Influenzae Serotype Research Articles

Structural and functional characterization of neuraminidase-like molecule N10 derived from bat influenza A virus

The recent discovery of the unique genome of influenza virus H17N10 in bats raises considerable doubt about the origin and evolution of influenza A viruses. It also identifies a neuraminidase (NA)-like protein, N10, that is highly divergent from the nine other well-established serotypes of influenza A NA (N1-N9). The structural elucidation and functional characterization of influenza NAs have illustrated the complexity of NA structures, thus raising a key question as to whether N10 has a special structure and function. Here the crystal structure of N10, derived from influenza virus A/little yellow-shouldered bat/Guatemala/153/2009 (H17N10), was solved at a resolution of 2.20 Å. Overall, the structure of N10 was found to be similar to that of the other known influenza NA structures. In vitro enzymatic assays demonstrated that N10 lacks canonical NA activity. A detailed structural analysis revealed dramatic alterations of the conserved active site residues that are unfavorable for the binding and cleavage of terminally linked sialic acid receptors. Furthermore, an unusual 150-loop (residues 147-152) was observed to participate in the intermolecular polar interactions between adjacent N10 molecules of the N10 tetramer. Our study of influenza N10 provides insight into the structure and function of the sialidase superfamily and sheds light on the molecular mechanism of bat influenza virus infection.

Insights into Avian Influenza Virus Pathogenicity: the Hemagglutinin Precursor HA0 of Subtype H16 Has an Alpha-Helix Structure in Its Cleavage Site with Inefficient HA1/HA2 Cleavage

With a new serotype (H17) of hemagglutinin (HA) recently being discovered, there are now 17 serotypes (H1 to H17) of influenza A viruses in total. It is believed that HA is initially expressed as a precursor of HA0 and then cleaved into HA1 and HA2, forming a disulfide bond-linked complex, for its full function. Structural data show that a loop structure exists in the cleavage site between HA1 and HA2, and this flexible loop is crucial for the efficient cleavage of HA0. Here, the crystal structures of H16 (a low-pathogenicity avian influenza virus) in their HA0 form (H16HA0) have been solved at 1.7-Å and 2.0-Å resolutions. To our surprise, an α-helix element in the cleavage site which inserts into the negatively charged cavity with the key residue R329 hidden behind the helix was observed. In vitro trypsin cleavage experiments demonstrated inefficient cleavage of H16HA0 under both neutral and low-pH conditions. The results provide new insights into influenza A virus pathogenicity; both the relatively stable α-helix structure in the flexible cleavage loop and inaccessibility of the cleavage site likely contribute to the low pathogenicity of avian influenza A virus. Furthermore, compared to all of the HAs whose structures have been solved, H16 is a good reference for assigning the HA subtypes into two groups on the basis of the three-dimensional structure, which is consistent with the phylogenetic grouping. We conclude that in light of the current H16HA0 structure, the natural α-helix element might provide a new opportunity for influenza virus inhibitor design.

Evolving epidemiology of invasive Haemophilus infections in the post-vaccination era: results from a long-term population-based study

Historically, Haemophilus influenzae (Hi) serotype b (Hib) caused most invasive Haemophilus infections worldwide, mainly in children. In 1989 routine childhood vaccination against Hib was initiated in Iceland. We conducted a population-based study of all patients in the country with Haemophilus spp. isolated from sterile sites (n = 202), from 1983 to 2008. Epidemiology, clinical characteristics of the infections and serotypes of the isolates were compared during the pre-vaccination (1983–1989) and post-vaccination era (1990–2008). Following the vaccination, the overall incidence of Hib decreased from 6.4 to 0.3/100 000 per year (p <0.05) whereas the incidence did not change significantly for infections caused by Haemophilus sensu lato not serotype b, hereafter referred to as non-type b Hi (0.9 vs 1.2, respectively). The most frequent diagnosis prior to 1990 was meningitis caused by Hib, which was subsequently replaced by pneumonia and bacteraemia caused by non-type b Hi. Most commonly, non-type b Hi were non-typeable (NTHi; 40/59), followed by Hi serotype f (14/59) and Hi serotype a (3/59). Pregnancy was associated with a markedly increased susceptibility to invasive Haemophilus infections (RR 25.7; 95% CI 8.0–95.9, p <0.0001) compared with non-pregnant women. The case fatality rate for Hib was 2.4% but 14% for non-type b Hi, highest at the extremes of age. Hib vaccination gives young children excellent protection and decreases incidence in the elderly due to herd effect in the community. Replacement with other species or serotypes has not been noted. Pregnant women are an overlooked risk group.

Immune response and interaction of avian influenza serotype H9N2 and Newcastle disease vaccines in Broilers

Immune response and interaction of avian influenza serotype H9N2 and Newcastle disease vaccines in Broilers

A study of the variation in genetic diversity of Haemophilus Influenzae serotype b (Hib) strains in the UK between 1987 and 2010 using multilocus variable-number tandem repeat analysis (MLVA): Category: Lesson in Microbiology & Infection Control

A study of the variation in genetic diversity of Haemophilus Influenzae serotype b (Hib) strains in the UK between 1987 and 2010 using multilocus variable-number tandem repeat analysis (MLVA): Category: Lesson in Microbiology & Infection Control

Studies on prevalence of h7 and h9 serotypes of avian influenza in commercial layer and broilers in Karachi, Pakistan

Studies on prevalence of h7 and h9 serotypes of avian influenza in commercial layer and broilers in Karachi, Pakistan

Detection of bacterial pathogens in Mongolia meningitis surveillance with a new real-time PCR assay to detect Haemophilus influenzae

Since the implementation of Haemophilus influenzae (Hi) serotype b vaccine, other serotypes and non-typeable strains have taken on greater importance as a cause of Hi diseases. A rapid and accurate method is needed to detect all Hi regardless of the encapsulation status. We developed 2 real-time PCR (rt-PCR) assays to detect specific regions of the protein D gene (hpd). Both hpd assays are very specific and sensitive for detection of Hi. Of the 63 non-Hi isolates representing 21 bacterial species, none was detected by the hpd #1 assay, and only one of 2 H. aphrophilus isolates was detected by the hpd #3 assay. The hpd #1 and #3 assays detected 97% (229/237) and 99% (234/237) of Hi isolates, respectively, and were superior for detection of both typeable and non-typeable Hi isolates, as compared to previously developed rt-PCR targeting ompP2 or bexA. The diagnostic sensitivity and specificity of these rt-PCR assays were assessed on cerebrospinal fluid specimens collected as part of meningitis surveillance in Ulaanbaatar, Mongolia. The etiology (Neisseria meningitidis, Hi, and Streptococcus pneumoniae) of 111 suspected meningitis cases was determined by conventional methods (culture and latex agglutination), previously developed rt-PCR assays, and the new hpd assays. The rt-PCR assays were more sensitive for detection of meningitis pathogens than other classical methods and improved detection from 50% (56/111) to 75% (83/111). The hpd #3 assay identified a non-b Hi that was missed by the bexA assay and other methods. A sensitive rt-PCR assay to detect both typeable and non-typeable Hi is a useful tool for improving Hi disease surveillance especially after Hib vaccine introduction.

Effectiveness of the 2009 seasonal influenza vaccine against pandemic influenza A(H1N1)2009 in healthcare workers in New Zealand, June-August 2009

There is uncertainty whether the 2009 seasonal influenza vaccination influences the risk of infection with the 2009 pandemic influenza A(H1N1) virus. This issue was investigated in 548 healthcare workers from Capital and Coast District Health Board, Wellington, New Zealand, presenting with influenza-like illness during the influenza pandemic between June and August 2009. All workers completed an assessment sheet and had a nasopharyngeal swab tested by real-time RT-PCR. The risk of pandemic influenza A(H1N1) infection associated with the 2009 seasonal inactivated trivalent influenza vaccine was determined by logistic regression, with adjustment for potential confounding variables. In 96 workers pandemic influenza A(H1N1) RNA was detected and 452 tested negative. The multivariate analysis did not show any effect of vaccination on PCR-confirmed influenza A(H1N1)2009 infection (odds ratio 1.2, 95% confidence interval 0.7–1.9, p=0.48). We conclude that 2009 seasonal influenza vaccination had no protective effect against influenza A(H1N1)2009 infection amongst healthcare workers. To protect against further waves of the current pandemic influenza or future pandemics in which the influenza virus is antigenically distinct from contemporary seasonal influenza viruses, it would be necessary to vaccinate with a specific pandemic influenza vaccine, or a seasonal influenza vaccine that includes the pandemic influenza serotype.

Complicated Meningitis caused by a rare serotype of Haemophilus influenzae in Portugal

We report a case of meningitis due to Haemophilus influenzae serotype d strain in an infant. As far as we know, this is the first report of a serotype d strain, responsible for childhood invasive disease in Europe, demonstrating an emerging of H. influenzae non-b serotype, in the post-vaccination era.

Development of a capture ELISA to determine kinetics of soluble CD25 following in vitro and in vivo stimulation of duck peripheral blood monocytes

In humans and other mammals, the α-chain of interleukin-2 (IL-2) receptor (CD25) is induced and expressed on the cell surface after lymphocyte activation and is released from the membrane of activated cells as a smaller soluble form (sCD25). However, little is known about avian sCD25. In the present study, we developed an antigen capture enzyme-linked immunosorbent assay (AC-ELISA) to detect serum sCD25 in ducks, and we used flow cytometry (FCM) to analyze the frequency of CD25 + cells in the peripheral blood of ducks infected with H9N2 or H5N1 avian influenza virus (AIV) or serotype II Riemerella anatipestifer (RA). Using the AC-ELISA, duck sCD25 molecules were detected in the supernatant and lysates of concanavalin A (Con A)-stimulated peripheral blood mononuclear cells (PBMC), and in the serum of ducks infected with H5N1 virus and RA. However, no sCD25 was detected in the serum of H9N2 AIV-infected ducks. FCM analysis revealed that CD25 + cells were upregulated within the PBMC of RA-infected ducks throughout the experiment until death, while in the PBMC of H9N2- and H5N1 AIV-infected ducks, the frequency of CD25 + cells increased in the early stage of infection and then returned to a lower level. Our findings confirm that the dynamics of sCD25 and CD25 + cells are different in the peripheral blood of ducks infected with H9N2 virus, H5N1 virus, and RA.

Vector Insert-Targeted Integrative Antisense Expression System for Plasmid Stabilization

Some DNA vaccine and gene therapy vector-encoded transgenes are toxic to the E. coli plasmid production host resulting in poor production yields. For plasmid products undergoing clinical evaluation, sequence modification to eliminate toxicity is undesirable because an altered vector is a new chemical entity. We hypothesized that: (1) insert-encoded toxicity is mediated by unintended expression of a toxic insert-encoded protein from spurious bacterial promoters; and (2) that toxicity could be eliminated with antisense RNA-mediated translation inhibition. We developed the pINT PR PL vector, a chromosomally integrable RNA expression vector, and utilized it to express insert-complementary (anti-insert) RNA from a single defined site in the bacterial chromosome. Anti-insert RNA eliminated leaky fluorescent protein expression from a target plasmid. A toxic retroviral gag pol helper plasmid produced in a gag pol anti-insert strain had fourfold improved plasmid fermentation yields. Plasmid fermentation yields were also fourfold improved when a DNA vaccine plasmid containing a toxic Influenza serotype H1 hemagglutinin transgene was grown in an H1 sense strand anti-insert production strain, suggesting that in this case toxicity was mediated by an antisense alternative reading frame-encoded peptide. This anti-insert chromosomal RNA expression technology is a general approach to improve production yields with plasmid-based vectors that encode toxic transgenes, or toxic alternative frame peptides.

Necrotising fasciitis complicating Haemophilus influenzae type b epiglottitis in an adult

To present the first reported case of Haemophilus influenzae type b epiglottitis leading to necrotising fasciitis. Case report and review of the literature regarding the association of necrotising fasciitis with Haemophilus influenzae infection and with epiglottitis. A previously well, 64-year-old woman presented with epiglottitis, and subsequently developed necrotising fasciitis of her chest wall. The cause of both infections was Haemophilus influenzae serotype b. This organism has frequently been implicated in epiglottitis, but has not previously been reported to cause simultaneous necrotising fasciitis. The patient became critically ill, requiring intensive care management, but following surgical debridement and antibiotic treatment she made a full recovery. Although increasingly uncommon, clinicians must continue to be proficient in the diagnosis and management of epiglottitis, and to be aware of its full range of possible complications. This case report highlights a previously unknown and potentially fatal complication of Haemophilus influenzae type b epiglottitis.

Necrotizing Fasciitis of the Extremity Caused by Haemophilus influenzae Serotype b in a Healthy Adult

Haemophilus influenzae is a rare cause of necrotizing fasciitis with only a few previously reported cases. We describe the case of a 44-year-old man who had necrotizing fasciitis of the right lower extremity develop after intramuscular injections of paracetamol on his right buttock. The causative organism isolated was Haemophilus influenzae serotype b. This unusual infection was treated with repeated débridement and culture-guided antibiotics. There was no recurrence of infection at last followup 1 year after the initial surgery. Wound coverage was achieved with the use of a full-thickness skin graft after application of platelet-rich plasma and a dermal regeneration template. This resulted in a cosmetically acceptable appearance of the involved extremity for our patient.

Human influenza: a virus classification using a probabilistic neural network

PurposeThe traditional method to distinguish the serotype of influenza A virus was based on the antigen reaction of HA and NA with their antibodies. The antibody of specific subtype virus was difficult to get and the reaction was not easy to be done. To be a complementation to the traditional classification methods based on the serological reaction, aims to present a novel method for viral classification.Design/methodology/approachThe similarity values of all subtype HA genes in vector space were considered and classified using a probabilistic neural network (PNN). The PNN model was trained by the 132 viral sequences in the training set and the classification quality was examined using 28 viral sequences in the testing set.FindingsA novel technique for the serotype classification of influenza A virus isolated from human was proposed in the paper. The system achieved 100 per cent accuracy with all serotypes of human influenza A virus.Research limitations/implicationsThe time for the large‐scale calculations of the average similarity based on multisequence alignment is the main limitation.Practical implicationsThis is a supplementation to the traditional virology research.Originality/valueThe novel classification method based on the similarity of viral nucleotide sequences and the PNN model would be useful for the epidemic supervision and prevention.

B Cells Promote Resistance to Heterosubtypic Strains of Influenza via Multiple Mechanisms

Immunity to heterosubtypic strains of influenza is thought to be mediated primarily by memory T cells, which recognize epitopes in conserved proteins. However, the involvement of B cells in this process is controversial. We show in this study that influenza-specific memory T cells are insufficient to protect mice against a lethal challenge with a virulent strain of influenza in the absence of B cells. B cells contribute to protection in multiple ways. First, although non-neutralizing Abs by themselves do not provide any protection to challenge infection, they do reduce weight loss, lower viral titers, and promote recovery of mice challenged with a virulent heterosubtypic virus in the presence of memory T cells. Non-neutralizing Abs also facilitate the expansion of responding memory CD8 T cells. Furthermore, in cooperation with memory T cells, naive B cells also promote recovery from infection with a virulent heterosubtypic virus by generating new neutralizing Abs. These data demonstrate that B cells use multiple mechanisms to promote resistance to heterosubtypic strains of influenza and suggest that vaccines that elicit both memory T cells and Abs to conserved epitopes of influenza may be an effective defense against a wide range of influenza serotypes.

Invasive Infections Caused by Haemophilus influenzae Serotypes in Twelve Canadian IMPACT Centers, 1996–2001

Haemophilus influenzae type b (Hib) immunization has changed the epidemiology of pediatric bacterial invasive disease. We describe the epidemiology of H. influenzae invasive infections in 12 Canadian pediatric tertiary care [Immunization Monitoring Program, ACTive (IMPACT)] centers during the era of universal immunization against this pathogen. Children with positive cultures for H. influenzae serotypes a to f (Hia to Hif) and nontypable H. influenzae from sterile sites were identified from the laboratory records at 12 IMPACT centers from January 1, 1996 to December 31, 2001. Hospital records were retrospectively reviewed for demographic and clinical information. Of 166 H. influenzae cases, 58 (35%) were caused by Hib, 89 (54%) by non-b serotypes, and 19 (11%) were not serotyped. The non-b serotypes included: 25 Hia (28%), 4 Hid (4%), 2 Hie (2%), 11 Hif (12%), and 47 were nontypable isolates (53%). For patients with Hib and Hia infection, meningitis was the most common presentation, accounting for 40% and 52% respectively, whereas the most common presentation for nontypable serotypes was pneumonia, seen in 43% of cases. Epiglottitis was associated mainly with Hib. Aboriginal ethnicity was an important risk factor for Hia cases, accounting for 76% of patients with infections caused by this serotype. Mean duration of hospitalization, need for admission to a pediatric intensive care unit, and case fatality rates were similar for the cases because of Hib, Hia, Hif, and nontypable serotypes. In 1996-2001, two-thirds of H. influenzae invasive disease in the 12 IMPACT centers was caused by non-b serotypes, which were associated with significant morbidity and mortality.

Synthetic multi‐epitope peptides identified in silico induce protective immunity against multiple influenza serotypes

Influenza causes yearly epidemics of mild disease and, occasionally, pandemics with millions of fatalities. Currently, no vaccine is effective against all influenza strains. Analysis of influenza sequences from animal and human isolates using CLUSTALW and a novel proprietary epitope prediction algorithm identified six conserved T cell-reactive regions in several proteins. Immunisation of transgenic mice with a preparation of these six regions as chemically synthesised peptides (FLU-v) induced a specific HLA-A*0201-mediated CD8(+) T cell response. This T cell population also reacted against human cells infected with three non-related influenza strains, confirming that the identified regions contain epitopes naturally presented by infected human cells and conserved amongst non-related viruses. Moreover, FLU-v immunisation significantly increased survival of transgenic mice against lethal challenge with influenza. Overall, FLU-v represents a promising influenza vaccine candidate, obviating the need for yearly vaccinations and allowing the stockpiling and initiation of a worldwide vaccination program in advance of a pandemic outbreak.

Hemagglutinin (HA) Proteins from H1 and H3 Serotypes of Influenza A Viruses Require Different Antigen Designs for the Induction of Optimal Protective Antibody Responses as Studied by Codon-Optimized HA DNA Vaccines

Effective antibody responses provide crucial immunity against influenza virus infection. The hemagglutinin (HA) protein is the major target of protective antibody responses induced by viral infection and by vaccination with both inactivated and live-attenuated flu vaccines, but knowledge about the optimal designs of protective HA antigens from different flu serotypes is still limited. In this study, we have significantly improved the immunogenicity of HA-expressing DNA vaccines by using codon-optimized HA sequences for either an H1 serotype (A/NewCal/20/99) or an H3 serotype (A/Panama/2007/99) human influenza A virus and then used these constructs as model antigens to identify the optimal HA antigen designs to elicit high-level protective antibody responses. Two forms of HA antigen, a wild-type, full-length HA and a secreted form with transmembrane (TM) domain-truncated HA, were produced. Both forms of HA DNA vaccines, from either H1 or H3 serotypes, were able to elicit high levels of HA-specific immunoglobulin G responses in immunized rabbits as measured by enzyme-linked immunosorbent assay. Interestingly, the abilities of H1 HA and H3 HA antigens to elicit hemagglutination inhibition (HI) and neutralizing antibody (NAb) responses differ. For the H1 HA antigens, the full-length HA induced significantly higher HI and NAb responses than did the TM-truncated HA. For the H3 HA antigen, both the full-length HA and TM-truncated HA induced high levels of HI and NAb responses. These data indicate that H1 and H3 antigens have different expression requirements for the induction of an optimal protective antibody response and that the structure integrity of HA antigens is critical for eliciting type-specific protective antibody responses. Our findings will have an important impact on future subunit-based flu vaccine development.

Comparison of two slide agglutination serotyping methods and PCR-based capsule typing for the characterization of Haemophilus influenzae serotypes

Ninety-three nasopharyngeal Haemophilus influenzae isolates were serotyped by two slide agglutination methods (SAST 1 and SAST 2) and the results compared with those obtained by capsule type-specific PCR. SAST 1 presented a low correlation with results obtained by PCR (75.2%) while SAST 2 showed a better agreement with the molecular technique results (93.5%). These findings suggest that SAST 2 could be an alternative method for adequate detection of H.influenzae type b.

Carriage and serotypes of haemophilus influenza in young children attending a kindergarten in Hangzhou

目的 了解健康儿童不同月份流感嗜血杆菌的携带状况及血清分型.方法对2002年9月至2003年6月杭州某甲级幼儿园2～5岁的入托儿童随机进行每周1次的咽拭子培养,用V因子、X因子试验鉴定流感嗜血杆菌,用玻片凝集法进行血清分型,用纸片扩散法进行药敏试验.结果连续10个月中受检儿童848例,217例(占25.6%)携带流感嗜血杆菌,其中1月份的携带率最高,达34.8%,6月份的携带率最低,为12.6%,儿童在12月、1月、2月、3月4个月中的总携带率显著高于其他6个月(χ2=10.95,P<0.001).217例携带者共分离到223株流感嗜血杆菌,不可分型菌株180株,占80.7%;可分型菌株占19.3% (43株),包括a型4株,c型2株,d型35株,e型1株和f型1株,未分离到b型流感嗜血杆菌.61株(27.4%)菌株产生β内酰胺酶,28.6%的菌株对氨苄西林耐药,且不可分型菌株对氨苄西林的耐药率显著高于可分型菌株(χ2=4.19,P<0.05).结论本研究中健康儿童对流感嗜血杆菌的携带率以12月、1月、2月和3月份为高,可分型流感嗜血杆菌以d型占绝对优势,新型菌苗的研制已成为一项紧迫的任务。