculative, enhanced poultry import controls and virologic surveillance efforts may help curb the entry and spread of new HPAI viral genes.

The robustness of a recently developed diagnostic microarray for influenza, the MChip, was evaluated with 16 historic subtype H1N1 influenza A viruses (A/H1N1), including A/Brevig Mission/1/1918. The matrix gene segments from all 16 viruses were successfully detected on the array. An artificial neural network trained with temporally related A/H1N1 viruses identified A/Brevig Mission/1/1918 as influenza virus A/H1N1 with 94% probability.

Before 2003, only occasional case reports of human H7 influenza virus infections occurred as a result of direct animal-to-human transmission or laboratory accidents; most of these infections resulted in conjunctivitis. An increase in isolation of avian influenza A H7 viruses from poultry outbreaks and humans has raised concerns that additional zoonotic transmissions of influenza viruses from poultry to humans may occur. To better understand the pathogenesis of H7 viruses, we have investigated their ability to cause disease in mouse and ferret models. Mice were infected intranasally with H7 viruses of high and low pathogenicity isolated from The Netherlands in 2003 (Netherlands/03), the northeastern United States in 2002-2003, and Canada in 2004 and were monitored for morbidity, mortality, viral replication, and proinflammatory cytokine production in respiratory organs. All H7 viruses replicated efficiently in the respiratory tracts of mice, but only Netherlands/03 isolates replicated in systemic organs, including the brain. Only A/NL/219/03 (NL/219), an H7N7 virus isolated from a single fatal human case, was highly lethal for mice and caused severe disease in ferrets. Supporting the apparent ocular tropism observed in humans following infection with viruses of the H7 subtype, both Eurasian and North American lineage H7 viruses were detected in the mouse eye following ocular inoculation, whereas an H7N2 virus isolated from the human respiratory tract was not. Therefore, in general, the relative virulence and cell tropism of the H7 viruses in these animal models correlated with the observed virulence in humans.

In previous work, a simple diagnostic DNA microarray that targeted only the matrix gene segment of influenza A (MChip) was developed and evaluated with patient samples. In this work, the analytical utility of the MChip for detection and subtyping of an emerging virus was evaluated with a diverse set of A/H5N1 influenza viruses. A total of 43 different highly pathogenic A/H5N1 viral isolates that were collected from diverse geographic locations, including Vietnam, Nigeria, Indonesia, and Kazakhstan, representing human, feline, and a variety of avian infections spanning the time period 2003-2006 were used in this study. A probabilistic artificial neural network was developed for automated microarray image interpretation through pattern recognition. The microarray assay and subsequent subtype assignment by the artificial neural network resulted in correct identification of 24 "unknown" A/H5N1 positive samples with no false positives. Analysis of a data set composed of A/H5N1, A/H3N2, and A/H1N1 positive samples and negative controls resulted in a clinical sensitivity of 97% and a clinical specificity of 100%.

Global surveillance of influenza is critical for improvements in disease management and is especially important for early detection, rapid intervention, and a possible reduction of the impact of an influenza pandemic. Enhanced surveillance requires rapid, robust, and inexpensive analytical techniques capable of providing a detailed analysis of influenza virus strains. Low-density oligonucleotide microarrays with highly multiplexed “signatures” for influenza viruses offer many of the desired characteristics. However, the high mutability of the influenza virus represents a design challenge. In order for an influenza virus microarray to be of utility, it must provide information for a wide range of viral strains and lineages. The design and characterization of an influenza microarray, the FluChip-55 microarray, for the relatively rapid identification of influenza A virus subtypes H1N1, H3N2, and H5N1 are described here. In this work, a small set of sequences was carefully selected to exhibit broad coverage for the influenza A and B viruses currently circulating in the human population as well as the avian A/H5N1 virus that has become enzootic in poultry in Southeast Asia and that has recently spread to Europe. A complete assay involving extraction and amplification of the viral RNA was developed and tested. In a blind study of 72 influenza virus isolates, RNA from a wide range of influenza A and B viruses was amplified, hybridized, labeled with a fluorophore, and imaged. The entire analysis time was less than 12 h. The combined results for two assays provided the absolutely correct types and subtypes for an average of 72% of the isolates, the correct type and partially correct subtype information for 13% of the isolates, the correct type only for 10% of the isolates, false-negative signals for 4% of the isolates, and false-positive signals for 1% of the isolates. In the overwhelming majority of cases in which incomplete subtyping was observed, the failure was due to the nucleic acid amplification step rather than limitations in the microarray.

Since 1997, outbreaks of highly pathogenic (HP) H5N1 and circulation of H9N2 viruses among domestic poultry in Asia have posed a threat to public health. To better understand the extent of transmission of avian influenza viruses (AIV) to humans in Asia, we conducted a cross-sectional virologic study in live bird markets (LBM) in Hanoi, Vietnam, in October 2001. Specimens from 189 birds and 18 environmental samples were collected at 10 LBM. Four influenza A viruses of the H4N6 ( n = 1), H5N2 ( n = 1), and H9N3 ( n = 2) subtypes were isolated from healthy ducks for an isolation frequency of over 30% from this species. Two H5N1 viruses were isolated from healthy geese. The hemagglutinin (HA) genes of these H5N1 viruses possessed multiple basic amino acid motifs at the cleavage site, were HP for experimentally infected chickens, and were thus characterized as HP AIV. These HA genes shared high amino acid identities with genes of other H5N1 viruses isolated in Asia during this period, but they were genetically distinct from those of H5N1 viruses isolated from poultry and humans in Vietnam during the early 2004 outbreaks. These viruses were not highly virulent for experimentally infected ducks, mice, or ferrets. These results establish that HP H5N1 viruses with properties similar to viruses isolated in Hong Kong and mainland China circulated in Vietnam as early as 2001, suggest a common source for H5N1 viruses circulating in these Asian countries, and provide a framework to better understand the recent widespread emergence of HP H5N1 viruses in Asia.

During the 2001-2002 influenza season, human influenza A (H1N2) reassortant viruses were detected globally. The hemagglutinin (HA) of these H1N2 viruses was similar to that of the A/New Caledonia/20/99 (H1N1) vaccine strain both antigenically and genetically, while their neuraminidase (NA) was antigenically and genetically related to that of recent human influenza H3N2 reference viruses such as A/Moscow/10/99. All six internal genes of the H1N2 reassortants originated from an H3N2 virus. After being detected only in eastern Asia during the past 10 years, Influenza B/Victoria/2/87 lineage viruses reappeared in many countries outside of Asia in 2001. Additionally, reassortant influenza B viruses possessing an HA similar to that of B/Shandong/7/97, a recent B/Victoria/2/87 lineage reference strain, and an NA closely related to that of B/Sichuan/379/99, a recent B/Yamagata/16/88 lineage reference strain, were isolated globally and became the predominant influenza B epidemic strain. The current influenza vaccine is expected to provide good protection against H1N2 viruses because it contains A/New Caledonia/20/99 (H1N1) and A/Panama/2007/99 (H3N2) like viruses whose H1 HA or N2 NA are antigenically similar to those of recent circulating H1N2 viruses. On the other hand, widespread circulation of influenza B Victoria lineage viruses required inclusion of a strain from this lineage in influenza vaccines for the 2002-2003 season.

Reassortant influenza A viruses bearing the H1 subtype of hemagglutinin (HA) and the N2 subtype of neuraminidase (NA) were isolated from humans in the United States, Canada, Singapore, Malaysia, India, Oman, Egypt, and several countries in Europe during the 2001-2002 influenza season. The HAs of these H1N2 viruses were similar to that of the A/New Caledonia/20/99(H1N1) vaccine strain both antigenically and genetically, and the NAs were antigenically and genetically related to those of recent human H3N2 reference strains, such as A/Moscow/10/99(H3N2). All 6 internal genes of the H1N2 reassortants examined originated from an H3N2 virus. This article documents the first widespread circulation of H1N2 reassortants on 4 continents. The current influenza vaccine is expected to provide good protection against H1N2 viruses, because it contains the A/New Caledonia/20/99(H1N1) and A/Moscow/10/99(H3N2)-like viruses, which have H1 and N2 antigens that are similar to those of recent H1N2 viruses.

Background: In 1997, highly pathogenic avian influenza A H5N1 viruses caused 18 human cases of respiratory illness and six deaths in Hong Kong. The genes of the H5N1 viruses isolated from humans were derived from avian influenza viruses, with no evidence of reassortment with human influenza viruses. The molecular determinants of human pathogenesis were not evident. Methods: The BALB/c mouse was used as a mammalian model to evaluate the biological and molecular basis of human H5N1 virus pathogenesis. Molecular correlates of H5N1 virus pathogenicity for mice were sought by analyzing the nucleotide sequence of human H5N1 viruses. Results: Based on high and low lethality for mice, the pathogenicity phenotype of 15 human H5N1 viruses was characterized; nine viruses displayed a high, five a low, and one an intermediate pathogenicity phenotype. H5N1 viruses with a high pathogenicity phenotype replicated in multiple solid organs and caused depletion of peripheral blood leukocytes. Sequence analysis determined that five specific amino acids in four proteins correlated with pathogenicity in mice. Conclusions: Alone or in combination, these specific residues are the likely determinants of virulence of human H5N1 influenza viruses in this mammalian model.

Since 1998, H3N2 viruses have caused epizootics of respiratory disease in pigs throughout the major swine production regions of the U.S. These outbreaks are remarkable because swine influenza in North America had previously been caused almost exclusively by H1N1 viruses. We sequenced the full-length protein coding regions of all eight RNA segments from four H3N2 viruses that we isolated from pigs in the Midwestern U.S. between March 1998 and March 1999, as well as from H3N2 viruses recovered from a piglet in Canada in January 1997 and from a pig in Colorado in 1977. Phylogenetic analyses demonstrated that the 1977 Colorado and 1997 Ontario isolates are wholly human influenza viruses. However, the viruses isolated since 1998 from pigs in the Midwestern U.S. are reassortant viruses containing hemagglutinin, neuraminidase and PB1 polymerase genes from human influenza viruses, matrix, non-structural and nucleoprotein genes from classical swine viruses, and PA and PB2 polymerase genes from avian viruses. The HA proteins of the Midwestern reassortant swine viruses can be differentiated from those of the 1995 lineage of human H3 viruses by 12 amino acid mutations in HA1. In contrast, the Sw/ONT/97 virus, which did not spread from pig-to-pig, lacks 11 of these changes.

Research on absenteeism has led to the development of models that emphasize individual-level variables of satisfaction and job involvement but fail to recognize the social framework of absenteeism. Most studies investigate chiefly blue-collar workers in fairly routinized jobs; few studies cross industrial and occupational lines to white-collar or professional work. Our social model examines absenteeism in terms of alternatives available to employees each day and the consequences of absenteeism for workers and for employers. Beyond psychological and aggregated demographic variables, we consider the role of occupational and organizational processes and point to needs for cross-industry and cross-occupational studies of absenteeism.

The study examined the importance of different bases of legitimacy of authority upon conformity and performance of temporary employees engaged in a realistic technical task. Task supervisors varied in apparent technical and administrative expertise. The contingency of rewards upon performance was systematically varied to separate the impact of expected reward contingency from variations in bases of legitimacy. Subjects were hired as temporary workers in a laboratory experiment to test these propositions, and to build upon an earlier laboratory study by Evan and Zelditch.

Do communities recognize and respond to deprofessionalized models of police service? This study tracks newspaper coverage of police and safety issues during an experimental foot patrol program. Content analysis of nonnews items confirms that police are evaluated more positively, depicted as more active but not made more visible overall after the first full year of program operation. The need for more community-level evidence of police impact is considered.

Swine influenza viruses (SIV) have been recognized as important pathogens for pigs and occasional human infections with swine origin influenza viruses (SOIV) have been reported. Between1990 and 2010, a total of twenty seven human cases of SOIV infections have been identified in the United States. Six viruses isolated from1990 to 1995 were recognized as classical SOIV (cSOIV) A(H1N1). After 1998, twenty-one SOIV recovered from human cases were characterized as triple reassortant (tr_SOIV) inheriting genes from classical swine, avian and human influenza viruses. Of those twenty-one tr_SOIV, thirteen were of A(H1N1), one of A(H1N2), and seven of A(H3N2) subtype. SOIV characterized were antigenically and genetically closely related to the subtypes of influenza viruses circulating in pigs but distinct from contemporary influenza viruses circulating in humans. The diversity of subtypes and genetic lineages in SOIV cases highlights the importance of continued surveillance at the animal–human interface.

To determine the genetic and antigenic relatedness as well as the cross-protective immunity of human H1N1 and avian H5N1 influenza virus neuraminidase (NA), we immunized rabbits with either a baculovirus-expressed recombinant NA from A/Beijing/262/95 (BJ/262) H1N1 or A/Hong Kong/483/97 (HK/483) H5N1 virus. Cross-reactive antibody responses were evaluated by multiple serological assays and cross-protection against H5N1 virus challenge was evaluated in mice. In a neuraminidase inhibition (NI) test, the antisera exhibited substantial inhibition of NA activity of the homologous virus, but failed to inhibit the NA activity of heterologous virus. However, these antisera exhibited low levels of cross-reactivity measured by plaque size reduction, replication inhibition, single radial hemolysis, and ELISA assays. Passive immunization with HK/483 NA-specific antisera significantly reduced virus replication and disease, and afforded almost complete protection against lethal homologous virus challenge in mice. However, passive immunization with BJ/262 (H1N1) NA-specific antisera was ineffective at providing cross-protection against lethal H5N1 virus challenge and only slightly reduced weight loss. Substantial amino acid variation among the NA antigenic sites was observed between BJ/262 and HK/483 virus, which was consistent with the lack of cross-reactive NI activity by the antibody and limited cross-protective immunity in mice. These results show a strong correlation between the lack of cross-protective immunity and low structural similarities of NA from a human seasonal H1N1 virus and an avian H5N1 influenza virus.