Influenza Virus Inactivation for Studies of Antigenicity and Phenotypic Neuraminidase Inhibitor Resistance Profiling

Introduction of a new influenza virus in humans urges quick analysis of its virological and immunological characteristics to determine the impact on public health and to develop protective measures for the human population. At present, however, the necessity of executing pandemic influenza virus research under biosafety level 3 (BSL-3) high-containment conditions severely hampers timely characterization of such viruses. We tested heat, formalin, Triton X-100, and β-propiolactone treatments for their potencies in inactivating human influenza A(H3N2) and avian A(H7N3) viruses, as well as seasonal and pandemic A(H1N1) virus isolates, while allowing the specimens to retain their virological and immunological properties. Successful heat inactivation coincided with the loss of hemagglutinin (HA) and neuraminidase (NA) characteristics, and β-propiolactone inactivation reduced the hemagglutination titer and NA activity of the human influenza virus 10-fold or more. Although Triton X-100 treatment resulted in inconsistent HA activity, the NA activities in culture supernatants were enhanced consistently. Nonetheless, formalin treatment permitted the best retention of HA and NA properties. Triton X-100 treatment proved to be the easiest-to-use influenza virus inactivation protocol for application in combination with phenotypic NA inhibitor susceptibility assays, while formalin treatment preserved B-cell and T-cell epitope antigenicity, allowing the detection of both humoral and cellular immune responses. In conclusion, we demonstrated successful influenza virus characterization using formalin- and Triton X-100-inactivated virus samples. Application of these inactivation protocols limits work under BSL-3 conditions to virus culture, thus enabling more timely determination of public health impact and development of protective measures when a new influenza virus, e.g., pandemic A(H1N1)v virus, is introduced in humans.Host switching of viruses from animals to humans may result in an epidemic among humans and can be particularly dangerous for the new, immunologically naïve host. Examples are the introduction of human immunodeficiency virus, severe acute respiratory syndrome coronavirus, and pandemic influenza A viruses in humans. In particular, avian influenza A virus subtypes H5N1, H9N2, and H7N7 have been transmitted directly to humans in the past decade, exhibiting the zoonotic potential of influenza viruses (4, 11, 19, 25). Moreover, the recent introduction of swine origin influenza A(H1N1)v virus in humans initiated the first influenza pandemic of the 21st century (16, 35). Introduction of a new influenza virus in humans urges quick analysis of its virological and immunological characteristics to assist in the determination of the impact on public health and the development of protective measures. At present, however, the necessity of executing pandemic influenza virus research under biosafety level 3 (BSL-3) high-containment conditions hampers timely characterization of such viruses.Several virological and immunological assays are used for the characterization of a virus and the immune response induced. For antigenic characterization of influenza viruses, hemagglutination assays and hemagglutination inhibition (HI) assays are the “gold standard” tests. In addition, since the global emergence of antiviral-resistant influenza viruses is becoming an increasing problem, the characterization of influenza virus susceptibilities to the neuraminidase (NA) inhibitors (NAIs) oseltamivir and zanamivir is a clinical necessity (2, 9, 13, 17, 23). For investigating the immune response against influenza viruses, the HI assay determines protective humoral responses (8). Finally, in addition to HI assay results, assessment of the human T-cell responses against influenza virus infection has been reported previously to provide an important marker of protection (3, 10, 22). Until now, these assays have been performed mostly by applying live virus, hence necessitating the use of BSL-3 conditions for studying (potential) pandemic influenza virus. Although numerous studies of virus inactivation, e.g., by means of virucidal compounds, UV light, or gamma irradiation treatment, have been performed, these studies have not comprehensively documented the preservation of influenza virus protein function and antigenic characteristics following inactivation (5-7, 14, 18). Specifically, these studies have not addressed whether inactivated virus can be used for phenotypic determination of susceptibilities to NAIs and for characterization of T-cell responses.In this study, we evaluated the inactivation of influenza viruses of human, avian, and swine origins by heat, formalin, Triton X-100, or β-propiolactone (β-PL) and the retention of hemagglutinin (HA) and NA glycoprotein functions and antigenic integrity. The optimal procedures have been used to demonstrate the proof of principle in antiviral susceptibility assays, antigenic characterization, and T-cell response assays with both seasonal and pandemic influenza A(H1N1) viruses.     

Flexible Label-Free Quantitative Assay for Antibodies to Influenza Virus Hemagglutinins

During the initial pandemic influenza H1N1 virus outbreak, assays such as hemagglutination inhibition and microneutralization provided important information on the relative protection afforded by the population''s cross-reactivity from prior infections and immunizations with seasonal vaccines. However, these assays continue to be limited in that they are difficult to automate for high throughput, such as in pandemic situations, as well as to standardize between labs. Thus, new technologies are being sought to improve standardization, reliability, and throughput by using chemically defined reagents rather than whole cells and virions. We now report the use of a cell-free and label-free flu antibody biosensor assay (f-AbBA) for influenza research and diagnostics that utilizes recombinant hemagglutinin (HA) in conjunction with label-free biolayer interferometry technology to measure biomolecular interactions between the HA and specific anti-HA antibodies or sialylated ligands. We evaluated f-AbBA to determine anti-HA antibody binding activity in serum or plasma to assess vaccine-induced humoral responses. This assay can reveal the impact of antigenic difference on antibody binding to HA and also measure binding to different subtypes of HA. We also show that the biosensor assay can measure the ability of HA to bind a model sialylated receptor-like ligand. f-AbBA could be used in global surveillance laboratories since preliminary tests on desiccated HA probes showed no loss of activity after >2 months in storage at room temperature, indicating that the same reagent lots could be used in different laboratories to minimize interlaboratory assay fluctuation. Future development of such reagents and similar technologies may offer a robust platform for future influenza surveillance activities.Vaccination, the cornerstone of public health intervention, helps prevent influenza morbidity and mortality. Effective vaccines induce protective immunity which is correlated with the presence of virus-specific antibodies (Abs) in serum that are directed against the external coat proteins of the virion, hemagglutinin (HA) and, to a lesser extent, neuraminidase (NA). HA is the principal antigen on the viral surface, and neutralizing antibodies are usually directed to hypervariable epitopes located in or near the HA receptor-binding site (RBS) and act to prevent host infection by blocking virus binding to the host cell (17). For this reason, induction of HA-specific antibodies that interfere with virus entry is used as a correlate of vaccine protective efficacy.Influenza viruses are characterized by their rapid antigenic change as a result of their high mutation frequency. Therefore, the composition of influenza virus vaccines requires frequent updates (every 2 years on average for H3N2) to match their antigenicity as closely as possible to that of the variant viruses most prevalent in the population (25). Analysis of antibody responses that correlate with protective immunity to influenza virus vaccination is an important element in the assessment of the potential impact of viral antigenic drift. The hemagglutination inhibition (HI) test is the most widely used serological test for the detection of anti-influenza virus antibodies (13, 36) and is used routinely to determine the serological outcome of vaccinations. The assay itself is technically simple but difficult to automate and standardize. In addition, interpretation of results can be affected by virus passage, antibody source (species), and variability between red blood cells from different species (19, 36).The continuing but sporadic human infections with H5N1 avian influenza viruses reported since 1997 have revealed that HI assays are less sensitive in detecting antibodies against avian influenza viruses (2, 16, 23, 24, 28) than are alternative assays such as the microneutralization (MN) test (24). The MN test, however, is technically very demanding and is currently performed only as a reference test on a small number of serum specimens. As virus-neutralizing activity of antiserum is mediated in part by blocking virus-receptor interactions, the results of the HI test often correlate well with those of the MN test. Recent international laboratory network studies showed large intra- and interlaboratory assay variations for both the HI and MN assays (26), although for H5N1, such variability can be reduced through the availability of an antibody standard (27). Despite limited reproducibility between labs, the HI and MN tests still provide the best available data to inform global surveillance and aid in decisions to update the seasonal influenza virus vaccine composition. Indeed, their use in recent months has been critical in assessing immunity to the pandemic H1N1 virus in the human population (4, 20). It is clear that new technologies and assays are urgently needed to improve sensitivity, accuracy, and sample throughput, as well as reproducibility between labs. In addition, assays need to be flexible and adaptable to be able to analyze antibody responses to emerging viruses from sources other than avian populations (12).Here, we report on a flexible label-free and cell-free assay to determine the relative functional avidity of polyclonal serum antibodies binding to the major virion coat protein, HA. The flu antibody biosensor assay (f-AbBA) presented here utilizes an established recombinant baculovirus expression system for producing HA (32) in conjunction with label-free biolayer interferometry (BLI) technology from Fortebio Inc., an optical technique that analyzes the interference pattern of white light reflected from a layer of immobilized protein on the tip of a fiber optic biosensor (1, 7). Macromolecules binding to the biosensor tip produce an increase in optical thickness at the biosensor tip, which results in a wavelength shift (measured in nanometers) that can be followed in real time, allowing one to determine binding kinetics. BLI technology minimizes interference from extraneous materials present in solution. Only molecules that bind to or dissociate from the biosensor surface produce a signal change. Sample preparation time is reduced because crude mixtures such as cell lysates, patient serum, or hybridoma supernatants can be assayed. The platform is designed to simultaneously analyze eight samples as a dip-and-read format using commercially available 96-well plates. We have used it to measure the biomolecular interactions of recombinant HA with reactive antibodies from patient sera in real time, without the need for secondary reagents for detection. Results have good correlation with existing serological methods but with improved sensitivity and reproducibility. By comparing binding to different HAs, we can determine the polyclonal specificity, while limited studies with human sera from prior vaccination studies reveal a linear correlation between binding observed with the biosensor and corresponding HI titers as well as some cross-reactivity with the 2009 pandemic H1N1 virus HA. Initial stability tests suggest that reagents may be prepared and desiccated on probes and stored at ambient temperature for extended periods of time without loss of function, offering different laboratories the chance to utilize the same reagents and thus improve interlaboratory assay variation.     

Microscopic Observation Drug Susceptibility Assay for Rapid Diagnosis of Lymph Node Tuberculosis and Detection of Drug Resistance

In this study, 132 patients with lymphadenopathy were investigated. Fifty-two (39.4%) were diagnosed with tuberculosis (TB). The microscopic observation drug susceptibility (MODS) assay provided rapid (13 days), accurate diagnosis (sensitivity, 65.4%) and reliable drug susceptibility testing (DST). Despite its lower sensitivity than that of other methods, its faster results and simultaneous DST are advantageous in resource-poor settings, supporting the incorporation of MODS into diagnostic algorithms for extrapulmonary TB.     

Simple Procedure for Drug Susceptibility Testing of Mycobacterium tuberculosis Using a Commercial Colorimetic Assay

 The aim of this study was to evaluate a simple method using a commercial colorimetric assay (Alamar Blue Oxidation-Reduction Indicator; Accumed, USA) in a microtiter format for testing the susceptibility of 94 strains of Mycobacterium tuberculosis to isoniazid, rifampicin, ethambutol and streptomycin. The method makes use of one critical concentration of each drug, and the results are available within 8–10 days. Overall, 97.1% agreement with the proportion method was obtained. Full agreement was obtained for isoniazid and rifampicin. The method is simple to perform, permits visual reading of results, and is practicable for laboratories with limited resources.     

Radioimmunoprecipitation Assay for Quantitation of Serum Antibody to the Hemagglutinin of Type A Influenza Virus

A double-antibody radioimmunoprecipitation (RIP) assay has been developed to provide a sensitive and specific measure of antibody to hemagglutinins of H3N2 influenza viruses. Chloramine T was used to radiolabel purified hemagglutinins to high specific activity without loss of antigenicity. The purity of the labeled hemagglutinin was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, which also established that both the HA(1) and HA(2) polypeptides were iodinated. Radiolabeled hemagglutinins with a specific activity that did not exceed 12 muCi/mug of protein could be maintained for up to 30 days at -70 degrees C in the presence of supplemental protein. The RIP assay was compared with conventional methods, hemagglutination inhibition and viral neutralization tests, using H3N equine 1 hybrid viruses for determining serum antihemagglutinin antibody titers. The geometric mean titers for human convalescent sera after infection with A/England/72 virus were 118, 161, and 18,822 for hemagglutination inhibition, viral neutralization, and RIP tests, respectively, and the three tests demonstrated significant rises in antihemagglutinin antibody titers with equal efficiency. In general, a positive correlation existed between antihemagglutinin antibody titers determined by these three procedures; however, the antibody level determined by RIP assay for each individual could not be related to hemagglutination inhibition or viral neutralization titers by a constant factor. A similar lack of a constant relationship was found by using hyperimmune guinea pig antisera, which suggests that the RIP assay can detect antibody populations that exhibit differing efficiencies for inhibition of viral hemagglutination and replication.     

Influenza A Virus Neuraminidase Enhances Meningococcal Adhesion to Epithelial Cells through Interaction with Sialic Acid-Containing Meningococcal Capsules

The underlying mechanisms of the epidemiological association between influenza virus infections and Neisseria meningitidis invasive infections are not fully understood. Here we report that adhesion of N. meningitidis to human Hec-1-B epithelial cells is enhanced by influenza A virus (IAV) infection. A potential role of the viral neuraminidase (NA) in facilitating meningococcal adhesion to influenza virus-infected epithelial cells was examined. Expression of a recombinant IAV NA in Hec-1-B human epithelial cells increased the adhesion of strains of N. meningitidis belonging to the sialic acid-containing capsular serogroups B, C, and W135 but not to the mannosamine phosphate-containing capsular serogroup A. Adhesion enhancement was not observed with an inactive NA mutant or in the presence of an NA inhibitor (zanamivir). Furthermore, purified IAV NA was shown to cleave sialic acid-containing capsular polysaccharides of N. meningitidis. On the whole, our findings suggest that a direct interaction between the NA of IAV and the capsule of N. meningitidis enhances bacterial adhesion to cultured epithelial cells, most likely through cleavage of capsular sialic acid-containing polysaccharides. A better understanding of the association between IAV and invasive meningococcal infections should help to set up improved control strategies against these seasonal dual viral-bacterial infections.Neisseria meningitidis is commonly found in the human naso-oropharynx, among other commensal bacterial species. Asymptomatic carriers represent about 10% of the population (41). In Europe and North America, cases of invasive infection leading to meningococcal disease (MD), mainly septicemia and meningitis, occur sporadically. The annual incidence of MD varies between 0.3 and 4.35 per 100,000 inhabitants (36). Both bacterial virulence factors (32) and host susceptibility factors (34, 38) contribute to the development of invasive infections, but the exact mechanisms involved remain largely unknown. The serogroups of N. meningitidis, which are defined by the nature of the capsular polysaccharide, are distributed differently among carried and disease-associated isolates (42). Serogroups B and C (whose capsules are composed of polymers of sialic acids) and serogroups Y and W135 (whose capsules are composed of repeated units of sialic acid with d-glucose and d-galactose, respectively [4]) are prominent in MD in Europe and North America. Serogroup A, whose capsule is composed of α1,6-linked N-acetylmannosamine-1-phosphate (18), is most frequently involved in MD epidemics in Africa.A number of clinical observations of MD occurring in patients with influenza have been reported (6, 13, 43). Epidemiological studies clearly showed a spatiotemporal association between influenza and N. meningitidis invasive infections (2, 14, 25). Recent data from the French National Reference Center for Meningococci and from the Sentinelles Network confirmed the overlap between the winter peaks of incidence of bacteriologically confirmed MD cases and influenza-like illnesses during the period from 2000 to 2008 (Fig. ​(Fig.1),1), in agreement with data published previously (14).Open in a separate windowFIG. 1.Invasive meningococcal infections and influenza-like illnesses recorded in France by the Reference Center for Meningococci and the Sentinelles Network from January 2000 to May 2008. Reporting of invasive meningococcal infections is mandatory. All invasive meningococcal isolates in France are sent to the National Reference Center for Meningococci for full characterization and typing. The general practitioners of the Sentinelles Network report on influenza-like illnesses on a weekly basis by sending patient deidentified data via the Internet to a GIS database (11). The monthly incidence of MD (right axis) and the weekly incidence of influenza-like illnesses (left axis) during the period of January 2000 to May 2008 are represented on the same graph.The mechanisms by which influenza virus infection may favor bacterial superinfection with N. meningitidis, as well as with staphylococci, pneumococci, streptococci, and Haemophilus influenzae, have been investigated using cellular and animal models (15, 19, 22, 33). Virus-induced immune dysregulation, such as impairment of phagocytic functions or alterations in the production of cytokines, can be involved (12). In mice convalescing from influenza A virus (IAV) infection, the production of interleukin-10 in the lungs enhanced susceptibility to meningococcal (3) and to pneumococcal (37) superinfection. Influenza virus infection can also enhance bacterial adhesion by disrupting the respiratory epithelium or by increasing the accessibility or expression of membrane receptors (26). Conflicting results about the effects of influenza virus infection on meningococcal adherence to epithelial cells have been reported. One study reported that meningococci bind IAV-infected epithelial cells more efficiently than uninfected cells (10), whereas another concluded that coinfection with influenza B virus does not affect the association of meningococci with cultured human nasopharyngeal mucosa (29). Adhesion to epithelial cells in the nasopharynx is the early step enabling N. meningitidis to colonize the upper respiratory tract before it possibly goes through the epithelial barrier into the blood to induce bacteremia and reaches the meningeal spaces to induce meningitis. The adhesion process is associated with a downregulation of the capsule (9). We hypothesized that sialic acid-containing capsules could be a substrate for the neuraminidase (NA) of IAV and that this direct virus-bacterium interaction could play a major role in enhancing meningococcal adhesion to the respiratory epithelium. This hypothesis was tested on isolates belonging to various serogroups of N. meningitidis, using an in vitro model of adhesion to epithelial cells transiently expressing IAV-derived recombinant NAs.     

Plaque Assay for Ebola Virus

A plaque assay for Ebola virus is reported. The procedure has real potential for future research, although it is less sensitive than indirect fluorescent-antibody and mouse inoculation tests.     

Microplate alamarBlue Assay for Paracoccidioides Susceptibility Testing

CLSI method M27-A3 is not available for use with dimorphic fungi, such as those of the Paracoccidioides genus. In this study, we developed a microdilution method and added the alamarBlue reagent to test the responses of Paracoccidioides brasiliensis and Paracoccidioides lutzii against amphotericin B and itraconazole antifungals. The test proved to be sensitive, practical, and inexpensive and can be used to monitor the activity of low-growth microorganisms and their response to various drugs.     

Diagnostic Accuracy and Reproducibility of WHO-Endorsed Phenotypic Drug Susceptibility Testing Methods for First-Line and Second-Line Antituberculosis Drugs

In an effort to update and clarify policies on tuberculosis drug susceptibility testing (DST), the World Health Organization (WHO) commissioned a systematic review evaluating WHO-endorsed diagnostic tests. We report the results of this systematic review and meta-analysis of the diagnostic accuracy and reproducibility of phenotypic DST for first-line and second-line antituberculosis drugs. This review provides support for recommended critical concentrations for isoniazid and rifampin in commercial broth-based systems. Further studies are needed to evaluate critical concentrations for ethambutol and streptomycin that accurately detect susceptibility to these drugs. Evidence is limited on the performance of DST for pyrazinamide and second-line drugs.     

H1N2新亚型流感病毒神经氨酸酶基因来源的进一步研究

对流感病毒Ｈ１Ｎ２亚型重组株Ａ／哈防／１／８８、Ａ／哈防／１２／９２以及Ｈ３Ｎ２亚型病毒株Ａ／雅防／２／８７、Ａ／京防／５７／８９８和Ａ／粤防／１／９２神经氨酸酶（ＮＡ）基因核苷酸全序列的测定，进一步弄清了Ａ／哈防／１／８８病毒株的ＮＡ基因确来自当时人群中流行的Ｈ３Ｎ２亚型病毒株，很可能是来自Ａ／雅防／２／８７类病毒株；而Ａ／哈防／１２／９２病毒株的ＮＡ基因可能是与Ａ／哈防／１／８８病毒株的ＮＡ基     

Detection of Nonhemagglutinating Influenza A(H3) Viruses by Enzyme-Linked Immunosorbent Assay in Quantitative Influenza Virus Culture

To assess the efficacy of novel antiviral drugs against influenza virus in clinical trials, it is necessary to quantify infectious virus titers in respiratory tract samples from patients. Typically, this is achieved by inoculating virus-susceptible cells with serial dilutions of clinical specimens and detecting the production of progeny virus by hemagglutination, since influenza viruses generally have the capacity to bind and agglutinate erythrocytes of various species through their hemagglutinin (HA). This readout method is no longer adequate, since an increasing number of currently circulating influenza A virus H3 subtype (A[H3]) viruses display a reduced capacity to agglutinate erythrocytes. Here, we report the magnitude of this problem by analyzing the frequency of HA-deficient A(H3) viruses detected in The Netherlands from 1999 to 2012. Furthermore, we report the development and validation of an alternative method for monitoring the production of progeny influenza virus in quantitative virus cultures, which is independent of the capacity to agglutinate erythrocytes. This method is based on the detection of viral nucleoprotein (NP) in virus culture plates by enzyme-linked immunosorbent assay (ELISA), and it produced results similar to those of the hemagglutination assay using strains with good HA activity, including A/Brisbane/059/07 (H1N1), A/Victoria/210/09 (H3N2), other seasonal A(H1N1), A(H1N1)pdm09, and the majority of A(H3) virus strains isolated in 2009. In contrast, many A(H3) viruses that have circulated since 2010 failed to display HA activity, and infectious virus titers were determined only by detecting NP. The virus culture ELISA described here will enable efficacy testing of new antiviral compounds in clinical trials during seasons in which nonhemagglutinating influenza A viruses circulate.