The 2017-2018 North American influenza season caused more hospitalizations and deaths than any year since the 2009 H1N1 pandemic. The majority of recorded influenza infections were caused by A(H3N2) viruses, with most of the virus's North American diversity falling into the A2 clade. Within A2, we observe a subclade which we call A2/re that rose to comprise almost 70 per cent of A(H3N2) viruses circulating in North America by early 2018. Unlike most fast-growing clades, however, A2/re contains no amino acid substitutions in the hemagglutinin (HA) segment. Moreover, hemagglutination inhibition assays did not suggest substantial antigenic differences between A2/re viruses and viruses sampled during the 2016-2017 season. Rather, we observe that the A2/re clade was the result of a reassortment event that occurred in late 2016 or early 2017 and involved the combination of the HA and PB1 segments of an A2 virus with neuraminidase (NA) and other segments a virus from the clade A1b. The success of this clade shows the need for antigenic analysis that targets NA in addition to HA. Our results illustrate the potential for non-HA drivers of viral success and necessitate the need for more thorough tracking of full viral genomes to better understand the dynamics of influenza epidemics.

Seasonal influenza virus A/H3N2 is a major cause of death globally. Vaccination remains the most effective preventative. Rapid mutation of hemagglutinin allows viruses to escape adaptive immunity. This antigenic drift necessitates regular vaccine updates. Effective vaccine strains need to represent H3N2 populations circulating one year after strain selection. Experts select strains based on experimental measurements of antigenic drift and predictions made by models from hemagglutinin sequences. We developed a novel influenza forecasting framework that integrates phenotypic measures of antigenic drift and functional constraint with previously published sequence-only fitness estimates. Forecasts informed by phenotypic measures of antigenic drift consistently outperformed previous sequence- only estimates, while sequence-only estimates of functional constraint surpassed more comprehensive experimentally-informed estimates. Importantly, the best models integrated estimates of both functional constraint and either antigenic drift phenotypes or recent population growth.

The emergence of severe acute respiratory syndrome (SARS) underscored the importance of influenza detection and response in China. From 2004, the Chinese National Influenza Center (CNIC) and the United States Centers for Disease Control and Prevention (USCDC) initiated Cooperative Agreements to build capacity in influenza surveillance in China. From 2004 to 2014, CNIC and USCDC collaborated on the following activities: 1) developing human technical expertise in virology and epidemiology in China; 2) developing a comprehensive influenza surveillance system by enhancing influenza-like illness (ILI) reporting and virological characterization; 3) strengthening analysis, utilization and dissemination of surveillance data; and 4) improving early response to influenza viruses with pandemic potential. Since 2004, CNIC expanded its national influenza surveillance and response system which, as of 2014, included 408 laboratories and 554 sentinel hospitals. With support from USCDC, more than 2500 public health staff from China received virology and epidemiology training, enabling > 98% network laboratories to establish virus isolation and/or nucleic acid detection techniques. CNIC established viral drug resistance surveillance and platforms for gene sequencing, reverse genetics, serologic detection, and vaccine strains development. CNIC also built a bioinformatics platform to strengthen data analysis and utilization, publishing weekly on-line influenza surveillance reports in English and Chinese. The surveillance system collects 200,000-400,000 specimens and tests more than 20,000 influenza viruses annually, which provides valuable information for World Health Organization (WHO) influenza vaccine strain recommendations. In 2010, CNIC became the sixth WHO Collaborating Centre for Influenza. CNIC has strengthened virus and data sharing, and has provided training and reagents for other countries to improve global capacity for influenza control and prevention. The collaboration's successes were built upon shared mission and values, emphasis on long-term capacity development and sustainability, and leadership commitment.

Influenza A(H3N2) viruses evade human immunity primarily by acquiring antigenic changes in the haemagglutinin (HA). HA receptor-binding features of contemporary A(H3N2) viruses hinder traditional antigenic characterization using haemagglutination inhibition and promote selection of HA mutants. Thus, alternative approaches are needed to reliably assess antigenic relatedness between circulating viruses and vaccines. We developed a high content imaging-based neutralization test (HINT) to reduce antigenic mischaracterization resulting from virus adaptation to cell culture. Ferret reference antisera were raised using clinical specimens containing viruses representing recent vaccine strains. Analysis of viruses circulating during 2011-2018 showed that gain of an N158-linked glycosylation in HA was a molecular determinant of antigenic distancing between A/Hong Kong/4801/2014-like (clade 3C.2a) and A/Texas/50/2012-like viruses (clade 3C.1), while multiple evolutionary HA F193S substitution were linked to antigenic distancing from A/Switzerland/97152963/2013-like (clade 3C.3a) and further antigenic distancing from A/Texas/50/2012-like viruses. Additionally, a few viruses carrying HA T135K and/or I192T showed reduced neutralization by A/Hong Kong/4801/2014-like antiserum. Notably, this technique elucidated the antigenic characteristics of clinical specimens, enabling direct characterization of viruses produced in vivo, and eliminating in vitro culture, which rapidly alters the genotype/phenotype. HINT is a valuable new antigenic analysis tool for vaccine strain selection.

Influenza virologic surveillance is critical each season for tracking influenza circulation, following trends in antiviral drug resistance, detecting novel influenza infections in humans, and selecting viruses for use in annual seasonal vaccine production. We developed a framework and process map for characterizing the landscape of US influenza virologic surveillance into 5 tiers of influenza testing: outpatient settings (tier 1), inpatient settings and commercial laboratories (tier 2), state public health laboratories (tier 3), National Influenza Reference Center laboratories (tier 4), and Centers for Disease Control and Prevention laboratories (tier 5). During the 2015-16 season, the numbers of influenza tests directly contributing to virologic surveillance were 804,000 in tiers 1 and 2; 78,000 in tier 3; 2,800 in tier 4; and 3,400 in tier 5. With the release of the 2017 US Pandemic Influenza Plan, the proposed framework will support public health officials in modeling, surveillance, and pandemic planning and response.

The United States 2017-18 influenza season (October 1, 2017-May 19, 2018) was a high severity season with high levels of outpatient clinic and emergency department visits for influenza-like illness (ILI), high influenza-related hospitalization rates, and elevated and geographically widespread influenza activity across the country for an extended period. Nationally, ILI activity began increasing in November, reaching an extended period of high activity during January-February, and remaining elevated through March. Influenza A(H3N2) viruses predominated through February and were predominant overall for the season; influenza B viruses predominated from March onward. This report summarizes U.S. influenza activity* during October 1, 2017-May 19, 2018.(dagger).

Rapid evolution of influenza A(H3N2) viruses necessitates close monitoring of their antigenic properties so emergence and spread of antigenic drift variants can be rapidly identified. Changes in hemagglutinin (HA) acquired by contemporary A(H3N2) viruses hinder antigenic characterization by traditional methods, thus complicating vaccine strain selection. Sequence-based approaches have been used to infer virus antigenicity; however, they are time-consuming and mid-throughput. To facilitate virological surveillance and epidemiological studies, we have developed and validated a pyrosequencing approach that enables identification of six HA clades of contemporary A(H3N2) viruses. The identification scheme of H3 clade 3C.2, 3C.2a, 3C.2b, 3C.3, 3C.3a and 3C.3b viruses is based on the interrogation of five SNPs within three neighboring HA regions: 412-431; 465-481; and 559-571. Two bioinformatics tools, IdentiFire (Qiagen) and FireComb (developed in-house) were utilized to expedite pyrosequencing data analysis. The assay's analytical sensitivity was 10 focus forming units; and respiratory specimens with CT value < 34 typically produced good quality pyrograms. When applied to 120 A(H3N2) virus isolates and 27 respiratory specimens, the assay displayed 100% agreement with clades determined by HA sequencing coupled with phylogenetics. The multi-SNP analysis described here was readily adopted by another laboratory with pyrosequencing capabilities. Implementation of this approach enhanced virological surveillance and epidemiological studies from 2013-2016 when over 3000 A(H3N2) viruses were examined.

BACKGROUND: During the 2014-15 US influenza season, expanded genetic characterization of circulating influenza A(H3N2) viruses was used to assess the impact of genetic variability of influenza A(H3N2) viruses on influenza vaccine effectiveness (VE). METHODS: A novel pyrosequencing assay was used to determine genetic group based on hemagglutinin (HA) gene sequences of influenza A(H3N2) viruses from patients enrolled US Flu Vaccine Effectiveness network sites. Vaccine effectiveness was estimated using a test-negative design comparing vaccination among patients infected with influenza A(H3N2) viruses and uninfected patients. RESULTS: Among 9710 enrollees, 1868 (19%) tested positive for influenza A(H3N2); genetic characterization of 1397 viruses showed 1134 (81%) belonged to one HA genetic group (3C.2a) of antigenically drifted H3N2 viruses. Effectiveness of 2014-15 influenza vaccination varied by A(H3N2) genetic group from 1% (95% confidence interval [CI], -14% to 14%) against illness caused by antigenically drifted A(H3N2) group 3C.2a viruses versus 44% (95% CI, 16% to 63%) against illness caused by vaccine-like A(H3N2) group 3C.3b viruses. CONCLUSION: Effectiveness of 2014-15 influenza vaccination varied by genetic group of influenza A(H3N2) virus. Changes in hemagglutinin genes related to antigenic drift were associated with reduced vaccine effectiveness.

Cold-adapted influenza strains A/Leningrad/134/17/57 (H2N2) and B/USSR/60/69, originally developed in Russia, have been reliable master donors of attenuation for preparing live attenuated influenza vaccines (LAIV). The classical strategy for generating LAIV reassortants is robust, but has some disadvantages. The generation of reassortants requires at least 3 passages under selective conditions after co-infection; each of these selective passages takes six days. Screening the reassortants for a genomic composition traditionally starts after a second limiting dilution cloning procedure, and the number of suitable reassortants is limited. We developed a new approach to shorten process of preparing LAIV seed viruses. Introducing the genotyping of reassortants by pyrosequencing and monitoring sequence integrity of surface antigens starting at the first selective passage allowed specific selection of suitable reassortants for the next cloning procedure and also eliminate one of the group selective passage in vaccine candidate generation. Homogeneity analysis confirmed that reducing the number of selective passages didn't affect the quality of LAIV seed viruses. Finally, the two-way hemagglutination inhibition test, implemented for all the final seed viruses, confirmed that any amino acid substitutions acquired by reassortants during egg propagation didn't affect antigenicity of the vaccine. Our new strategy reduces the time required to generate a vaccine and was used to generate seasonal LAIVs candidates for the 2012/2013, 2014/2015, and 2015/2016 seasons more rapidly.

Here we define the epitopes on HA that are targeted by a group of 9 recombinant monoclonal antibodies (rmAbs) isolated from memory B cells of mice, immunized by infection with A(H1N1)pdm09 virus followed by a seasonal TIV boost. These rmAbs were all reactive against the HA1 region of HA, but display 7 distinct binding footprints, targeting each of the 4 known antigenic sites. Although the rmAbs were not broadly cross-reactive, a group showed subtype-specific cross-reactivity with the HA of A/South Carolina/1/18. Screening these rmAbs with a panel of human A(H1N1)pdm09 virus isolates indicated that naturally-occurring changes in HA could reduce rmAb binding, HI activity, and/or virus neutralization activity by rmAb, without showing changes in recognition by polyclonal antiserum. In some instances, virus neutralization was lost while both ELISA binding and HI activity were retained, demonstrating a discordance between the two serological assays traditionally used to detect antigenic drift.

Understanding the spatiotemporal patterns of emergence and circulation of new human seasonal influenza virus variants is a key scientific and public health challenge. The global circulation patterns of influenza A/H3N2 viruses are well characterized, but the patterns of A/H1N1 and B viruses have remained largely unexplored. Here we show that the global circulation patterns of A/H1N1 (up to 2009), B/Victoria, and B/Yamagata viruses differ substantially from those of A/H3N2 viruses, on the basis of analyses of 9,604 haemagglutinin sequences of human seasonal influenza viruses from 2000 to 2012. Whereas genetic variants of A/H3N2 viruses did not persist locally between epidemics and were reseeded from East and Southeast Asia, genetic variants of A/H1N1 and B viruses persisted across several seasons and exhibited complex global dynamics with East and Southeast Asia playing a limited role in disseminating new variants. The less frequent global movement of influenza A/H1N1 and B viruses coincided with slower rates of antigenic evolution, lower ages of infection, and smaller, less frequent epidemics compared to A/H3N2 viruses. Detailed epidemic models support differences in age of infection, combined with the less frequent travel of children, as probable drivers of the differences in the patterns of global circulation, suggesting a complex interaction between virus evolution, epidemiology, and human behaviour.

BACKGROUND: Telomeres provide a key mechanism for protecting the integrity of chromosomes and their attrition after cell division and during aging are evident in lymphocytes. However, the significance of telomere shortening in age-associated decline of immune function is unknown. METHODS: We selected 22 HLA-A2+ healthy older adults who have relatively short or long telomere lengths to compare their antibody response against the influenza vaccine, and their CD8+ T cell response against an influenza antigen. RESULTS: B cells from individuals with a robust antibody response to the influenza vaccine had significantly longer telomeres than those with a poor antibody response. Monocyte-derived antigen-presenting cells of both short and long telomere groups induced similar expansions of influenza M1-specific CD8+ T cells. Vaccination did not increase M1-specific CD8+ T cells in blood, however, M1-specific CD8+ T cells from the long telomere group exhibited significantly better expansion in vitro compared to those from the short telomere group. Finally, M1-specific CD8+ T cells that underwent more expansions had significantly longer telomeres compared to cells with fewer divisions. CONCLUSIONS: Telomere length is positively associated with a robust lymphocyte response and telomere attrition may contribute to the age-associated decline of adaptive immunity.

National U.S. influenza antiviral surveillance incorporates data generated by neuraminidase (NA) inhibition (NI) testing of isolates supplemented with NA sequence analysis; and pyrosequencing analysis of clinical specimens. Lack of established correlates for clinically relevant resistance to NA inhibitors (NAIs) hinders interpretation of NI assay data. Nonetheless, A(H3N2) viruses are commonly monitored for highly reduced or reduced inhibition in the NI assay and/or presence of NA markers, E119V, R292K and N294S. In 2012-2013, three drug resistant A(H3N2) viruses were detected by NI assay among isolates (n=1424); all showed highly reduced inhibition by oseltamivir and had E119V. In addition, one R292K variant was detected among clinical samples (n=1024) by a 3-target pyrosequencing assay. Overall, frequency of NAI resistance was low, 0.16% (4 of 2448). To screen for additional NA markers previously identified in viruses from NAI-treated patients, the pyrosequencing assay was modified to include Q136K, I222V, del245-248 and del247-250. The 7-target pyrosequencing assay detected NA variants carrying E119V, Q136 and del245-248 in an isolate from an oseltamivir-treated patient. Next, this assay was applied to clinical specimens collected from hospitalized patients and submitted for NI testing, but failed cell culture propagation. Of the 27 clinical specimens tested, 4 (15%) contained NA changes: R292K (n=2), E119V (n=1) and del247-250 (n=1). Recombinant NAs with del247-250 and del245-248, respectively, conferred highly reduced inhibition by oseltamivir, reduced inhibition by zanamivir, and normal inhibition by peramivir and laninamivir. Our results demonstrated the benefits of the 7-target pyrosequencing assay in conducting A(H3N2) antiviral surveillance and testing for clinical care.

We report characteristics of oseltamivir-resistant influenza A(H1N1)pdm09 viruses and patients infected with these viruses in the United States. During 2013-14, fifty-nine (1.2%) of 4,968 analyzed US influenza A(H1N1)pdm09 viruses had the H275Y oseltamivir resistance-conferring neuraminidase substitution. Our results emphasize the need for local surveillance for neuraminidase inhibitor susceptibility among circulating influenza viruses.

In February and September each year the World Health Organisation (WHO) recommends influenza viruses to be included in influenza vaccines for the forthcoming winters in the Northern and Southern Hemispheres respectively. These recommendations are based on data collected by National Influenza Centres (NIC) through the Global Influenza Surveillance and Response System (GISRS) and a more detailed analysis of representative and potential antigenically variant influenza viruses from the WHO Collaborating Centres for Influenza (WHO CCs) and Essential Regulatory Laboratories (ERLs). This article provides a detailed summary of the antigenic and genetic properties of viruses and additional background data used by WHO experts during development of the recommendations for the 2012 Southern Hemisphere influenza vaccine composition.

BACKGROUND: In early 2009, a novel influenza A(H1N1) virus that emerged in Mexico and United States rapidly disseminated worldwide. The spread of this virus caused considerable morbidity with over 18,000 recorded deaths. The new virus was found to be a reassortant containing gene segments from human, avian and swine influenza viruses. METHODS/RESULTS: The first case of human infection with A(H1N1)pdm09 in Pakistan was detected on 18(th) June 2009. Since then, 262 laboratory-confirmed cases have been detected during various outbreaks with 29 deaths (as of 31(st) August 2010). The peak of the epidemic was observed in December with over 51% of total respiratory cases positive for influenza. Representative isolates from Pakistan viruses were sequenced and analyzed antigenically. Sequence analysis of genes coding for surface glycoproteins HA and NA showed high degree of high levels of sequence identity with corresponding genes of regional viruses circulating South East Asia. All tested viruses were sensitive to Oseltamivir in the Neuraminidase Inhibition assays. CONCLUSIONS: Influenza A(H1N1)pdm09 viruses from Pakistan form a homogenous group of viruses. Their HA genes belong to clade 7 and show antigenic profile similar to the vaccine strain A/California/07/2009. These isolates do not show any amino acid changes indicative of high pathogenicity and virulence. It is imperative to continue monitoring of these viruses for identification of potential variants of high virulence or drug resistance.

BACKGROUND: Longitudinal data are limited about the circulating strains of influenza viruses and their public health impact in Indonesia. We conducted influenza surveillance among outpatients and hospitalized patients with influenza-like illness (ILI) across the Indonesian archipelago from 2003 through 2007. METHODOLOGY: Demographic, clinical data, and respiratory specimens were collected for 4236 ILI patients tested for influenza virus infection by RT-PCR and viral culture. PRINCIPAL FINDINGS: Influenza A and B viruses co-circulated year-round with seasonal peaks in influenza A virus activity during the rainy season (December-January). During 2003-2007, influenza viruses were identified in 20.1% (4236/21,030) of ILI patients, including 20.1% (4015/20,012) of outpatients, and 21.7% (221/1018) of inpatients. One H5N1 case was identified retrospectively in an outpatient with ILI. Antigenic drift in circulating influenza A and B virus strains was detected during the surveillance period in Indonesia. In a few instances, antigenically drifted viruses similar to the World Health Organization (WHO) vaccine strains were detected earlier than the date of their designation by WHO. CONCLUSIONS: Influenza A and B virus infections are an important cause of influenza-like illness among outpatients and hospitalized patients in Indonesia. While year-round circulation of influenza viruses occurs, prevention and control strategies should be focused upon the seasonal peak during rainy season months. Ongoing virologic surveillance and influenza disease burden studies in Indonesia are important priorities to better understand the public health impact of influenza in South-East Asia and the implications of influenza viral evolution and global spread.

BACKGROUND: Influenza causes yearly seasonal epidemics and periodic pandemics. Global systems have been established to monitor the evolution and impact of influenza viruses, yet regional analysis of surveillance findings has been limited. This study describes epidemiological and virological characteristics of influenza during 2006-2010 in the World Health Organization's Western Pacific Region. METHODOLOGY/PRINCIPAL FINDINGS: Influenza-like illness (ILI) and influenza virus data were obtained from the 14 countries with National Influenza Centres. Data were obtained directly from countries and from FluNet, the web-based tool of the Global Influenza Surveillance and Response System. National influenza surveillance and participation in the global system increased over the five years. Peaks in ILI reporting appeared to be coincident with the proportion of influenza positive specimens. Temporal patterns of ILI activity and the proportion of influenza positive specimens were clearly observed in temperate countries: Mongolia, Japan and the Republic of Korea in the northern hemisphere, and Australia, New Zealand, Fiji and New Caledonia (France) in the southern hemisphere. Two annual peaks in activity were observed in China from 2006 through the first quarter of 2009. A temporal pattern was less evident in tropical countries, where influenza activity was observed year-round. Influenza A viruses accounted for the majority of viruses reported between 2006 and 2009, but an equal proportion of influenza A and influenza B viruses was detected in 2010. CONCLUSIONS/SIGNIFICANCE: Despite differences in surveillance methods and intensity, commonalities in ILI and influenza virus circulation patterns were identified. Patterns suggest that influenza circulation may be dependent on a multitude of factors including seasonality and population movement. Dominant strains in Southeast Asian countries were later detected in other countries. Thus, timely reporting and regional sharing of information about influenza may serve as an early warning, and may assist countries to anticipate the potential severity and burden associated with incoming strains.

Influenza A virus reservoirs in animals have provided novel genetic elements leading to the emergence of global pandemics in humans. Most influenza A viruses circulate in waterfowl, but those that infect mammalian hosts are thought to pose the greatest risk for zoonotic spread to humans and the generation of pandemic or panzootic viruses. We have identified an influenza A virus from little yellow-shouldered bats captured at two locations in Guatemala. It is significantly divergent from known influenza A viruses. The HA of the bat virus was estimated to have diverged at roughly the same time as the known subtypes of HA and was designated as H17. The neuraminidase (NA) gene is highly divergent from all known influenza NAs, and the internal genes from the bat virus diverged from those of known influenza A viruses before the estimated divergence of the known influenza A internal gene lineages. Attempts to propagate this virus in cell cultures and chicken embryos were unsuccessful, suggesting distinct requirements compared with known influenza viruses. Despite its divergence from known influenza A viruses, the bat virus is compatible for genetic exchange with human influenza viruses in human cells, suggesting the potential capability for reassortment and contributions to new pandemic or panzootic influenza A viruses.

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.

Diagnostic testing for viral infections has evolved during the past decade. Documenting a seroconversion, or significant increase in antibody titer between paired acute-phase and convalescent-phase serum specimens, is a well-proven method for detecting acute viral infection but can be challenging, because blood sample collection is invasive, a second blood sample is required, and late collection of the acute-phase serum sample complicates interpretation. In contrast, polymerase chain reaction (PCR) assays are rapid and sensitive when performed on respiratory specimens collected early in the illness but can lack specificity because of amplicon contamination or presence of virus not etiologically linked to the illness. PCR assays have largely replaced serologic examination and culture for detecting viral pathogens in respiratory disease surveillance [1]. The added value of serologic examination in disease surveillance has not been fully assessed. | We compared serologic examination and PCR results among patients enrolled in a pneumonia etiology study in rural Thailand from September 2003 through August 2005 [2]. Patients who were hospitalized and met a broad case definition for respiratory disease were enrolled. We compared patients who had specimens (nasopharyngeal swab and serum) tested for adenovirus, human metapneumovirus (HMPV), influenza viruses A and B, parainfluenza viruses (PIVs) 1–3, and respiratory syncytial virus (RSV), as described elsewhere [2]. Conventional (first 18 months) and real-time (last 6 months) reverse-transcription PCR assays were used [3]. Influenza human serologic examination was conducted using hemagglutination inhibition test [4]. Adenovirus; PIVs 1, 2, and 3; HMPV; and RSV immunoglobulin G antibodies were tested using enzyme immunoassay [5–8]. A positive result was defined as a positive PCR test result and/or a ≥4-fold increase in antibody titer of the convalescent serum sample. We analyzed PIV 1 and 3 together, because the presence of cross-reactive epitopes on these related viruses complicates serologic discrimination. We compared proportions using McNemar’s χ2 statistic.

The recent influenza pandemic, caused by a novel H1N1 influenza A virus, as well as the seasonal influenza outbreaks caused by varieties of influenza A and B viruses, are responsible for hundreds of thousands of deaths worldwide. Few studies have evaluated the utility of real-time reverse transcription-PCR to detect influenza virus RNA from formalin-fixed, paraffin-embedded tissues obtained at autopsy. In this work, respiratory autopsy tissues from 442 suspect influenza cases were tested by real-time reverse transcription-PCR for seasonal influenza A and B and 2009 pandemic influenza A (H1N1) viruses and the results were compared to those obtained by immunohistochemistry. In total, 222 cases were positive by real-time reverse transcription-PCR, and of 218 real-time, reverse transcription-PCR-positive cases also tested by immunohistochemistry, only 107 were positive. Although formalin-fixed, paraffin-embedded tissues can be used for diagnosis, frozen tissues offer the best chance to make a postmortem diagnosis of influenza because these tissues possess nucleic acids that are less degraded and, as a consequence, provide longer sequence information than that obtained from fixed tissues. We also determined that testing of all available respiratory tissues is critical for optimal detection of influenza virus in postmortem tissues.