Induction of influenza (H5N8) antibodies by modified vaccinia virus Ankara H5N1 vaccine.

Aim:The aim of the current study was to evaluate the efficacy of a trivalent-inactivated oil-emulsion vaccine against challenge by different clades highly pathogenic avian influenza (HPAI) viruses including HPAI-H5N8 and the virulent genotype VII Newcastle disease virus (NDV) (vNDV).Materials and Methods:The vaccine studied herein is composed of reassortant AI viruses rgA/Chicken/Egypt/ME1010/2016 (clade 2.2.1.1), H5N1 rgA/Chicken/Egypt/RG-173CAL/2017 (clade 2.2.1.2), and “NDV” (LaSota NDV/CK/Egypt/11478AF/11); all used at a concentration of 108 EID50/bird and mixed with Montanide-ISA70 oil adjuvant. Two-week-old specific pathogen free (SPF) chickens were immunized subcutaneously with 0.5 ml of the vaccine, and hemagglutination inhibition (HI) antibody titers were monitored weekly. The intranasal challenge was conducted 4 weeks post-vaccination (PV) using 106 EID50/0.1 ml of the different virulent HPAI-H5N1 viruses representing clades 2.2.1, 2.2.1.1, 2.2.1.2, 2.3.4.4b-H5N8, and the vNDV.Results:The vaccine induced HI antibody titers of >6log2 against both H5N1 and NDV viruses at 2 weeks PV. Clinical protection against all HPAI H5N1 viruses and vNDV was 100%, except for HPAI H5N1 clade-2.2.1 and HPAI H5N8 clade-2.3.4.4b viruses that showed 93.3% protection. Challenged SPF chickens showed significant decreases in the virus shedding titers up to <3log10 compared to challenge control chickens. No virus shedding was detected 6 “days post-challenge” in all vaccinated challenged groups.Conclusion:Our results indicate that the trivalent H5ND vaccine provides significant clinical protection against different clades of the HPAI viruses including the newly emerging H5N8 HPAI virus. Availability of such potent multivalent oil-emulsion vaccine offers an effective tool against HPAI control in endemic countries and promises simpler vaccination programs.

To the Editor: From early 2013 (1) through November 2014, >460 human cases of laboratory-confirmed avian influenza A(H7N9) virus infection occurred in China. Although human-to-human transmission of subtype H7N9 virus is not common, evidence has been reported of probable transmission among several family clusters (2), between 2 household contacts (3), and between a doctor and an infected patient (4). Taken together, these observations suggest that family members, health care providers, and other close contacts (hereafter called contacts) of H7N9-infected persons may be at risk for infection. In China, national guidelines regarding H7N9-infected patients call for observation of contacts for 7 days after exposure for signs and symptoms of infection and, if any occur, collection of throat swab specimens for testing by molecular assays (5). The guidelines do not call for serologic testing. Because human avian influenza infections may be mild or asymptomatic, we sought to determine whether serologic testing would show evidence of H7N9 virus infection among contacts of infected persons during the 2013–2014 epidemic in China. Contacts were defined in accordance with China’s guidelines for prevention and control of human H7N9 virus infection (5,6). The institutional review board of Wuxi Center for Disease Control and Prevention, Wuxi, Jiangsu Province, China, reviewed and approved this study. During the epidemic, we recruited contacts of patients in Wuxi and collected throat swab specimens when signs or symptoms of infection developed; serum samples were collected 2–3 weeks later. Swab specimens were tested for H7N9 virus by using real-time reverse transcription PCR (7). Serum samples were tested for antibodies against hemagglutinin antigens of 3 avian influenza viruses (A/Anhui/1/2013 [H7N9], A/Anhui/1/2005 [H5N1]-RG5, and A/chicken/Jiangsu/1/00 [H9N2]) (8) by using a horse erythrocyte hemagglutination inhibition (HI) assay and against the hemagglutinin antigens of 2 seasonal influenza viruses (A/California/07/2009 [H1N1] and A/Victoria/210/2009 [H3N2]) by using a turkey erythrocyte HI assay. Serum samples with HI titers >1:40 against H7N9 virus were confirmed positive by microneutralization assay. Ten laboratory-confirmed human infections with H7N9 virus occurred in Wuxi during March 29, 2013–May 15, 2014. In total, 225 contacts of 7 H7N9-infected patients were enrolled in the study (Table); contacts included 30 family members; 177 health care workers (54 physicians, 119 nurses who provided patient care with standard precautions, 2 hospital attendants, and 2 nurse assistants who provided services related to patient care, safety, and comfort, including anxiety relief, and medical observation); and 18 other contacts (8 friends who visited the patient in the hospital, 2 patients who shared the same room, and 8 patients who shared the same hospital area). The contacts of 3 other H7N9-infected patients declined to participate in the study. Table Demographic characteristics and HI antibody titers against influenza subtype H7N9, H5N1, H9N2, H1N1, and H3N2 viruses among close contacts of avian influenza A(H7N9)–infected persons, China, 2013–2014* Serologic assay results showed that, 14–28 days after their earliest exposure to an H7N9-infected patient, 22 (9.8%) contacts had elevated HI antibody titers (>1:40) against H7N9 virus; titers were 1:40 for 17 contacts and 1:80 for 5 contacts. Positive results for all 22 serum samples were validated by microneutralization assay; 15 (68.2%) samples had microneutralization antibody titers of >1:10 against H7N9 virus antigen (Table). Of the contacts with an HI titer of >1:80 and microneutralization titer of >1:40, 3 were nurses, 1 was a nurse assistant, and 1 was a family member (a patient’s daughter). All 5 of these contacts had antibody titers of 1:80 to seasonal H1N1 virus, 3 had titer of 1:80, and 1 each had titer of 1:160 or 1:640. Of the 225 contacts, 108 had HI titers >1:80 against seasonal H3N2 virus (1:80 for 63 contacts, 1:160 for 27 contacts, 1:320 for 9 contacts, and >1:640 for 8 contacts). All contacts had influenza subtype H5N1 and H9N2 antibody titers of <1:80. A previous epidemiologic study (2) reported the medical monitoring of 2,657 contacts of H7N9-infected patients in mainland China and found that, for 28 of the contacts, respiratory symptoms developed within 7 days after monitoring began. Results of molecular assay testing of throat swab specimens for H7N9 virus were negative for all 28 contacts; the study did not include serologic testing. However, small serologic survey studies in Taiwan (9) and household contacts in mainland China (10) showed no evidence of human-to-human transmission among contacts. A limitation of our study is that we did not collect serum samples from all contacts of infected persons or from controls; therefore, we could not assess the possibility of false-positive results or asymptomatic infections. However, our findings of elevated levels of subtype H7N9 antibody among 6.7% of contacts during this epidemic in China offer evidence that human-to-human transmission of H7N9 virus may occur among contacts of infected persons. Technical Appendix: Cross-reaction of hemagglutination inhibition titers of control serum samples to virus strains used in this study. Click here to view.(40K, pdf)

A two-phase combined measles-rubella vaccine (MR) immunization schedule was introduced for age 1 and prior to primary school entry in Japan in April 2006. Further immunization was also introduced for 13 (Phase 3) and 18-year-old (Phase 4) cohorts for the 5-year period from April 2008 to March 2013. We surveyed Phases 3 and 4 MR immunization immunogenicity and safety. From August 2007 to December 2009, we conducted 3 Phase 3 and 15 Phase 4 immunizations. We then took paired serum samples (pre- and 4-6 weeks post-immunization), and measured measles antibody titers using hemagglutination inhibition (HI) and neutralizing test (NT), and rubella antibody titers using HI. Pre-positive measles HI antibody titer (> or = 8) was 72% (13/18) and pre-positive measles NT antibody titer (> or = 2) was 100% (18/18). Post-positive measles HI and NT antibody titers were 94% (17/18) and 100% (18/18). Mean post-immunization measles HI and NT antibody titers were significantly higher than pre-titers, with four-fold or greater increases seen in 9 (50%) and 6 (33%) subjects. Pre-positive rubella HI antibody titer (> or = 8) was 94% (17/18), and post-positive rubella HI antibody titer 100% (18/18). Mean post-immunization rubella HI antibody titer was significantly higher than pre-titer, with four-fold or greater increases seen in 8 subjects (44%). Paired HI antibody titers were measured in pre- and post-Phase 1 immunization for measles in 3 subjects and for rubella in 2 subjects. Those with post-Phase 1 measles HI antibody titers of 32, 64, and 128 yielded titers of 16, 8, and < 8 pre-Phase 3 or Phase 4 immunization, showing antibody reduction or seronegative conversion. Those with post-Phase 1 rubella HI antibody titers of 128 and 256 yielded titers of 64 and 32 in pre-Phase 4 immunization, showing antibody reduction. Seroconversion or four-fold or greater increases in titer were seen post-immunization in 60% (3/5) of these subjects. A clinical reaction survey of all subjects 4 weeks post-immunization, showed only 1 case of mild fever and no local or systemic adverse reactions such as generalized urticaria or anaphylaxis. In conclusion, Phases 3 and 4 MR immunogenicity was satisfactory.

IntroductionHemagglutination (HA) and hemagglutination inhibition (HI) assays are conventionally used for detection and identification of influenza viruses. HI assay is also used for detection of antibodies against influenza viruses. Primarily turkey or chicken erythrocytes [red blood cells (RBCs)] are used in these assays, as they are large, nucleated, and sediment fast, which makes it easy to determine the titer. Human influenza viruses agglutinate RBCs from chicken, human, and guinea pig, but not from horse. Human influenza viruses bind preferentially to sialic acid (SA) linked to galactose (Gal) by α 2, 6 linkage (SA α 2, 6-Gal), whereas avian influenza (AI) viruses bind preferentially to SA α 2, 3-Gal linkages. With this background, the present study was undertaken to study erythrocyte binding preferences and receptor specificities of AI viruses isolated from India.Materials and methodsA total of nine AI virus isolates (four subtypes) from India and three reference AI strains (three subtypes) were tested in HA and HI assays against mammalian and avian erythrocytes. The erythrocytes from turkey, chicken, goose, guinea pig and horse were used in the study. The receptor specificity determination assays were performed using goose and turkey RBCs. The amino acids present at 190 helix, 130 and 220 loops of the receptor-binding domain of the hemagglutinin protein were analyzed to correlate amino acid changes with the receptor specificity.ResultsAll tested highly pathogenic avian influenza (HPAI) H5N1 viruses reacted with all five types of RBCs in the HA assay; AI H9N2 and H5N2 viruses did not react with horse RBCs. For H5N1 viruses guinea pig and goose RBCs were best for both HA and HI assays. For H9N2 viruses, guinea pig, fowl and turkey RBCs were suitable. For other tested AI subtypes, avian and guinea pig RBCs were better. Eight isolates of H5N1, one H4N6 and one H7N1 virus showed preference to avian sialic acid receptors. Importantly, two isolates of HPAI H5N1, H9N2 and H11N1 viruses showed receptor specificity preference to both avian and mammalian sialic acid (α-2, 3 and α-2, 6) receptors.ConclusionsUse of different types of RBCs resulted in titer variations in HA and HI assays. This showed that RBCs giving optimum HA and HI titers would increase sensitivity of detection and would be more appropriate for identification and antigenic analysis of AI viruses. Analysis of 16 amino acids in the receptor-binding domain of the hemagglutinin of HPAI H5N1 viruses revealed that the only variation observed was in S221P amino acid position. Two H5N1 viruses showed S221P amino acid change, out of which only one H5N1 virus showed preference to α 2, 6 sialic acid receptor. One H5N1 virus isolate with amino acid S at 221 position, showed preference to α 2,3 as well as α 2,6 sialic acid receptors. This indicated that factor(s) other than S221P mutation in the hemagglutinin are probably involved in determining receptor specificity of H5N1 viruses. This is the first report of receptor specificity and erythrocyte binding preferences of AI viruses from India.

Objective To analyze the immunostimulatory effects of cyclic dinucleotides (CDN) on immune responses to a nasal spray influenza split virus vaccine and to evaluate its potential as a mucosal adjuvant. Methods A H1N1 influenza split virus vaccine combined with different CDN was used for mouse immunization. Each mouse was intranasally immunized twice with 4.5 μg of hemagglutinin (HA) and 10 μg of CDN with an interval of 21 d. Titers of hemagglutination inhibition (HI) antibodies in serum, secretory IgA (sIgA) in bronchoalveolar lavage fluid and IgG in serum were detected 21 d after the last immunization. Immunostimulatory activities of different CDN were compared. Effects of cyclic di-GMP (c-di-GMP) and chitosan (CSN) on the immunogenicity of H1N1 and H7N9 influenza split vaccines were analyzed and compared. H1N1 influenza split vaccine combined with c-di-GMP or CSN was used to immunize mice. Three weeks after the last immunization, these mice were challenged with 10 times the median lethal dose (LD50) of A/Puerto Rico/8/34 (H1N1) influenza virus. Survival rates of the mice were observed for 14 d. Results All three CDN induced high levels of HI antibodies and IgG in serum and sIgA in BALF. HI antibody seroconversion rates were also higher than those of the control groups. c-di-GMP was superior to CSN in enhancing the immunogenicity of H1N1 and H7N9 antigens as higher titers of HI antibodies in serum and sIgA in BALF were induced. Conclusions CDN could enhance the immunogenicity of influenza antigens with better efficacy than CSN adjuvant. Key words: Cyclic dinucleotide; Adjuvant; Chitosan; Immunogenicity

Impact of repeated yearly vaccination on immune responses to influenza vaccine in an elderly population

The H5 and H7N9 subtypes of highly pathogenic avian influenza viruses (HPAIVs) in China pose a serious challenge to public health and the poultry industry. In this study, a replication competent recombinant influenza A virus of the Í5N1 subtype expressing the H7 HA1 protein from a tri-cistronic NS segment was constructed. A heterologous dimerization domain was used to combine with the truncated NS1 protein of 73 amino acids to increase protein stability. H7 HA1, nuclear export protein coding region, and the truncated NS1 were fused in-frame into a single open reading frame via 2A self-cleaving peptides. The resulting PR8-H5-NS1(73)H7 stably expressed the H5 HA and H7 HA1 proteins, and exhibited similar growth kinetics as the parental PR8-H5 virus in vitro. PR8-H5-NS1(73)H7 induced specific hemagglutination inhibition (HI) antibody against H5, which was comparable to that of the combination vaccine of PR8-H5 and PR8-H7. The HI antibody titers against H7 virus were significantly lower than that by the combination vaccine. PR8-H5-NS1(73)H7 completely protected chickens from challenge with both H5 and H7 HPAIVs. These results suggest that PR8-H5-NS1(73)H7 is highly immunogenic and efficacious against both H5 and H7N9 HPAIVs in chickens.Highlights:- PR8-H5-NS1(73)H7 simultaneously expressed two HA proteins of different avian influenza virus subtypes.- PR8-H5-NS1(73)H7 was highly immunogenic in chickens.- PR8-H5-NS1(73)H7 provided complete protection against challenge with both H5 and H7N9 HPAIVs.

Intranasal DNA Vaccination Induces Potent Mucosal and Systemic Immune Responses and Cross-protective Immunity Against Influenza Viruses

The current epizootic of H5N1 highly pathogenic avian influenza (HPAI) in poultry is unprecedented in its virulence, extent and longevity, raising global concern that the virus could mutate into a form easily transmitted between humans and initiate an influenza pandemic. The ability to rapidly and accurately diagnose infections with novel influenza subtypes is crucial to minimizing morbidity and mortality in humans and reducing the potential for a pandemic. However, questions remain about how to ensure validity of the currently available diagnostics, optimize their availability and the potential offered by new technologies. To address these questions, during 19–20 February 2007, more than 40 scientists, clinicians, researchers and industry representatives from around the world came together for the first World Health Organization (WHO) Consultation on Diagnosis of H5N1 Avian Influenza Infections in Humans (summary available at http://www.who.int/csr/disease/avian_influenza/guidelines/diagnosis_consultation/en/index.html). The meeting was co-organized by the WHO Global Influenza Programme (GIP), the International Society for Influenza and other Respiratory Viruses (ISIRV) and the Foundation for Innovative New Diagnostics (FIND). This marked the first time public and private sectors met at length to discuss this important issue. An 'open forum' meeting style was adopted, and substantial time was allotted for discussion. Overall, the consultation addressed: The 'state of the art' for H5N1 diagnostics in humans. Considerations and gaps related to H5N1 diagnostic capacity. Collaborative ways forward and the roles of WHO, private industry and other stakeholders. This meeting summary will present the discussions and recommendations generally agreed by the consultation participants. Diagnostic tests (to identify influenza virus in clinical material, containing cells and secretions and tissues) are based either on growth of virus in culture or by direct detection of virus antigen or RNA. Virus may be amplified in embryonated chicken eggs or mammalian cell culture, and then subjected to further testing for identification. Serological techniques [e.g. haemagglutination inhibition (HI) or microneutralization (MN)] may also be used to identify the presence of antibody in the serum of exposed individuals, providing indirect evidence of infection. These basic techniques can be used for diagnosing infections both in humans and in animals. In general, antigenic or molecular screening is used to first identify influenza virus type (A or B). Then the specific subtype is identified based on either serological reactivity of two viral surface glycoproteins, haemagglutinin (HA) and neuraminidase (NA), or on molecular characterization of the genes coding for these two proteins. There are 16 recognized HA and nine recognized NA subtypes of influenza A viruses. Wild waterfowl are considered the natural reservoir for influenza A viruses, and all HA and NA subtypes of influenza A have been identified in birds. Currently, only two influenza A subtypes (H1N1 and H3N2) are circulating or appearing in humans, causing recurring human seasonal influenza epidemics. Since the start of the current H5N1 HPAI epizootic in 2003, the virus has caused disease in poultry and wild birds in at least 59 countries in Asia, Africa, and Europe (http://www.oie.int). Although to date H5N1 remains an avian virus, it can cross the species barrier, and human infections with the avian H5N1 virus have now been confirmed in 12 countries.† † http://www.who.int/csr/disease/avian_influenza/country/cases_table_2007_07_25/en/index.html In addition to global concern about disease and deaths in humans, there is also concern that the virus will mutate into a form easily transmitted between humans, initiating a pandemic. The ongoing exposure of humans in countries experiencing disease in animals and ensuing global pandemic concern have highlighted some gaps and challenges in human influenza diagnostics. Appropriate clinical management, including timely treatment of human H5N1 cases‡ ‡ http://www.who.int/medicines/publications/WHO_PSM_PAR_2006.6.pdf , as well as plans for containing an emerging influenza pandemic,§ § http://www.who.int/csr/disease/avian_influenza/guidelines/draftprotocol/en/index.html rely on the ability to rapidly and accurately diagnose the virus in humans. Ensuring that effective influenza diagnostic systems are in place globally could be extremely cost effective. For example, it has been shown that although laboratory diagnosis represents a small percentage of medical centre costs, it leverages 60–70% of all critical decisions, e.g. admission, discharge and drug therapy.1Diagnosis of H5N1 in humans is not yet achievable in the vast majority of diagnostic laboratories. One challenge to rapid and accurate diagnosis is the continual evolution of influenza viruses.2 The eight RNA gene segments of influenza A viruses mutate at different rates.3 Specifically, the HA and NA genes, on which diagnostics depend, have high mutation rates compared to the other genes. This rapid evolution in the H5N1 viruses isolated since 1997 has resulted in the emergence of genetically and antigenically distinct lineages (http://www.WHOweblink.org). The circulating H5N1 viruses can currently be grouped into many different clades with four clades including viruses that have infected humans in the following countries:4 Clade 1 Thailand, Vietnam, Cambodia, China Clade 2.1 Indonesia Clade 2.2 China, Iraq, Azerbaijan, Turkey, Egypt, Nigeria, Djibouti Clade 2.3 China, Laos, Vietnam A second major challenge to global diagnostic capability is the availability of healthcare infrastructure to rapidly diagnose H5N1 infection at the initial point of care (POC), as the virus is circulating in many regions that lack existing diagnostic capacity, even for seasonal influenza. In practice, diagnosis of viral infections is conducted in several different environments, each having specific features, and therefore having somewhat different test requirements (Table 1). The third challenge is the uncertainty about the demand for tests for emerging influenza strains over the next months and years. Because the course of the H5N1 epizootic in animals and associated infections in humans cannot be predicted, it is possible that demand will decrease if the epizootic begins to be controlled in animals. It is also possible that demand will increase rapidly if there is suspected human-to-human transmission and the pandemic phase increases. Therefore, questions of stockpiling, reagent/kit shelf life, production times, etc. must be considered. The actual technical 'know how' for influenza diagnosis is fairly advanced, though this has not yet translated into significant innovation in rapid detection in field settings. Improvements are continually being made in both antigenic and molecular techniques for antigen and antibody detection, including development of increasingly simple-to-use tests (e.g. dipstick tests). Simpler techniques are required for routine diagnostic screening and sero-epidemiological studies in the field. Despite technological advances, however, the accuracy of H5N1 diagnoses relies heavily on the quality of the specimens collected and their preparation. If samples are not collected from patients early in the course of their infection and/or from sites where the viral load is high, or if samples are not handled, stored, and transported appropriately, false-negative tests may result irrespective of the validity of the test used. Approaches to collecting, preserving and shipping specimen for the diagnosis of avian influenza A (H5N1) have been summarized in a WHO document previously and are available at http://www.who.int/csr/resources/publications/surveillance/WHO_CDS_EPR_ARO_2006_1/en/. The basic diagnostic approaches, including benefits and constraints, are described below. Virus culture in eggs is traditionally regarded as the gold standard for amplifying and detecting avian influenza viruses. Cell culture can also be used for amplification with several lines (e.g. primary monkey kidney, MDCK, HeLa, MRC-5 or LLC-MK2) available, using tube culture, shell vial or multi-well plates. The cytopathic effect in cell culture to identify positives is not always distinctive; sensitivity of cell lines can vary for different strains, and there can be variation in the relative diagnostic yield from different techniques. Once cultured, virus can be easily detected and identified using techniques such as haemadsorption, antigen detection by immunofluorescence, other immunossays or haemagglutination (http://www.diagnosticdocweblink.org). Increasingly, polymerase chain reaction (PCR) is being used directly on original clinical samples, eliminating this virus isolation step for the purpose of diagnosis (see below). However, virus isolation as part of the diagnostic approach has the additional benefit of providing strains for further characterization, and vaccine development. The need for BSL-3 containment (BSL-3 enhanced or BSL-4 in some countries) for isolation and/or amplification of the HPAI H5N1 viruses constrains the use of virus isolation for diagnosis of this virus in many laboratories. The MN assay remains the gold standard for serological diagnosis of H5N1 infection in humans.4 Other methods include HI with use of horse red blood cells, complement fixation, single-radial haemolysis and enzyme immuno assay. Conventional HI tests that use turkey or chicken RBC have poor sensitivity for the detection of antibodies to avian influenza viruses including H5N1. However, the HI assay using horse red blood cells may be a suitable alternative for sero-diagnosis of some avian viruses (e.g. H5N1) but this may not apply to all avian influenza subtypes, highlighting the fact that significant strain/subtype differences exist. The international body of knowledge for serological diagnosis of H5 subtype infections is growing but information on other subtypes (e.g. H7) is limited. Although the methods for serological diagnosis differ in various laboratories, WHO does provide a set of standard criteria for serological diagnosis of human infection of avian influenza infection, i.e. a person meeting clinical definition of H5N1 case and one of the following:¶ ¶ http://www.who.int/csr/disease/avian_influenza/guidelines/case_definition2006_08_29/en/index.html Serological confirmation with appropriately timed paired sera. Greater than fourfold rise in neutralization antibody titre for H5N1. An MN antibody titre for H5N1 ≥1:80. A positive result using a different serological assay (e.g. A horse RBC HI titre of ≥1:160 or greater or H5-specific western blot positive result). There can be considerable variability in results on consecutive serological testing. Thus, negative and positive controls must always be included and samples/studies with low titre cut-off points should be interpreted with caution. Nonspecific reactivity of samples can be a problem. Modification techniques (e.g. serum adsorption) may be necessary to remove cross-reactive antibodies, especially when human infection with a novel avian subtype (such as H5) is reported. Nonspecific cross reactivity in patients 60–70 years of age can be seen when using the MN test.5 It remains unclear whether the cross-reactivity might be associated with some degree of protection in humans.6 Novel serological assays based on the use of engineered viruses with H5 antigen may allow 'neutralization' of H5N1 viruses to be carried out in a BSL-2 setting.7 As antibody response to H5N1 virus appears only in the second week of illness, serological tests cannot be used to detect early stages of influenza infection. Current serological tests are therefore most useful to identify mild or asymptomatic infections and epidemiologically assess populations at risk of exposure, such as family members and contacts of H5N1 case-patients, healthcare workers or co-workers and individuals exposed to infected domestic or wild birds. However, there is not much sero-epidemiological information being systematically collected globally. Follow-up investigations on specific outbreaks have yielded some data8, 9 but the extent of human exposure to H5N1 remains largely unknown. Immunofluorescence assays (both direct and indirect) can be used for detection of H5N1 antigen in samples, but rely heavily on specimen quality. While rapid, these methods are also dependent on the quality of fluorescence reagents and the expertise of the person interpreting the results of the tests and have inherently low sensitivity. Enzyme immunoassays in a micro-plate format are not widely used for human influenza diagnostics but the immuno-assay principle has been adapted for rapid antigen detection (rapid diagnostic tests) by flow-through or lateral flow devices. Sensitivity and specificity of antigenic tests depend not only on the test technique, but also on factors like type of specimen analysed, quality of specimen and timing of specimen collection (related to viral shedding).10 Based on published data, sensitivities for detection of human influenza H1N1 or H3N2 in rapid diagnostic tests are approximately 70–75% while specificities are approximately 90–99%. It should be noted that sensitivity of such methods for direct detection of H5N1 has been disappointing so far. The analytical sensitivity of currently available antigen detection test kits for influenza A remains too low for reliable use as POC tests for direct detection of H5N1 virus in clinical specimens. But if the sensitivity of such methods can be enhanced, they may become useful for H5N1 rapid testing.11 The use of molecular techniques to identify specific gene sequences provides a sensitive method for diagnosis. Furthermore, their use can potentially reveal the genetic sequence of the virus which is useful for molecular epidemiology and provides other important characteristics of the virus, including antiviral resistance status, occurrence of genetic reassortment or presence of key virulence mutations. While some of this information can be obtained by direct sequencing of PCR-amplified viral cDNA, more detailed molecular analysis typically requires prior virus amplification by culture. PCR is used widely now, with thermocyclers and other requisite equipment available in many national laboratories throughout affected regions although maintenance of the assays requires regular update of generic information. The multiple test steps (extraction, amplification, detection) and reagent preparation are highly sensitive to minor changes and requires experienced personal working within good quality systems. In particular, the amplification reaction of viral nucleic acids makes it susceptible to cross-contamination, unless stringent measures to avoid such contamination are in place.12 'Chip technology', which includes miniaturized approaches to genetic sequence detection may also allow simple, automated, rapid and economical PCR testing on a large scale, but automated systems are still expensive, and availability of a POC chip platform is at least 4 years away. Numerous sophisticated chip approaches to detection are available but all ultimately depend upon binding to specified virus sequences. As the viral mutation rate is high, it is important for all these approaches that constant surveillance of viral genetic sequence variations occurs, allowing adjustments to primers and probes. PCR can also be performed in a multiplex format for a panel of respiratory pathogens that is relevant to the differential diagnosis of AI and viral pneumonia (e.g. influenza B, parainfluenza 1, 2 and 3, respiratory syncytial virus, metapneumovirus, adenovirus, coronaviruses, mycoplasma and chlamydiae). A clinically and/or epidemiologically credible alternative diagnosis is useful in excluding AI. Closed tube real-time (RT) PCR systems that utilize fluorescent detectors are now widely available in a variety of formats including portable ones easily used in the field or for POC analysis. These show promise, but remain expensive for provincial or local laboratories and even though off the shelf reagents are available for detection of H5N1 strains, training of personnel and suitable laboratory environments are still crucial. Other molecular strategies are under development for rapid identification of influenza infections. For example, microarray and proteomic analysis of peripheral blood leucocytes or serum, respectively, may, in future, identify host response markers (e.g. gene response profiles, acute phase proteins, cytokines or other immune regulators) that may provide useful diagnostic signatures characteristic of groups of aetiological agents. During the consultation, a myriad of technical, political, economic and cultural issues were discussed. The following three general points emerged as being key to optimizing H5N1 diagnostics globally. In general, current technologies are adequate for the detection and characterization of diagnostic samples at the reference laboratory level, though advances in speed and miniaturization are occurring. There is however an acute need for field and POC tests that are relatively simple, sensitive and specific enough for use at referral hospitals and primary healthcare facilities. Such tests need to be to detect and between currently circulating strains of both avian influenza and seasonal influenza and enough to genetic changes in the For POC screening the sensitivity should be as high as possible to and tests should be The sensitivity of currently used rapid tests for H5N1 disease is from in the 1997 to in the and sensitivity does not always clinical sensitivity of diagnostic However, the poor clinical sensitivity of current POC tests for detecting H5N1 is not to a poor sensitivity for detecting H5N1 virus to human influenza but the poor analytical sensitivity for detecting influenza viral antigen in Furthermore, the of test also on the of the disease for test sensitivity and the positive for test will be and negative will be when influenza is rapid POC diagnostic with high sensitivity tests must be where it is for and shipping specimens to laboratories in the This may new techniques to be that into the challenges at many POC in affected In general, the ability to rapidly and accurately including human influenza has in though issues remain that the of many techniques. Appropriate collection may be including viral collection and and The specimens for virus detection have been summarized in the relevant WHO load in different clinical specimens in patients with H5N1 disease that are to and that respiratory specimens (e.g. are to be than respiratory specimens. There are with of specimens as well as A chain may be in and of and systems may not have been previously and the may not be may to to lack of and uncertainty of are to expensive when available, and may with a shelf and kits may or protection from which cannot be and may be of high (e.g. become or when may when have an even more shelf for may be In there is specific national to the reagents and There may be a lack of experienced lack of for training and a lack of training There may not be of the various assays and their use and (e.g. including of the different rapid detection While these may be and other emerging disease and public the need for diagnostic for influenza. of equipment is of an than is the lack of infrastructure to including for of the equipment and technical as well as international and may be in some and may not be possible in some laboratories, risk of of samples and risk of human human protection equipment may not be available, or may be used to training or (e.g. of protection level, of to and other equipment may be for the of new tests and reference strains for their quality are on a global level, from industry to assays and diagnostic As an international standard for H5N1 diagnostic test though has not yet been a relatively influenza gene (such as the infections with influenza A subtype can still be identified even in the of ongoing virus However, for identification of virus the reagents in diagnostic tests on either molecular sequences or must be continually to the currently circulating false-negative results can be and kits must therefore be easily to changes to allow detection of emerged strains and reagents should be continually identified by and be available WHO Influenza testing for both genes (e.g. to detect all influenza A strains with subtype specific tests the haemagglutinin of human and avian subtypes, one can avoid false-negative results of variations in the viral However, timely availability of viral and genetic sequence is a major to the and of reference reagents and Thus, ongoing surveillance of H5N1 viruses in animals and humans and global of are ultimately crucial to diagnostic test development and the validity of tests used. The Influenza influenza in countries and a of and laboratories by Currently, the H5N1 controls H5 H5 RNA and H5N1 has that one set and is not suitable for all and some diagnostic have specific Therefore, within Europe it is that different of reference reagents should be available, and primers and must be on each Currently, the WHO for Influenza at the for and in the provides domestic for its PCR influenza including of assays to laboratories and to other public laboratories, and of positive H5N1 to public laboratories in the at The and reagents are also available to international public laboratories. for human diagnostic test differ countries and from and to also vary different countries and some countries more than a to new diagnostic techniques. countries and/or for their although these may be time and expensive to especially for new technologies. International of requirements for could countries by providing both and industry a set of recognized should be based on risk and to public and be and International for including can be by following studies different laboratories. The of WHO International for avian influenza diagnosis should be to and subtype it may not be possible to international for H5N1 reagents and the of may need to be Serological test results are highly between laboratories. In to be to H5N1 results from different assays or laboratories, assays an standard may be more than an response can be and variation could be compared and Currently, the WHO is with including and on a virus neutralization to between laboratories H5 from show that the laboratories using and HI assays to test for H3N2 of the laboratories could not than results in In a of a quality for influenza virus detection and was by for Diagnostics in with the for Diagnostics of and some national reference laboratories. from within various sectors (e.g. reference laboratories, laboratories, and public were from that positives were Other challenges remain in detecting and of influenza virus, in influenza and influenza quality remain crucial to and document adequate and should be An important is the of positive H5N1 clinical samples for test from other for and should be and the various of samples, use of considered. International should be As the of avian influenza infections in humans it is important to that H5N1 remains a disease of animals. Although the for influenza testing are somewhat the and of diagnostic test techniques are for and human In the currently circulating strains in animals are still that will most as the virus has not yet adapted to humans. Therefore, the of of tests and as well as technical personnel in human and diagnostic laboratories should be As diagnosis of AI in animals is made on specimens where viral load is high and a only requires a animals from a to be confirmed as AI for relevant the sensitivity of POC tests is stringent that it is for diagnosis of human infection. surveillance and is to the risk factors for human infection with ongoing of the public sectors with the sectors and studies at the (e.g. birds in where H5N1 has poultry workers and poultry at should be national should not on laboratory but should in the collection of the surveillance in to strategies of and to the and of WHO and the public in to direct its and development for influenza diagnostics in the of the uncertainty of for H5 and influenza diagnostics. is considerable time and in new and diagnostic approaches and technologies for from use of to of methods to large or multiplex assay The public benefits when industry is in to ongoing and can by the required diagnostic analytical sensitivity and other clinical It should also be recognized that for of diagnostic clinical specimens of viral load to be in clinical are important in test and may in fact be to clinical specimens from patients which are a and and may be with multiple specimen should be to from such clinical specimens for test under the WHO International Health will be to rapid detection of human infections with influenza viruses in the now influenza reference laboratories, with of culture and influenza could diagnostic testing where national is The WHO Global Influenza Programme and its of Influenza and can a in this in by providing training and technical and global public benefit from of the following the of local and the for influenza testing at POC and in referral hospitals in regions and at risk development and of rapid, sensitive and specific POC screening tests for H5N1 infections in humans. collection of virus from animals and humans and their to reference laboratories in to be to currently circulating influenza strains and update tests the of reference laboratories in providing technical kits and reference should be between public and and available international of reagents and clinical are to from such specimens for test a global of avian influenza viruses in with development of the international of the of international for H5N1 diagnostic criteria and for all for new and including use of samples requirements a WHO working to the next steps in of reagents global of avian influenza viruses and for of H5N1 diagnostic tests

In general, avian influenza (AI) vaccines protect chickens from morbidity and mortality and reduce, but do not completely prevent, replication of wild AI viruses in the respiratory and intestinal tracts of vaccinated chickens. Therefore, surveillance programs based on serological testing must be developed to differentiate vaccinated flocks infected with wild strains of AI virus from noninfected vaccinated flocks in order to evaluate the success of vaccination in a control program and allow continuation of national and international commerce of poultry and poultry products. In this study, chickens were immunized with a commercial recombinant fowlpox virus vaccine containing an H5 hemagglutinin gene from A/turkey/Ireland/83 (H5N8) avian influenza (AI) virus (rFP-H5) and evaluated for correlation of immunological response by hemagglutination inhibition (HI) or agar gel immunodiffusion (AGID) tests and determination of protection following challenge with a high pathogenicity AI (HPAI) virus. In two different trials, chickens immunized with the rFP-H5 vaccine did not develop AGID antibodies because the vaccine lacks AI nucleoprotein and matrix genes, but 0%-100% had HI antibodies, depending on the AI virus strain used in the HI test, the HI antigen inactivation procedure, and whether the birds had been preimmunized against fowlpox virus. The most consistent and highest HI titers were observed when using A/turkey/Ireland/83 (H5N8) HPAI virus strain as the beta-propiolactone (BPL)-inactivated HI test antigen, which matched the hemagglutinin gene insert in the rFP-H5 vaccine. In addition, higher HI titers were observed if ether or a combination of ether and BPL-inactivated virus was used in place of the BPL-inactivated virus. The rFP-H5 vaccinated chickens survived HPAI challenge and antibodies were detected by both AGID and HI tests. In conclusion, we demonstrated that the rFP-H5 vaccine allowed easy serological differentiation of infected from noninfected birds in vaccinated populations of chickens when using standard AGID and HI tests.

Serologic evidence of influenza A(H1N1)pdm09 virus infection in northern sea otters.

Effect of omega-3 rich diet on the response of Japanese quails (Coturnix coturnix japonica) infected with Newcastle disease virus or avian influenza virus H9N2

BackgroundPre-existing antibodies to influenza virus neuraminidase may provide protection against infection influenza viruses containing novel hemagglutinin (HA). The aim of our study was to evaluate serum neuraminidase-inhibiting (NI) antibodies against А/California/07/2009(H1N1) [H1N1/2009pdm] and А/New Caledonia/20/1999(H1N1) [H1N1/1999] influenza viruses in relation with the age of participants and hemagglutination-inhibition (HI) antibody levels. Anti-H1N1/2009pdm neuraminidase and anti-H1N1/1999 neuraminidase antibody levels were measured in total 219 serum samples from Russian healthy peoples of various ages examined before and a year after pandemic strain appearance. We adjusted peroxidase-linked lectin micro-procedure to measure NI antibody titers using the reassortant A/H7N1 influenza viruses based on A/equine/Prague/1/56(H7N7). Also, HI antibody titers were estimated against H1N1/2009pdm, H1N1/1999 and a panel of seasonal A/H1N1 influenza viruses.ResultsIn sera samples collected during the fall of 2010, mean titers of specific HI and NI antibodies to H1N1/2009pdm were 2–2.1 times lower than antibody levels against H1N1/1999. Of the 163 individuals examined, 58 (35.6%) had NI anti-H1N1/2009pdm antibody titers > 1:20, compared to 93 (57.1%) who had NI anti-H1N1/1999 antibody titers > 1:20. There were low correlations between HI and NI antibody levels against either H1N1/1999 or H1N1/2009pdm in the same serum samples. The 24 adults born between 1957 and 1977 expressed very low levels of NI antibodies to A/H1N1 influenza viruses. Persons with low HI anti-H1N1/2009pdm titers but positive to seasonal A/H1N1 demonstrated significantly higher NI anti-A/H1N1 antibody titers than unexposed subjects. In 2005 cross-reactive NI anti-H1N1/2009pdm antibody titers > 1:20 were detected among 7.1% of young people.ConclusionsOur study confirmed that contact with seasonal influenza viruses may have contributed to generating the cross-reacting anti-H1N1/2009pdm NI antibodies which were detected in the sera of 18-20 years old people examined before the pandemic virus active circulation. The lowest levels of antibodies to the neuraminidase of N1 subtype were in the group of participants born during the circulation of influenza A/H2N2 or A/H3N2 viruses. The low correlation between HI and NI antibody titers suggests that NI antibody detection can be used as an additional test to evaluate the immune response after influenza infections or immunizations.

Viral Tropism and the Pathogenesis of Influenza in the Mammalian Host

Identifying hemagglutination inhibition (HI) antibody titers as a key immune correlate of protection (CoP) is crucial for developing, licensing, and monitoring the ongoing effectiveness of new influenza vaccines. Using a new statistical methodology, we explored the link between an inactivated quadrivalent influenza vaccine's impact on HI antibody titers and its effectiveness against A/H1N1-associated influenza illness. We utilized data from a phase 3, observer-blind, randomized, controlled trial in children aged 6-35 months to assess HI antibody titers as an immune CoP. The assessment used a statistical method developed within a causal inference framework and a new information-theoretic metric of surrogacy, the so-called individual causal association (ICA). The 75% and 85% uncertainty intervals of the ICA are 0.5511-0.8282 and 0.3632-0.8684, respectively, indicating a substantial reduction in the uncertainty about the vaccine's effect on the absence of infection when its impact on the HI antibody titers is known. The evaluation yielded evidence supporting the validity of HI antibody titers as a CoP for influenza infection.