Hemagglutinin Inhibition Test Research Articles

Discovery of Avian Paramyxoviruses APMV-1 and APMV-6 in Shorebirds and Waterfowl in Southern Ukraine.

Emerging RNA virus infections are a growing concern among domestic poultry industries due to the severe impact they can have on flock health and economic livelihoods. Avian paramyxoviruses (APMV; avulaviruses, AaV) are pathogenic, negative-sense RNA viruses that cause serious infections in the respiratory and central nervous systems. APMV was detected in multiple avian species during the 2017 wild bird migration season in Ukraine and studied using PCR, virus isolation, and sequencing. Of 4090 wild bird samples collected, mostly from southern Ukraine, eleven isolates were grown in ovo and identified for APMV serotype by hemagglutinin inhibition test as: APMV-1, APMV-4, APMV-6, and APMV-7. To build One Health's capacity to characterize APMV virulence and analyze the potential risks of spillover to immunologically naïve populations, we sequenced virus genomes in veterinary research labs in Ukraine using a nanopore (MinION) platform. RNA was extracted and amplified using a multiplex tiling primer approach to specifically capture full-length APMV-1 (n = 5) and APMV-6 (n = 2) genomes at high read depth. All APMV-1 and APMV-6 fusion (F) proteins possessed a monobasic cleavage site, suggesting these APMVs were likely low virulence, annually circulating strains. Utilization of this low-cost method will identify gaps in viral evolution and circulation in this understudied but important critical region for Eurasia.

The determination of haemagglutinin influenza antibodies in the Polish population in the epidemic season 2020/2021 during the SARS-CoV-2 pandemic

The aim of the study was to prove the level of antibodies against haemagglutinin in the sera of people from seven age groups in the epidemic season 2020/2021 in Poland to determine the differentiation of the antibody level and the protection rate depending on age. The level of anti-haemagglutinin antibodies was established by haemagglutinin inhibition test (HAI). A total of 700 randomly selected sera from people belonging to 7 different age groups were tested. The results confirmed the presence of antibodies against the following influenza antigens: A/Guangdong-Maonan/SWL1536/2019 (H1N1)pdm09-like virus, A/Hong Kong/2671/2019 (H3N2)-like virus, B/Washington/02/2019 (B/Victoria lineage)-like virus and B/Phuket/3073/2013 (B/Yamagata lineage)-like virus. The level of haemagglutinin antibodies varied between the studied age groups, with the highest values in the 5-9 age group and the lowest in the 0-4 age group. It was also proven that the protection rate was the highest for the A/Hong Kong/2671/2019(H3N2)-like virus antigen, which exceeded the protection level in the 5 age groups. Considering the very low percentage of people vaccinated in the epidemic season 2020/2021 in Poland, which amounted to only 6.1%, the results should be interpreted as the immune system's response to an infection with influenza virus.

The validation of Haemagglutinin Inhibition test for the detection of antibodies against canine influenza virus in a guinea pig serum model

The validation of Haemagglutinin Inhibition test for the detection of antibodies against canine influenza virus in a guinea pig serum model

Development of a cELISA for effective detection of the antibody against H7 subtype of avian influenza virus

H7 avian influenza viruses (AIVs) normally circulated among birds before. From 1996 to 2012, human infections with H7 AIVs (H7N2, H7N3, and H7N7) were reported in Canada, Italy, Mexico, the Netherlands, the United Kingdom and the USA. Until March 2013, human infections with H7N9 AIVs were reported in China. Since then, H7N9 AIVs have continued to circulate in both humans and birds. Therefore, the detection of antibodies against the H7 subtype of AIVs has become an important topic. In this study, a competitive enzyme-linked immunosorbent assay (cELISA) method for the detection of antibody against H7 AIVs was established. The optimal concentration of antigen coating was 5 μg mL−1, serum dilution was 1/10, and enzyme-labeled antibody was 1/3 000. To determine the cut-off value of cELISA, percent inhibition (PI) was determined by using receiver operating characteristic (ROC) curve analysis in 178 AIVs negative samples and 368 AIVs positive serum samples (n=546). When PI was set at 40%, the specificity and sensitivity of cELISA were 99.4 and 98.9%, respectively. This method could detect the antibodies against H7Nx (N1–N4, N7–N9) AIVs, and showed no reaction with AIVs of H1–H6 and H8–H15 subtypes or common avian viruses such as Newcastle disease virus (NDV), Infectious bronchitis virus (IBV) and Infectious bursal disease virus (IBDV), exhibiting good specificity. This method showed a coincidence rate of 98.56% with hemagglutinin inhibition (HI) test. And the repeatability experiment revealed that the coefficients of variation (CV) of intra- and inter-batch repetition were all less than 12%. The data indicated that the cELISA antibody-detection method established in this study provided a simple and accurate technical support for the detection of a large number of antibody samples of H7-AIV.

Re-current Epizootics of Highly Pathogenic Avian Influenza in Nigeria: Status of Vaccination as Alternate control

Epizootic of Highly Pathogenic Avian Influenza subtype H5N1 in Nigeria was successfully contained during the first wave that lasted from 2006 to 2008 without the use of vaccine. Re-current and more severe outbreak was witnessed in 2015 and there are suspicions that some farmers may have resorted to vaccination to prevent infections in their flocks. We investigate evidence of vaccination in farms and the status of vaccination as alternate control for HPAI in Nigeria. The study was carried out in a cross section of 24 commercial poultry farms in four States in South West and North Central Nigeria. Five hundred and one sera collected randomly were screened by agar gel immunodiffusion (AGID) assay for antibody to group specific influenza A nucleoprotein. One hundred and eight sera obtained from five H5N1 infected poultry farms were also concurrently screened. Reactive sera were further analysed by Hemagglutinin Inhibition (HI) test against H5 antigen using 1% suspension of pooled washed chicken red blood cells. Only 8 out of 501 sera (1.6%) had evidence of influenza A antibody. All of the 8 samples were from one farm with 20 samples collected representing 40% seroconversion at farm level. Three out of those sera were positive for H5 at HI titer of 3log2. All other sera including those obtained from HPAI infected farms were negative for influenza antibody. This study confirms limited antibody response to avian influenza subtype H5 most likely due to vaccination in one commercial flock. Vaccination against avian influenza by farmers desperate to protect their investments may lead to unregulated and suboptimal application of vaccines requiring farmers’ and stakeholders’ engagement to forestall negative impact.
 Keywords: Avian influenza; Control measures; Recurrent outbreaks; Vaccination status.

Acute Influenza A virus outbreak in an enzootic infected sow herd: Impact on viral dynamics, genetic and antigenic variability and effect of maternally derived antibodies and vaccination.

Influenza A virus (IAV) is a highly contagious pathogen in pigs. Swine IAV (swIAV) infection causes respiratory disease and is thereby a challenge for animal health, animal welfare and the production economy. In Europe, the most widespread strategy for controlling swIAV is implementation of sow vaccination programs, to secure delivery of protective maternally derived antibodies (MDAs) to the newborn piglets. In this study we report a unique case, where a persistently swIAV (A/sw/Denmark/P5U4/2016(H1N1)) infected herd experienced an acute outbreak with a new swIAV subtype (A/sw/Denmark/HB4280U1/2017(H1N2)) and subsequently decided to implement a mass sow vaccination program. Clinical registrations, nasal swabs and blood samples were collected from four different batches of pigs before and after vaccination. Virus isolation, sequencing of the virus strain and hemagglutinin inhibition (HI) tests were performed on samples collected before and during the outbreak and after implementation of mass sow vaccination. After implementation of the sow mass vaccination, the time of infection was delayed and the viral load significantly decreased. An increased number of pigs, however, tested positive at two consecutive sampling times indicating prolonged shedding. In addition, a significantly smaller proportion of the 10-12 weeks old pigs were seropositive by the end of the study, indicating an impaired induction of antibodies against swIAV in the presence of MDAs. Sequencing of the herd strains revealed major differences in the hemagglutinin gene of the strain isolated before- and during the acute outbreak despite that, the two strains belonged to the same HA lineage. The HI tests confirmed a limited degree of cross-reaction between the two strains. Furthermore, the sequencing results of the hemagglutinin gene obtained before and after implementation of mass sow vaccination revealed an increased substitution rate and an increase in positively selected sites in the globular head of the hemagglutinin after vaccination.

Outbreaks of highly pathogenic avian influenza in zoo birds caused by HA clade 2.3.4.4 H5N6 subtype viruses in Japan in winter 2016.

In late 2016, two zoos, one in northern Japan and the other in central Japan, experienced highly pathogenic avian influenza (HPAI) outbreaks, in which multiple zoo birds were infected with H5N6 subtype HPAI virus (HPAIV). Here, we report an overview of these HPAI outbreaks. HPAIV infections were confirmed by virus isolation in three black swans (Cygnus atratus) and three snowy owls (Bubo scandiacus) kept in the Omoriyama Zoo hospital. At Higashiyama Zoo and Botanical Gardens, following the death of a black swan at a zoo pond, nine waterfowl, including two black swans, four cackling geese (Branta hutchinsii leucopareia), two mallards (Anas platyrhynchos), and a wigeon (Anas penelope), died after HPAIV infection in isolation facilities. Based on the presence of H5-specific antibodies in their sera, two surviving black swans and a surviving mallard at Higashiyama Zoo appeared to have HPAIV infection, although the virus was not isolated. The detectable levels of antibodies (≥10 HI) were maintained for at least 5-9months, as determined by haemagglutinin inhibition test. Isolation of two H5N6 subtype HPAIVs from an open-air pond where affected zoo birds were previously housed at Higashiyama Zoo strongly indicates that wild waterfowl associated with aquatic environments brought the virus to the zoo. The phylogenetic relationships of the 18 isolates indicated direct viral transmission among birds within each zoo. In both zoos, containment of suspected birds in isolation facilities might have allowed the virus spread among birds inside the facility. However, maintaining containment measures and strict sanitation procedures could facilitate successful physical containment and clearance of HPAIV in both zoos.

A systematic review and meta-analysis of cross-reactivity of antibodies induced by H7 influenza vaccine

ABSTRACTInoculation with vaccine is the major intervention currently used to prevent influenza infections. However, it will be a challenge to produce and implement a new vaccine when a novel highly pathogenic influenza virus emerges in humans as significant infections. H7 subtype influenza viruses have similar epitopes on hemagglutinin, which can induce cross-reactive antibodies. In this study, a meta-analysis of the cross-reactivity of antibodies induced by one H7 subtype influenza vaccine against other H7 subtypes was performed. Database search was conducted in PubMed, Cochrane Library, EMBASE, MEDLINE, Chinese Biological Medicine Database (CBM), and Wanfang. A total of 9 articles comprising 811 human subjects were included in this meta-analysis. All assessed H7 influenza vaccines induced vaccine strain-specific protective antibodies [seroconversion rate (SCR) = 0.74, 95% CI (0.65, 0.82); seroprotection rate (SPR) = 0.81, 95% CI (0.78, 0.83)]. All H7 influenza virus monovalent vaccines exhibited cross-reactivity tested by hemagglutinin inhibition test (HI), microneutralization test (MN) and immunosorbent assay (ELISA) to other H7 subtype viruses. H7N1, H7N3, H7N7, and H7N9 vaccines elicited cross-reactive antibodies against other H7 subtype influenza viruses [SCR = 0.66, 95% CI (0.50, 0.82); SPR = 0.79, 95% CI (0.67, 0.91)]. The pooled SCR (95%CI) of cross-reactivity of H7N1 and H7N3 vaccines were 0.88 (0.85, 0.91) and 0.40 (0.26, 0.54), respectively. The consolidated SPR (95%CI) of H7N1 and H7N7 vaccines were 0.89 (0.86, 0.92) and 0.93 (0.81, 1.06). All H7 vaccines induced cross-reactive antibodies against H7N9 viruses [SCR = 0.69, 95% CI (0.52, 0.86); SPR = 0.85, 95% CI (0.76, 0.94)]. H7 vaccines can be used to limit influenza infection when a new highly pathogenic H7 virus appears.

Inoculation of fowlpox viruses coexpressing avian influenza H5 and chicken IL-15 cytokine gene stimulates diverse host immune responses

Fowlpox virus (FWPV) has been used as a recombinant vaccine vector to express antigens from several important avian pathogens. Attempts have been made to improve vaccine strains induced-host immune responses by coexpressing cytokines. This study describes the construction of recombinant FWPV (rFWPV) strain FP9 and immunological responses in specific-pathogen-free (SPF) chickens, co-expressing avian influenza virus (AIV) H5 of A/Chicken/Malaysia/5858/2004, and chicken IL-15 cytokine genes. Expression of H5 (50 kD) was confirmed by western blotting. Anti-H5 antibodies, which were measured by the haemagglutinin inhibition test, were at the highest levels at Week 3 post-inoculation in both rFWPV/H5- and rFWPV/H5/IL-15-vaccinated chickens, but decreased to undetectable levels from Week 5 onwards. CD3+/CD4+ or CD3+/CD8+T cell populations, assessed using flow cytometry, were significantly increased in both WT FP9- and rFWPV/H5-vaccinated chickens and were also higher than in rFWPV/H5/IL-15- vaccinated chickens, at Week 2. Gene expression analysis using real time quantitative polymerase chain reaction (qPCR) demonstrated upregulation of IL-15 expression in all vaccinated groups with rFWPV/H5/IL-15 having the highest fold change, at day 2 (117±51.53). Despite showing upregulation, fold change values of the IL-18 expression were below 1.00 for all vaccinated groups at day 2, 4 and 6. This study shows successful construction of rFWPV/H5 co-expressing IL-15, with modified immunogenicity upon inoculation into SPF chickens.

Prevalence of Multiple Subtypes of Avian Influenza Virus Antibodies in Egg Yolks of Mallards ( Anas platyrhynchos) and White-winged Terns ( Chlidonias leucopterus) in the Northeastern Republic of China.

Wild birds are natural hosts of avian influenza viruses (AIV) and can transmit viruses to poultry and other species. To monitor the prevalence of AIV antibodies, 211 eggs from wild Mallards ( Anas platyrhynchos) and 177 from wild White-winged Terns ( Chlidonias leucopterus) were collected from Zhalong Wetland and Xianghai Wetland in northeastern Republic of China from April to September, 2016. A hemagglutinin inhibition test detected the presence of H1, H3, H5, and H7 subtype-specific antibodies. The prevalences of AIV antibodies of subtypes H1 and H3 were relatively high while the prevalences of H5 and H7 AIV subtype antibody were low. In Zhalong Wetland, the prevalence of H1 AIV subtype antibody in Mallards was the highest, with a percentage of 11.0%. Prevalence of all AIV subtype-specific antibodies in Mallards was higher than those in White-winged Terns.

Predictive markers for humoral influenza vaccine response in patients with common variable immunodeficiency

A subgroup of patients with common variable immunodeficiencies (CVIDs) responds to vaccination. The aim of this study was to try to identify predictive markers for those with a humoral immune response after influenza vaccination. Forty-eight patients with CVID (29 female and 19 male patients; mean age, 57.7years) were vaccinated with the A(H1N1) influenza vaccine Pandemrix (GlaxoSmithKline, Wavre, Belgium) and boosted after 1month. Blood samples were collected before each vaccination and 2months later. Patients with a 4-fold titer increase in results on the hemagglutinin inhibition test (≥1:40) were considered responders and compared with nonresponders for clinical, immunologic, and genetic markers. Eight (16.7%) patients responded to the vaccination. Asignificantly higher proportion of the responders, who showed a EUROclass SmB-Trnorm21norm profile (P=.03) with a post-germinal center B-cell pattern (P=.04) in blood, experienced enteropathies (P=.04) compared with nonresponders. On the other hand, bronchiectasis was found exclusively among nonresponders (n=7), as was autoimmune cytopenia (n=5). Nonresponders with a EUROclass SmB-Trnorm21low profile (P=.02) had a significantly greater prevalence of progressive antibody deficiency (P=.048) and, at diagnosis, a higher mean serum IgM level (P=.03), lower mean serum IgG1 level (P=.007), expansion of absolute counts of cytotoxic CD8+ T cells (P=.033), and increased proportion of memory CD8+ T cells (P=.044) in blood. CVID-associated HLA markers were not detected in responders (P=.03). About one fifth of the patients with CVIDs achieved protective antibody levels after A(H1N1) vaccination and selected clinical, and immunologic markers were identified thatmight predict a positive outcome of influenza vaccination. Patients with CVID should be offered vaccination also against seasonal influenza because of the potential severity of the infection and risk for bacterial complications.

The Protective Effects of the A/ZJU01/ PR8/2013 Split H7N9 Avian Influenza Vaccine Against Highly Pathogenic H7N9 in BALB/c Mice

Background/Aims: Since the first case of novel H7N9 infection was reported, China has experienced five epidemics of H7N9. During the fifth wave, a highly pathogenic H7N9 strain emerged. In order to assess whether the H7N9 vaccine based on A/Zhejiang/DTID-ZJU01/2013(H7N9) was effective in protecting against highly pathogenic H7N9, we conducted this study. Methods: Groups of mice were immunized twice by intraperitoneal injection with 500 µl of either split vaccine alone or MF59-adjuvanted vaccine. Serum was collected 2 weeks after the second vaccine booster. The hemagglutinin inhibition test was conducted on vaccine seed and highly pathogenic H7N9 to evaluate the neutralization of highly pathogenic H7N9. We also immunized mice and challenged them with highly pathogenic H7N9. Mice were observed for illness, weight loss, and death at 1 week and 2 weeks post-infection. Then, the mice were sacrificed and lungs were removed. Antibody responses were assessed and pathological changes in the lung tissue were evaluated. Results: The ability of serum to neutralize highly pathogenic H7N9 was reduced. In mice, highly pathogenic H7N9 was more virulent than A/Zhejiang/DTID-ZJU01/2013(H7N9). After challenge with highly pathogenic H7N9, all mice died while mice challenged with A/Zhejiang/DTID-ZJU01/2013(H7N9) all recovered. The A/ZJU01/PR8/2013 split H7N9 avian influenza vaccine was able to protect against infection with highly pathogenic H7N9 in mice, with or without MF59. Moreover, H7N9 vaccine adjuvanted with MF59 produced high antibody levels, which lead to better protection. Conclusions: The A/ZJU01/PR8/2013 split H7N9 avian influenza vaccine based on A/Zhejiang/DTID-ZJU01/2013(H7N9) is effective in protecting against highly pathogenic H7N9. H7N9 vaccine adjuvanted with MF59 offers better protection against infection with highly pathogenic H7N9. In order to make the H7N9 vaccine applicable to humans, further clinical trials are required to evaluate MF59 adjuvanted vaccine. Meanwhile, the vaccine strain should be updated based on the highly pathogenic H7N9 gene sequence.

An oligothiophene compound neutralized influenza A viruses by interfering with hemagglutinin

Background and ObjectivesRecently influenza pandemic outbreaks were caused by emerging H5N1, H7N9 and H1N1 viruses. However, virucidal disinfectants are mainly unspecific and toxic. It is tactical to discover specific virucidal compounds. MethodsThe inhibitory potency was determined in H5N1 pseudovirus system; Interactions of compounds with hemagglutinin (HA) were detected with surface plasmon resonance (SPR) and further calculated with molecular docking. Virucidal effect was also estimated in influenza virus A/Puerto Rico/8/34(H1N1). Prevention efficacy was further estimated in mice model. ResultsOligothiophene compound 4sc was potently virucidal against H5N1 pseudovirus with selective index>1169 (IC50=0.17±0.01μM). Pseudovirus assay revealed 4sc may interact with HA. However, HA inhibition test indicated 4sc did not interact with receptor pocket in HA. SPR detection revealed 4sc interacted directly with HA and its HA2 subunits. Molecular docking analysis revealed that 4sc interacted with the cavity of HA2 stem region and HA1-HA2 interface which consist of 7 residues: L22, K262, G472 and F1102 in HA2; M241, E251 and N271 in HA1. 4sc also potently and irreversibly neutralized PR8 (H1N1) virus, causing 105.06±0.26 fold decrease of virus titer after exposure for 10min. 4sc blocked PR8 transmission to MDCK cells. Amazingly, virucidal effect of 4sc was not significantly reduced even at 4°C. Furthermore, 4sc blocked viral transmission to mice. ConclusionOligothiophene compound 4sc is a novel selective virucide of influenza virus, which blocks entry by interfering viral hemagglutinin. Due to promising safety profile and stable virucidal effect at 4°C, 4sc may be useful in disinfecting H5N1 and H1N1 influenza virus.

Circulation of Antibodies Against Influenza Virus Hemagglutinins in the 2014/2015 Epidemic Season in Poland.

The aim of this study was to determine the level of anti-hemagglutinin antibodies in the serum of people in different age-groups during the 2014/2015 epidemic influenza season in Poland. A total of 1050 sera were tested. The level of anti-hemagglutinin antibodies was determined using the hemagglutinin inhibition test. The results provided information on the incidence of circulating A/California/7/2009(H1N1)pdm09, A/Texas/50/2012(H3N2), and B/Massachusetts/2/2012 viruses. The level of antibodies against influenza differed between age-groups. The protection rate was the highest for the antigen B/Massachusetts/2/2012, with the decreasing order of values in the following age-groups: ≥65 years (76.7 %), 15-25 years (72.7 %), and 0-4 years (62.0 %). The average values of the protection rate in other age-groups were as follows: 43.3 % in 22-64 years, 40% in 5-9 years, and 39.3 % in 45-64 years of age, while the lowest value of 22.7 % was in 10-14 years old subjects. In the 2014/2015 epidemic season in Poland only were 3.6 % of the population vaccinated. That is why the presented results could be interpreted as a response of the immune system of patients after infection caused by influenza virus.

Prospective study of avian influenza H9 infection in commercial poultry farms of Punjab Province and Islamabad Capital Territory, Pakistan.

A prospective study was conducted from November 2013 to February 2014 to estimate the spatial clustering; cumulative incidence and risk factors associated with avian influenza (AI) subtype H9 infection on commercial poultry farms of Pakistan. A total of 400 farms were enrolled and followed during the study period. Among these, 109 farms submitted samples suspected for AI to the laboratory, and only 47 farms were confirmed positive by hemagglutinin inhibition (HI) test. Data was collected from these 109 farms about their demography, management, and biosecurity practices. The cumulative incidence of H9N2 was 11.75 % (95 % confidence interval (CI) 8.76–15.23). The highest number of cases (40.42 %) was reported in January. One most likely cluster (p = 0.009, radius = 4.61 km) occurred in the Kasur district. Multivariable logistic regression analyses showed that the presence of wild birds on the farms (odds ratio (OR) = 16.18; 95 % CI 3.94–66.45) was independently associated with H9N2 infection. Cleaning of cages before delivery on farm (OR = 0.16; 95 % CI = 0.06–0.47), presence of a footbath at the entrance of farm (OR = 0.24; 95 % CI 0.08–0.79), and changing of gloves (OR = 0.33; 95 % CI 0.11–0.99) were protective factors against H9N2 infection. Reducing the exposure to risk factors and adapting biosecurity measures may reduce the risk of AI H9N2 infection on commercial poultry farms in Pakistan.

Broad range of inhibiting action of novel camphor-based compound with anti-hemagglutinin activity against influenza viruses in vitro and in vivo

Influenza virus continues to remain one of the leading human respiratory pathogens causing significant morbidity and mortality around the globe. Due to short-term life cycle and high rate of mutations influenza virus is able to rapidly develop resistance to clinically available antivirals. This makes necessary the search and development of new drugs with different targets and mechanisms of activity. Here we report anti-influenza activity of camphor derivative 1,7,7-trimethylbicyclo[2.2.1]heptan-2-ylidene-aminoethanol (camphecene). In in vitro experiments it inhibited influenza viruses A(H1, H1pdm09, H3 and H5 subtypes) and B with EC50’s lying in micromolar range. Due to low cytotoxicity it resulted in high selectivity indices (74–661 depending on the virus). This effect did not depend on susceptibility or resistance of the viruses to adamantane derivatives amantadine and rimantadine. The compound appeared the most effective when added at the early stages of viral life cycle (0–2h p.i.). In direct hemagglutinin inhibition tests camphecene was shown to decrease the activity of HA’s of influenza viruses A and B. The activity of camphecene was further confirmed in experiments with influenza virus-infected mice, in which, being used orally by therapeutic schedule (once a day, days 1–5 p.i.) it decreased specific mortality of animals infected with both influenza A and B viruses (highest indices of protection 66.7% and 88.9%, respectively). Taken together, these results are encouraging for further development of camphecene-based drug(s) and for exploration of camphor derivatives as highly prospective group of potential antivirals.

Potency of an inactivated influenza vaccine prepared from A/duck/Hong Kong/960/1980 (H6N2) against a challenge with A/duck/Vietnam/OIE-0033/2012 (H6N2) in mice.

H6 influenza viruses are prevalent in domestic and wild birds in Eurasian countries and have been isolated from pigs and a human. To prepare for an influenza pandemic, we have established an influenza virus library consisting of more than 1,300 influenza virus strains, including 144 combinations of 16 hemagglutinin and 9 neuraminidase subtypes. H6 viruses in the library were classified into Early, Group II, Group III, and W312 sublineages and the North America lineage on the basis of their phylogenetic features. Chicken antisera to A/duck/Hong Kong/960/1980 (H6N2) of the Early sublineage broadly reacted with viruses of different sublineages in a hemagglutinin inhibition test. A whole inactivated virus particle vaccine was prepared from A/duck/Hong Kong/960/1980 (H6N2) which was stocked in the influenza virus library. The potency of this vaccine against A/duck/Vietnam/OIE-0033/2012 (H6N2), which belongs to a different sublineage, was evaluated in mice. The test vaccine was sufficiently potent to induce an immune response that reduced the impact of disease caused by a challenge with A/duck/Vietnam/OIE-0033/2012 (H6N2) in mice. The present results indicate that the whole inactivated virus particle vaccine prepared from a virus strain in the influenza virus library is useful as a vaccine against pandemic influenza.

Avian Influenza A(H7N9) Virus Screening in Patients with Fever and Flu-Like Symptoms in a Tertiary Hospital in an Area with Confirmed Cases

Novel avian influenza A(H7N9) virus was isolated in fatal patients in Yangtze River Delta of China in March 2013. We aimed to screen the virus in febrile patients in a tertiary hospital in an area with confirmed cases. Throat-swab specimens collected from consecutive patients with fever (≥38°C) and flu-like symptoms from April 15 to April 25, 2013 were subjected to detect novel avian influenza A(H7N9) virus with real-time PCR. The clinical outcomes in the patients and close contacts were followed up. Of total 200 patients screened, one (0.5%) was positive for avian influenza A(H7N9) virus and 199 others were negative. The infected patient experienced respiratory failure and had diffuse infiltrates in the right lower lobe in chest CT images. He received symptomatic and antibacterial treatments as well as oseltamivir. His condition was substantially improved within three days after admission; avian influenza A(H7N9) virus was not detected after 5 days' antiviral therapy. The hemagglutinin inhibition test showed that the serum titers against avian influenza A(H7N9) virus increased from <1∶20 at the early phase to 1∶80 at the convalescent phase. Follow-up of 23 close contacts showed that none of them developed fever and other symptoms within two weeks. Our findings suggest that although the infection rate of avian influenza A(H7N9) virus in patients with fever and flu-like symptoms is rare, the screening is valuable to rapidly define the infection, which will be critical to improve the clinical outcomes.

Protection from Clinical Disease Against Three Highly Virulent Strains of Newcastle Disease Virus After In Ovo Application of an Antibody–Antigen Complex Vaccine in Maternal Antibody–Positive Chickens

Worldwide, Newcastle disease (ND) remains one of the most economically important diseases of poultry. Current vaccination strategies for commercial poultry include the use of inactivated and live ND vaccines that typically induce protection against virulent field viruses. Here, we tested the efficacy of an antigen-antibody complex (AAC) ND vaccine delivered in ovo. Commercial maternal antibody-positive broiler chickens (Gallus domesticus) were vaccinated in ovo with an AAC vaccine composed of live B1-LaSota Newcastle disease virus (NDV) complexed with NDV-specific antiserum, and then they were challenged at weekly intervals after hatch. Challenge viruses included three exotic ND disease (END) viruses: the neurotropic strain Texas GB NDV-92-01 (TxGB) and two viscerotropic isolates, one isolate from the 2002-2003 outbreak in California (California 2002 isolate S212676 [CA]) and the other isolate from a 1997 END outbreak in South Korea (South Korea 94-147 [SK]). Results demonstrate that maternal antibody was able to provide approximately 50% protection in either vaccinated or control chickens at 7 days of age after TxGB challenge. However, with challenge at > or = 14 days, most control birds died, whereas all AAC-vaccinated birds were protected. Challenge with the CA or SK viruses in chickens at 28 days of age resulted in 100% protection of vaccinated birds, whereas all control birds died. In addition, AAC-vaccinated birds displayed decreased incidence of viral shedding in oral and cloacal swabs than control birds. Antibody titers were significantly (P < 0.05) higher in vaccinated chickens, as determined by enzyme-linked immunosorbent assay and hemagglutinin-inhibition tests, than in nonvaccinated controls. Together, these results demonstrate the efficacy of AAC vaccines delivered in ovo to protect commercial poultry.

Pandemic H1N1 Influenza Virus in Chilean Commercial Turkeys with Genetic and Serologic Comparisons to U.S. H1N1 Avian Influenza Vaccine Isolates

Beginning in April 2009, a novel H1N1 influenza virus caused acute respiratory disease in humans, first in Mexico and then around the world. The resulting pandemic influenza A H1N1 2009 (pH1N1) virus was isolated in swine in Canada in June 2009 and later in breeder turkeys in Chile, Canada, and the United States. The pH1N1 virus consists of gene segments of avian, human, and swine influenza origin and has the potential for infection in poultry following exposure to infected humans or swine. We examined the clinical events following the initial outbreak of pH1N1 in turkeys and determined the relatedness of the hemagglutinin (HA) gene segments from the pH1N1 to two H1N1 avian influenza (AI) isolates used in commercial turkey inactivated vaccines. Overall, infection of turkey breeder hens with pH1N1 resulted in -50% reduction of egg production over 3-4 weeks. Genetic analysis indicated one H1N1 AI vaccine isolate (Alturkey/North Carolina/17026/1988) contained approximately 92% nucleotide sequence similarity to the pH1N1 virus (A/Mexico/4109/2009); whereas, a more recent AI vaccine isolate (A/ swine/North Carolina/00573/2005) contained 75.9% similarity. Comparison of amino acids found at antigenic sites of the HA protein indicated conserved epitopes at the Sa site; however, major differences were found at the Ca2 site between pH1N1 and A/ turkey/North Carolina/127026/1988. Hemagglutinin-inhibition (HI) tests were conducted with sera produced in vaccinated turkeys in North Carolina to determine if protection would be conferred using U.S. AI vaccine isolates. HI results indicate positive reactivity (HI titer > or = 5 log2) against the vaccine viruses over the course of study. However, limited cross-reactivity to the 2009 pH1N1 virus was observed, with positive titers in a limited number of birds (6 out of 20) beginning only after a third vaccination. Taken together, these results demonstrate that turkeys treated with these vaccines would likely not be protected against pH1N1 and current vaccines used in breeder turkeys in the United States against circulating H1N1 viruses should be updated to ensure adequate protection against field exposure.