Influenza virus detection in clinical specimens

The authors compared the results of influenza A (H1N1) and influenza A (H3N2) virus detection in nasopharyngeal swabs from flu patients by molecular hybridization (MH), ELISA, virus isolation and seroconversion. Using the immunofluorescence (IF) technique influenza virus was detected in cell suspensions from the first chick embryo passage. Altogether 63 swabs from various epidemic seasons were separated into 3 groups according to specimen sampling and storage. It was shown that influenza virus RNA could be found in 16 out of 22 swab specimens (72%) stored at -70 degrees C without thawing and that ELISA revealed the influenza virus antigen in 19 cases (86%); in contrast, IF was positive in 6 (27%) and virus isolation in 5 (22%) cases only. However, the positive rate of MH decreased to 9% in 21 swab specimens repeatedly thawed and stored at -20 degrees C and was completely negative after prolonged storage of repeatedly thawed samples. Despite these conditions, ELISA was still successful in both latter sample groups (71-80%). For specificity control, 29 samples coming from patients with influenza B virus and other respiratory virus diseases (adeno- and respiratory syncytial virus) were used.     

The Local Origin of the Febrile Response Induced in Ferrets During Respiratory Infection with a Virulent Influenza Virus

Intranasal infection of ferrets with a virulent Clone (7a) of the recombinant influenza virus A/PR/8/34—A/England/939/69 (H₃N₂) produced a fever approximately 24 h in duration beginning about 29 h after infection. The origin of this fever has been investigated as an indication of what might happen in influenza in man.The systemic production of fever by virus interaction with phagocytes in the reticuloendothelial system appeared unlikely because insufficient virus escaped into the bloodstream. Ten half-hourly i.v. injections of 10⁸ 50%0 Egg-Bit Infectious Doses (EBID₅₀) of virus were needed to produce a fever of short duration (3-8 h). Yet, after the intranasal infection, which results in the 24 h fever, the total virus content in the nasal mucosa was less than 10⁸ EBID₅₀ before the onset of fever and only reached 10^8.5 EBID₅₀ for 4 h during fever. Also, just before or during the fever produced by intranasal infection, influenza virus antigens could not be detected by fluorescent antibody in the spleens of the animals but were detected in animals receiving a single bloodstream injection of 10⁸ EBID₅₀ of virus.Fever is more likely to result from release of leucocyte pyrogen by virus-phagocyte interaction in the upper respiratory tract. A pyrogen active in ferrets with the characteristics of leucocyte (endogenous) pyrogen was produced by incubating influenza virus with ferret peripheral phagocytes in vitro. A pyrogen with similar properties was released by incubation of nasal inflammatory cells collected from infected febrile ferrets and many of the cells were shown by fluorescent antibody to have interacted with influenza virus.     

重庆市2009-2010年流行性感冒流行特征及监测结果分析

目的分析2009-2010年重庆市流感流行特征和监测结果,为控制流感提供科学依据。方法收集重庆市流感疫情资料、流感样病例监测资料、病原学资料和暴发疫情资料,并对资料进行分析。结果 2009-2010年重庆市出现流感流行,流行高峰集中在2009年9～10月,流行优势毒株为新甲型H1N1亚型,2010年1月以来则以季节性B型为主。城区监测点病毒分离阳性率显著高于农村监测点（χ2=133.04,P〈0.001）。聚集和暴发疫情主要集中在学校（96%）,流感疫情高发时间和病原学检测结果与监测情况一致。结论甲型H1N1流感在2009年出现流行并成为优势株,进入2010年B型流感活动加强,应及时加强流感样病例监测和病原学监测工作,防止出现流行。     

Influenza viruses in cell cultures

Summary Calf kidney cells were found to be susceptible to seven representative strains of influenza virus (two type A, two type A₁, one type A₂, one type B, and the virus of Swine influenza).A quantal assay system, based on cytopathic effect, haemadsorption, or haemagglutination, has been developed. It is at least as sensitive as assay in whole eggs. One strain of virus, FM 1, required passage in calf kidney cells before attaining maximal infectivity for them.     

Evaluation of Alere i Influenza A&B for Rapid Detection of Influenza Viruses A and B

Rapid and accurate diagnosis of influenza is important for infection control, as well as for patient management. Alere i Influenza A&B is an isothermal nucleic acid amplification-based integrated system for detection and differentiation of influenza virus A and influenza virus B. The performance of the Alere i Influenza A&B was screened using frozen nasopharyngeal-swab specimens collected in viral transport medium (VTM) that were originally tested fresh with the FilmArray Respiratory Panel (RP) assay during the 2012–2013 influenza outbreak. In total, 360 VTM specimens were selected for Alere i Influenza A&B testing: 40 influenza virus A H1N1-2009 (influenza virus A-1), 40 influenza virus A H3N2 (influenza virus A-3), 37 influenza virus A “equivocal” or “no subtype detected” (influenza virus A-u), 41 influenza virus B, and 202 influenza virus-negative specimens, as initially determined by the FilmArray RP assay. The Alere assay showed sensitivities of 87.2%, 92.5%, 25.0%, and 97.4% for influenza virus A-1, influenza virus A-3, influenza virus A-u, and influenza virus B, respectively, after discordant resolution by Prodesse ProFLU+ PCR. The specificities were 100% for both influenza virus A and influenza virus B. In general, the Alere i Influenza A&B provided good sensitivity, although the assay did show poorer sensitivity with samples determined to have low influenza virus A titers by Prodesse ProFlu+ PCR (a mean real-time PCR threshold cycle [C_T] value of 31.9 ± 2.0), which included the majority of the samples called influenza virus A “equivocal” or “no subtype detected” by a single BioFire FilmArray RP test. The integrated, rapid, and simple characteristics of the Alere i Influenza A&B assay make it a potential candidate for point-of-care testing, with a test turnaround time of less than 15 min.     

Influenza virus replication in human alveolar macrophages

Studies with animal models suggest that alveolar macrophages may be important cells in some respiratory virus infections, but little is known about the role of these cells in virus infections in man. In this study human alveolar macrophages were obtained by fibreoptic bronchoscopy and infected in vitro with a variety of influenza viruses. After infection with the NWS strain of influenza virus the haemagglutinin and nucleoprotein viral antigens were demonstrated in >90% of cells at 24 h by immunofluorescence with specific antisera. There was no cytopathic effect at this time, and no virus release was detected by plaque assay of culture fluids on MDCK cells. Alveolar macrophages were also infected with a human vaccine strain (H₁N₁) of influenza virus and with two recent isolates (H₁N₁ and H₃N₂). In each case viral nucleoprotein antigen was produced in 10–20% of the cells by 24 h postinfection, but there was no release of infectious virus. There was no cytopathic effect and the phagocytosis of IgG-coated latex beads was unimpaired 24 h after in vitro infection.     

Outcome of Influenza Infection: Effect of Site of Initial Infection and Heterotypic Immunity

An infection established throughout the total respiratory tract of mice with a highly lung adapted influenza virus (H0N1) led to death from viral pneumonia. The 50% lethal dose (LD₅₀) was approximately the same as the 50% infectious dose (ID₅₀). An infection with the same virus initiated in the nasal mucosa spread to the trachea and lungs over a 3- to 5-day period but was not lethal except at very high infecting doses. The LD₅₀ was 30,000 times the ID₅₀. Mice that had recovered from a prior infection with A/PC/73(H3N2) demonstrated enhanced recovery (heterotypic immunity) when challenged with A/PR/8/34(H0N1). Heterotypically immune mice infected while anesthetized with this potentially lethal virus stopped shedding virus from the nose, trachea, and lungs by day 7 and recovered. Heterotypically immune mice, infected awake, stopped shedding virus from the nose by day 5, and, in fact, the virus did not spread to the trachea or lungs. Thus, some of the variation in the severity of influenza infections may be explained by two factors: the site of initial infection and previous infection with heterotypic influenza virus.     

A Study of Influenza 2017–2018 Outbreak in Andhra Pradesh,India

Background and Objectives: Influenza virus is a typical human pathogen causing serious respiratory illness resulting in significant mortality throughout the globe. Andhra Pradesh witnessed the first case of influenza A H1N1 in India from Hyderabad (now in Telangana) on May 16, 2009. In the recent past, Andhra Pradesh witnessed exponential increase in the number of confirmed cases of influenza infection. In this study, we present the salient features of the recent outbreak of influenza during 2017–2018 in the state of Andhra Pradesh, first of its kind after the division of the state. Materials and Methods: Clinically, suspected cases of influenza-like illness received in the Virus Research and Diagnostic Laboratory, Department of Microbiology, Sri Venkateswara Institute of Medical Sciences (SVIMS), Tirupati, from January 2017 to May 2018 were included in the study. The samples were tested for influenza A, influenza A (H1N1) pdm09, influenza A (H3N2), influenza B, influenza B/Yamagata and influenza B/Victoria. Results: A total of 1286 samples were received for testing. The positive samples were influenza A unsubtypable (109), influenza A (H1N1) pdm09 (356), influenza A (H3N2) (38) and influenza B (19; Victoria - 2, Yamagata - 17). There was no significant difference in positivity between genders with 260 (49.81%) females and 262 (50.19%) males being positive. Conclusion: The outbreak started in the late monsoon (January) of 2017 and had two peaks; one in summer months and another in winter months. Influenza B virus was reported from December 2017 to May 2018. Age groups ≤5 years and 6–18 years had higher positivity as compared to other age groups. Regular surveillance programmes are required for assessing the trends of influenza infections due to various subtypes and to plan timely and adequate steps for preventing the spread to larger vulnerable population.     

Morphological and Cytochemical Characterization of Cells Infiltrating Mouse Lungs After Influenza Infection

To initiate evaluation of the cell-mediated immunological response to influenza virus in a major site of disease, lung cells were obtained by transpleural lavage from lungs of uninfected mice and from those infected 3 or 6 days previously with 5 50% mouse infectious doses (MID₅₀) of avirulent (P3) or virulent (P9) influenza A Hong Kong (H3N2) virus. The number of cells recovered by lavage was dependent on the dose, time after inoculation, and the type of virus used for inoculation. Although lavage pools were shown to contain peripheral blood leukocytes, this contamination was shown to be consistently less than 5% of the total leukocytes harvested. Among the ca. 0.75 × 10⁶ lavage cells obtained from each uninfected mouse, about 90% were macrophages or lymphocytes in approximately equal proportion. T, B, and null (lyphocytes lacking theta or surface immunoglobulin markers) lymphocytes averaged 23, 9, and 7% of cells in these suspensions, respectively. After infection with either P3 or P9 virus, increased numbers of activated macrophages and lymphoblasts were observed. The major change during P3 infection was an increase in absolute numbers of null lymphocytes. In contrast, during P9 infection, T and B lymphocytes and macrophages progressively increased in absolute numbers while null cells decreased. These data suggest that cell-mediated immunological responses to influenza virus occur in the lung during infection, but that the responses to virulent and avirulent variants may differ both qualitatively and quantitatively.     

Influenza viruses in cell cultures

Summary The multiplication of seven influenza strains (2 of Type A, 2 of Type. A₁, 1 of Type A₂, 1 of Type B, and 1 of swine influenza) was followed in cultures of calf kidney cells maintained in a synthetic medium.Although none of the viruses had been previously passed in cell cultures, six of seven strains produced infective progeny in quantity, the yield per cell being as high as from the allantois of chick embryos.The rate of multiplication was slower than in eggs (doubling times ranging between 1.7 and 4 hours), and peak titres were attained on the third day after an inoculum of 100 ID₅₀.The virus released into the medium had an infectivity-to-haemagglutinin ratio comparable to virus grown in the allantois; cell-bound virus had an ID/HA ratio about tenfold lower.Part of the experiments reported in this paper were performed at the Department of Microbiology, John Curtin School of Medical Research, Australian National University, Canberra, Australia. Work carried out at Marburg was supported by the Deutsche Forschungsgemeinschaft.     

Dendritic and T Cell Response to Influenza is Normal in the Patients with X-Linked Agammaglobulinemia

Introduction

Influenza virus is a potential cause of severe disease in the immunocompromised. X-linked agammaglobulinemia (XLA) is a primary immunodeficiency characterized by the lack of immunoglobulin, B cells, and plasma cells, secondary to mutation in Bruton??s tyrosine kinase (Btk) gene. Btk is expressed in both B and dendritic cells (DC). However, little is known about the immune response of DC and T cells to influenza virus in XLA patients.

Methods

The in vitro maturation and antigen presenting function of monocyte-derived immature DC (imDC) from 12 XLA patients and 23 age-matched normal controls in response to influenza virus were examined. Influenza virus-specific CD4 and CD8 T cell responses in the patients and controls were further determined after administration of inactivated trivalent influenza vaccine.

Results

imDC from XLA patients had normal maturation based on major histocompatibility complex (MHC)-I, MHC-II, CD83 and CD86 expression, and interferon (IFN)-?? and interleukin-12 production upon influenza virus stimulation. They also had a normal capacity to induce allogeneic T cell proliferation in response to influenza virus. TIV was well tolerated in XLA patients. Influenza virus-specific CD4⁺IFN-??⁺ and CD8⁺ IFN-??⁺ T cells and HLA-A2/M1_58?C66-tetramer⁺ CTLs could be induced by TIV in XLA patients, and the levels and duration of maintaining these virus-specific cells in XLA patients are comparable to that in normal controls.

Conclusion

We demonstrated for the first time that XLA patients have fully competent DC and T cell immune responses to influenza virus. TIV is safe and could be an option for providing T cell-mediated protection against influenza virus infection in XLA patients.     

Influenza virus infection of newborn rats: Virulence of recombinant strains prepared from a cold-adapted,attenuated parent

Summary Infant rats were infected with one of a series of influenza A viruses. The growth of viruses in the turbinates or lungs, and the ability of virus infection to potentiate a subsequent bacterial infection by Haemophilus influenzae (HIb), were measured. The three virus strains known to be virulent for man grew to relatively high titres-of 10^5.0–10^6.8 EBID₅₀/ml in the turbinates of infant rats at 48 hours post-infection, and virus infection enhanced subsequent systemic infection following intranasal inoculation of rats with HIb. In contrast, influenza virus A/Ann Arbor/6/60—P17 and the three recombinant viruses prepared from this strain, all of which are attenuated for man, replicated to significantly lower titres of 10^2.6–10^4.1 EBID₅₀/ml in infant rats turbinates, and failed to promote systemic infection by HIb to the same degree. The results, together with those of previous studies, suggest that the behaviour of influenza viruses in infant rats may be an indication for virus virulence for man, and thus provide a test which could facilitate the development of live, attenuated virus vaccines.With 3 Figures     

Influenza virus infection of mouse lymphoblasts alters the binding affinity of anti-H-2 antibody: Requirement for viral neuraminidase

The requirement for histocompatibility in the T lymphocyte killing of virus-infected cells has led us to investigate the effect of influenza virus infection on mouse cell surface histocompatibility (H-2) antigens. Monoclonal anti-H-2 antibody made it possible to develop equilibrium binding conditions for the assay of H-2 antigen-antibody interactions on intact cells. Scatchard analysis of anti-H-2 binding with normal and virus-infected cells yielded linear curves indicating homogeneity of the interaction at varied concentrations of antibody through saturating levels. The estimated number of 2 × 10⁵ - 5 × 10⁵ H-2 antigenic sites per mouse lymphoblast does not appear to change during the course of influenza virus infection. However, the K_a (binding affinity constant) of anti-H-2 binding is rapidly elevated by virus infection (“0” time), continues to increase for 3 h post infection, then decreases. Control cells, treated with normal egg allantoic fluid, show no change in K_a during similar incubation. This change in K_a requires the presence of active viral neuraminidase. Thermal denaturation of the neuraminidase of the virus particles abolishes their ability to induce K_a alteration, even though hemagglutinin activity is retained. Treatment of cells with neuraminidase of bacterial origin led to an elevation of K_a, but did not mimic the viral effect in time dependence and magnitude of peak responses. The time-dependent lowering of K_a from peak values appeared to relate to virus replication, since UV light-inactivated virus-induced K_a elevation, but did not produce the typical K_a decline at 4-5 h post infection. The changes in K_a of anti-H-2 binding during influenza infection reflects a virus- induced alteration of the H-2 molecule or its environment in the host cell membrane. The molecular basis of this change and its relation to H-2-restricted recognition of influenza virus-infected cells by cytotoxic T cells requires further study.     

Influenza B Virus NB Glycoprotein Is a Component of the Virion

The influenza B virus NB glycoprotein is abundantly expressed at the surface of virus-infected cells. NB spans the membrane once and has an 18 amino acid ectodomain, a 22 amino acid transmembrane domain, and a 60 amino acid cytoplasmic tail. The NB N-terminal ectodomain contains two asparagine residues that are modified by the addition of N-linked carbohydrate chains, which become further modified by the addition of polylactosaminoglycan. We have now shown that NB is also modified by addition of palmitic acid. To determine if NB is incorporated into virions, metabolic labeling, immunoblotting, and immunogold electron microscopy techniques were used. NB was identified in virions grown in MDCK cells or in embryonated chicken eggs in two forms: (a) NB modified by addition of polylactosaminoglycan (NB_pl), and (b) a cleaved species (NB_c) that has a smaller molecular weight than unglycosylated NB (NB₁₂). Proteinase K digestion of purified virions converted NB_plto NB_c. Examination of virions purified by isopycnic centrifugation by electronmicroscopy and immunogold staining, using an affinity-purified antibody raised to a peptide derived from the NB cytoplasmic tail, showed staining for NB in influenza B virions. Quantification of the amount of NB in purified virions using two unrelated biochemical methods indicated there are on average approximately 15–100 molecules of NB per virion. Although the number of NB molecules incorporated on average into an influenza B virus particle is small, this finding is reminiscent of the number of molecules (14–68 monomers) found on average of the M₂integral membrane protein of influenza A virus.     

Influenza diagnosis and vaccination in Poland

In Poland between several thousand and several million cases of influenza and suspected influenza cases are registered, depending on the epidemic season. A variety of methods are available for the detection of the influenza viruses responsible for respiratory infection starting with the isolation of the virus in chick embryos or in cell lines such as MDCK, VERO, etc., and finishing with a variety of modifications of the classical PCR molecular biology such as PCR multiplex and Real-Time. The most effective way to combat influenza is through vaccination. Regular vaccination is one of the few steps that may be taken to protect individuals, especially in high-risk groups, from the potential and serious complications of influenza. In many countries, including Poland, despite the recommendations, the rate of vaccination against influenza is still low in all age groups. In the epidemic season 2011/2012, the level of distribution of the seasonal influenza vaccines was 4.5% of the population.     

Influenza virus emitted by naturally-infected hosts in a healthcare setting

BackgroundThe emergence of novel respiratory viruses such as avian influenza A(H7N9) virus and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) highlights the importance of understanding determinants of transmission to healthcare workers (HCWs) and the public.ObjectivesWe aim to determine the viral content of the air emitted by symptomatic inpatients or long-term care residents with laboratory-confirmed influenza virus infection (emitters), and in the breathing zones of healthcare workers who attend to them.DesignA prospective pilot study of patients with laboratory-confirmed influenza virus infection was undertaken. Air within 1 m of the patient was sampled using a high volume air sampler. In addition, a lower volume air sampler was placed <1 m from the patient, with another >1 m from the patient. Viral RNA was recovered from the samplers and submitted for quantitative real time PCR. In addition, personal button samplers were provided to HCWs.ResultsThe air emitted by 15 participants with laboratory-confirmed influenza virus infection was sampled. Of the patients infected with influenza A, viral RNA was recovered from the air emitted by 9/12 patients using the low-volume sampler; no viral RNA was detected from air emitted by patients with influenza B (n = 3). Influenza virus RNA was recovered from one HCW’s sampler.ConclusionsPatients with respiratory virus infection emit virus into the air which disperses to >1 m and may reach the breathing zone of a HCW. This pilot study highlights the feasibility and importance of conducting a larger-scale study to identify determinants of exposure and transmission from patient to HCW.     

Genetic divergence of Influenza A(H3N2) amino acid substitutions mark the beginning of the 2016–2017 winter season in Israel

BackgroundInfluenza vaccine composition is reevaluated each year due to the frequency and accumulation of genetic changes that influenza viruses undergo. The beginning of the 2016–2017 influenza surveillance period in Israel has been marked by the dominance of influenza A(H3N2).ObjectivesTo evaluate the type, subtype, genetic evolution and amino acid substitutions of influenza A(H3N2) viruses detected among community patients with influenza-like illness (ILI) and hospitalized patients with respiratory illness in the first weeks of the 2016–2017 influenza season.Study designRespiratory samples from community patients with influenza-like illness and from hospitalized patients underwent identification, subtyping and molecular characterization. Hemagglutinin sequences were compared to the vaccine strain, phylogenetic tree was created, and amino acid substitutions were determined.ResultsInfluenza A(H3N2) predominated during the early stages of the 2016–2017 influenza season. Noticeably, approximately 20% of community patients and 36% of hospitalized patients, positive for influenza₃), received the 2016–2017 influenza vaccine. The influenza A(H3N2) viruses demonstrated genetic divergence from the vaccine strain into three separate subgroups within the 3C.2a clade. One resembled the new 3C.2a1 subclade, one resembled the recently proposed 3C.2a2 subclade and the other was not previously described. Diversity was observed within each subgroup, in terms of additional amino acid substitutions.ConclusionsCharacterization of the 2016–2017 A(H3N2) influenza viruses is imperative for determining the future influenza vaccine composition.