H5 Subtype Viruses Research Articles

Highly Pathogenic and Low Pathogenic Avian Influenza H5 Subtype Viruses in Wild Birds in Ukraine.

There have been three waves of highly pathogenic avian influenza (HPAI) outbreaks in commercial, backyard poultry, and wild birds in Ukraine. The first (2005-2006) and second (2008) waves were caused by H5N1 HPAI virus, with 45 outbreaks among commercial poultry (chickens) and backyard fowl (chickens, ducks, and geese) in four regions of Ukraine (AR Crimea, Kherson, Odesa, and Sumy Oblast). H5N1 HPAI viruses were isolated from dead wild birds: cormorants (Phalacrocorax carbo) and great crested grebes (Podiceps cristatus) in 2006 and 2008. The third HPAI wave consisted of nine outbreaks of H5N8 HPAI in wild and domestic birds, beginning in November 2016 in the central and south regions (Kherson, Odesa, Chernivtsi, Ternopil, and Mykolaiv Oblast). H5N8 HPAI virus was detected in dead mute swans (Cygnus olor), peacocks (Pavo cristatus) (in zoo), ruddy shelducks (Tadorna ferruginea), white-fronted geese (Anser albifrons), and from environmental samples in 2016 and 2017. Wide wild bird surveillance for avian influenza (AI) virus was conducted from 2006 to 2016 in Ukraine regions suspected of being intercontinental (north-south and east-west) flyways. A total of 21 511 samples were collected from 105 species of wild birds representing 27 families and 11 orders. Ninety-five avian influenza (AI) viruses were isolated (including one H5N2 LPAI virus in 2010) from wild birds with a total of 26 antigenic hemagglutinin (HA) and neuraminidase (NA) combinations. Fifteen of 16 known avian HA subtypes were isolated. Two H5N8 HPAI viruses (2016-2017) and two H5N2 LPAI viruses (2016) were isolated from wild birds and environmental samples (fresh bird feces) during surveillance before the outbreak in poultry in 2016-2017. The Ukrainian H5N1, H5N8 HPAI, and H5N2 LPAI viruses belong to different H5 phylogenetic groups. Our results demonstrate the great diversity of AI viruses in wild birds in Ukraine, as well as the importance of this region for studying the ecology of avian influenza.

An avian influenza virus H6N1 outbreak in commercial layers: case report and reproduction of the disease

ABSTRACTAn outbreak of low pathogenic avian influenza (LPAI) subtype H6N1 (intravenous pathogenicity index = 0.11) infection occurred in four productive brown layer flocks on three farms in the Netherlands within a period of two months. The farms were located at a maximum distance of 4.6 km from each other. The infections were associated with egg production drops up to 74%, pale eggshells and persisting high mortality up to 3.2% per week. Three flocks were slaughtered prematurely as they were not profitable anymore. Newcastle disease, infectious bronchitis, egg drop syndrome and Mycoplasma gallisepticum infections could very likely be excluded as cause of or contributor to the condition in the field. Also, the anticoccidial drug nicarbazin, which can cause egg production drops and eggshell decolouration, was not detected in eggs from affected flocks. Furthermore, post mortem examinations revealed no lesions indicative of bacterial infection. Moreover, bacteriological analysis of hens was negative. The condition was reproduced in commercial brown layers after intratracheal inoculation with virus isolates from affected flocks. It is concluded that the LPAI H6N1 virus is very likely the only cause of the disease. An overview of main manuscripts published since 1976 describing non-H5 and non-H7 avian influenza (AI) virus infections in chickens and their biological significance is included in the present study, in which once more is shown that not only high pathogenic AI virus subtypes H5 and H7 can be detrimental to flocks of productive layers, but also non-H5 and non-H7 LPAI viruses (H6N1 virus).RESEARCH HIGHLIGHTSLPAI H6N1 can be detrimental to productive layersDetrimental effects are severe egg drop and persistent high mortalityLPAI H6N1 virus outbreak seems to be self-limiting

Influenza A Prevalence and Subtype Diversity in Migrating Teal Sampled Along the United States Gulf Coast.

Wild birds in the order Anseriformes are important reservoirs for influenza A viruses (IAVs); however, IAV prevalence and subtype diversity may vary by season, even at the same location. To better understand the ecology of IAV during waterfowl migration through the Gulf Coast of the United States (Louisiana and Texas), surveillance of blue-winged (Spatula discors) and American green-winged (Anas carolinensis) teal was conducted. The surveillance was done annually during the spring (live capture; 2012-17) and fall (hunter harvested; 2007-17) at times inferred to coincide with northward and southward movements, respectively, for these waterfowl species. During spring migration, 266 low pathogenicity (LP) IAV positive samples were recovered from 7547 paired cloacal-oropharyngeal (COP) samples (prevalence, 3.5%; annual range, 1.3%-8.4%). During fall migration, 650 LP IAV-positive samples were recovered from 9493 COP samples (prevalence, 6.8%; annual range, 0.4%-23.5%). Overall, 34 and 20 different IAV subtypes were recovered during fall and spring sampling, respectively. Consistent with previous results for fall migrating ducks, H3 and H4 hemagglutinin (HA) subtypes were most common; however, H4 subtype viruses predominated every year. This is in contrast to the predominance of LP H7 and H10 HA subtype viruses during spring. The N6 and N8 neuraminidase subtypes, which were usually associated with H4, were most common during fall; the N6 subtype was not recovered in the spring. These consistent seasonal trends in IAV subtype detection in teal are currently not understood and highlight the need for further research regarding potential drivers of spatiotemporal patterns of infection, such as population immunity.

Highly Pathogenic and Low Pathogenic Avian Influenza H5 Subtype Viruses in Wild Birds in Ukraine.

There have been three waves of highly pathogenic avian influenza (HPAI) outbreaks in commercial, backyard poultry, and wild birds in Ukraine. The first (2005-2006) and second (2008) waves were caused by H5N1 HPAI virus, with 45 outbreaks among commercial poultry (chickens) and backyard fowl (chickens, ducks, and geese) in four regions of Ukraine (AR Crimea, Kherson, Odesa, and Sumy Oblast). H5N1 HPAI viruses were isolated from dead wild birds: cormorants (Phalacrocorax carbo) and great crested grebes (Podiceps cristatus) in 2006 and 2008. The third HPAI wave consisted of nine outbreaks of H5N8 HPAI in wild and domestic birds, beginning in November 2016 in the central and south regions (Kherson, Odesa, Chernivtsi, Ternopil, and Mykolaiv Oblast). H5N8 HPAI virus was detected in dead mute swans (Cygnus olor), peacocks (Pavo cristatus) (in zoo), ruddy shelducks (Tadorna ferruginea), white-fronted geese (Anser albifrons), and from environmental samples in 2016 and 2017. Wide wild bird surveillance for avian influenza (AI) virus was conducted from 2006 to 2016 in Ukraine regions suspected of being intercontinental (north-south and east-west) flyways. A total of 21 511 samples were collected from 105 species of wild birds representing 27 families and 11 orders. Ninety-five avian influenza (AI) viruses were isolated (including one H5N2 LPAI virus in 2010) from wild birds with a total of 26 antigenic hemagglutinin (HA) and neuraminidase (NA) combinations. Fifteen of 16 known avian HA subtypes were isolated. Two H5N8 HPAI viruses (2016-2017) and two H5N2 LPAI viruses (2016) were isolated from wild birds and environmental samples (fresh bird feces) during surveillance before the outbreak in poultry in 2016-2017. The Ukrainian H5N1, H5N8 HPAI, and H5N2 LPAI viruses belong to different H5 phylogenetic groups. Our results demonstrate the great diversity of AI viruses in wild birds in Ukraine, as well as the importance of this region for studying the ecology of avian influenza.

Prevalence and Phylogenetics of H9n2 in Backyard and Commercial Poultry in Pakistan.

Surveillance of H9N2 is currently focused on areas central to the commercial poultry industry. This study determined the prevalence of H9N2 virus in commercial and backyard poultry flocks in Punjab Province, Pakistan. Oral and tracheal swabs were collected from commercial and backyard poultry from January 2015 through June 2016. Antisera against H5, H7, H9, and Newcastle disease viruses were used for virus identification. Molecular confirmation was made by reverse transcription PCR. Avian influenza virus subtypes H5 and H7 were not detected. The H9N2 virus was isolated in 5.7% of 905 tested flocks (5-10 birds/flock). Prevalence in commercial and backyard poultry was 6.7% of 687 flocks and 2.7% of 218 flocks, respectively. Hemagglutinin and neuraminidase-gene-based phylogenetic analysis of commercial and backyard poultry isolates showed 100% homology. Within sublineage B2 of Pakistan, identity among most recent isolates (2015) was 100%, compared to 75%-99% identity with previously isolated viruses (2010-12), indicating continued virus evolution. Most of the previously reported and currently studied viruses were isolated near the Pakistan-India border. Phylogenetic analysis showed that Pakistani and Indian isolates were closely related, indicating that avian influenza virus transmission may occur across this border.

Comparative In Vitro and In Vivo Analysis of H1N1 and H1N2 Variant Influenza Viruses Isolated from Humans between 2011 and 2016

Influenza A virus pandemics are rare events caused by novel viruses which have the ability to spread in susceptible human populations. With respect to H1 subtype viruses, swine H1N1 and H1N2 viruses occasionally cross the species barrier to cause human infection. Recently isolated from humans (termed variants), swine viruses were shown to display great genetic and antigenic diversity, hence posing considerable public health risk. Here, we utilized in vitro and in vivo approaches to provide characterization of H1 subtype variant viruses isolated since the 2009 pandemic and discuss the findings in context with previously studied H1 subtype human isolates. The variant viruses were well adapted to replicate in the human respiratory cell line Calu-3 and the respiratory tracts of mice and ferrets. However, with respect to hemagglutinin (HA) activation pH, the variant viruses had fusion pH thresholds closer to that of most classical swine and triple-reassortant H1 isolates rather than viruses that had adapted to humans. Consistent with previous observations for swine isolates, the tested variant viruses were capable of efficient transmission between cohoused ferrets but could transmit via respiratory droplets to differing degrees. Overall, this investigation demonstrates that swine H1 viruses that infected humans possess adaptations required for robust replication and, in some cases, efficient respiratory droplet transmission in a mammalian model and therefore need to be closely monitored for additional molecular changes that could facilitate transmission among humans. This work highlights the need for risk assessments of emerging H1 viruses as they continue to evolve and cause human infections.IMPORTANCE Influenza A virus is a continuously evolving respiratory pathogen. Endemic in swine, H1 and H3 subtype viruses sporadically cause human infections. As each zoonotic infection represents an opportunity for human adaptation, the emergence of a transmissible influenza virus to which there is little or no preexisting immunity is an ongoing threat to public health. Recently isolated variant H1 subtype viruses were shown to display extensive genetic diversity and in many instances were antigenically distinct from seasonal vaccine strains. In this study, we provide characterization of representative H1N1v and H1N2v viruses isolated since the 2009 pandemic. Our results show that although recent variant H1 viruses possess some adaptation markers of concern, these viruses have not fully adapted to humans and require further adaptation to present a pandemic threat. This investigation highlights the need for close monitoring of emerging variant influenza viruses for molecular changes that could facilitate efficient transmission among humans.

Stability of retroviral pseudotypes carrying the hemagglutinin of avian influenza viruses under various storage conditions

Retroviral pseudotypes are broadly used as safe instruments to mimic the structure and surface of highly pathogenic viruses. They have been employed for the discovery of new drugs, as diagnostic tools in vaccine studies, and part of serological assays. Because of their widespread use in research and their potential as tools for quality control, it is important to know their shelf life, stability, and best storage conditions. In this study, we produced pseudotypes carrying the lacZ reporter gene and the hemagglutinin (HA) of avian influenza virus subtypes H5 and H7 to investigate their stability under various storage conditions. We produced pseudotypes with titers of approximately 106 RLU/mL, which decreased to 105–106 RLU/mL after short-term storage at 4 °C (up to 4 weeks). Stability was maintained after long-term storage at −20 °C (up to 12 months), even under storage variations such as freeze-thaw cycles. We conclude that, although the titers decreased by 1 log10 under the different storage conditions, the remaining titers can be readily applicable in many techniques, such as neutralization assays. These findings show that large quantities of retroviral pseudotypes can be safely stored for short- or long-term use, allowing standardization and reduced variation in assays involving retroviral pseudotypes.

Compatibility between haemagglutinin and neuraminidase drives the recent emergence of novel clade 2.3.4.4 H5Nx avian influenza viruses in China.

Genetic reassortments between highly pathogenic avian influenza (HPAI) H5 subtype viruses with different neuraminidase (NA) subtypes have increased in prevalence since 2010 in wild birds and poultry from China. The HA gene slightly evolved from clade 2.3.4 to clade 2.3.4.4, raising the question of whether novel clade 2.3.4.4 HA broke the balance with N1 but is matched well with NAx to drive viral epidemics. To clarify the role of compatibility between HA and NA on the prevalence of H5Nx subtypes, we constructed 10 recombinant viruses in which the clade 2.3.4 or clade 2.3.4.4 HA genes were matched with different NA (N1, N2 and N8) genes and evaluated viral characteristics and pathogenicity. Combinations between clade 2.3.4 HA and N1 or between clade 2.3.4.4 HA and NAx, but not between clade 2.3.4.4 HA and N1, or between clade 2.3.4 HA and NAx, promoted viral growth, NA activity, thermostability, low-pH stability and pathogenicity in chicken and mice. These findings suggest that both clade 2.3.4 HA/N1 and clade 2.3.4.4 HA/NAx displayed a better match, which could promote the increased prevalence of clade 2.3.4 H5N1 AIV (prior to 2010) and clade 2.3.4.4 H5Nx AIV (since 2010) in China, respectively.

Development of an Influenza Rapid Diagnostic Kit Specific for the H7 Subtype.

Since the spring of 2013, human infections with H7N9 viruses have been detected in China. Some of these viruses have become highly pathogenic. Highly and low pathogenic avian influenza H7N9 viruses are currently co-circulating with the seasonal influenza A viruses H3N2 and H1N1pdm09. Prompt identification and isolation of H7N9 patients is one measure to prevent the spread of H7N9 virus and help prevent a pandemic. The majority of commercially available point-of-care rapid influenza diagnostic kits can differentiate between influenza A and B viruses, but cannot distinguish between H7N9 viruses and seasonal influenza A viruses. Accordingly, we have developed a rapid diagnostic kit specific for the H7 subtype that is accessible, easy to use. Although the detection limit of this H7 kit is one-tenth lower than that of a commercially available rapid influenza A and B diagnostic kit of similar design, except for the specificity of the monoclonal antibodies used, this kit is highly specific, detecting only H7-subtype influenza viruses, including the recent highly pathogenic H7N9 viruses from humans, and does not show any non-specific reactions with other HA subtypes. This H7 kit will be of value for the early detection of H7N9-infected patients.

Cross-reactive mouse monoclonal antibodies raised against the hemagglutinin of A/Shanghai/1/2013 (H7N9) protect against novel H7 virus isolates in the mouse model

Influenza viruses remain a major global public health risk. In addition to seasonal influenza viruses, epizootic influenza A H7 subtype viruses of both the Asian and North American lineage are of concern due to their pandemic potential. In China, the simultaneous occurrence of H7N9 zoonotic episodes and seasonal influenza virus epidemics could potentially lead to novel reassortant viruses with the ability to efficiently spread among humans. Recently, the H7N9 virus has evolved into two new lineages, the Pearl River Delta and the Yangtze River Delta clade. This development has also resulted in viruses with a polybasic cleavage site in the hemagglutinin that are highly pathogenic in avian species and have caused human infections. In addition, an outbreak of a highly pathogenic H7N8 strain was reported in the US state of Indiana in 2016. Furthermore, an H7N2 feline virus strain caused an outbreak in cats in an animal shelter in New York City in 2016, resulting in one human zoonotic event. In this study, mouse monoclonal antibodies previously raised against the hemagglutinin of the A/Shanghai/1/2013 (H7N9) virus were tested for their (cross-) reactivity to these novel H7 viruses. Moreover, the functionality of these antibodies was assessed in vitro in hemagglutination inhibition and microneutralization assays. The therapeutic and prophylactic efficacy of the broadly reactive antibodies against novel H7 viruses was determined in vivo in mouse passive transfer-viral challenge experiments. Our results provide data about the conservation of critical H7 epitopes and could inform the selection of pre-pandemic H7 vaccine strains.

Risk Factors for Avian Influenza H9 Infection of Chickens in Live Bird Retail Stalls of Lahore District, Pakistan 2009\u20132010

This study was conducted to identify risk factors associated with AIV infections in live bird retail stalls (LBRS) in Lahore District, Pakistan. A cross-sectional survey of LBRS was conducted from December 2009-February 2010 using two-stage cluster sampling based on probability proportional to size. A total of 280 oropharyngeal swab sample pools were collected from 1400 birds in 8 clusters and tested by qRT-PCR for the matrix (M) gene of type A influenza virus and HA gene subtypes H9, H5 and H7. Thirty-four (34) samples were positive for the M gene, of which 28 were also positive for H9. No sample was found positive for H5 or H7. Data for 36 potential risk factors, collected by questionnaire, were analyzed by survey-weighted logistic regression and prevalence odds ratios (OR) for associated risk factors were calculated. A final multivariable model identified three risk factors for H9 infection in LRBS, namely obtaining birds from mixed sources (OR 2.28, CI95%: 1.4–3.7), keeping birds outside cages (OR 3.10, CI95%: 1.4–7.0) and keeping chicken breeds other than broilers (OR 6.27, CI95%: 1.7–23.2). Sourcing birds from dealers/wholesalers, keeping birds inside cages and avoiding mixing different breeds in cages could reduce the risk of H9 infections in LRBS.

Broadly protective anti-hemagglutinin stalk antibodies induced by live attenuated influenza vaccine expressing chimeric hemagglutinin

The development of influenza vaccines that can provide broad protection against all drifted seasonal virus variants, zoonotic infections and emerging pandemic strains, has been a priority for two decades. Here we propose a strategy of inducing broadly-reactive anti-stalk antibody by sequential immunizations with live attenuated influenza vaccines (LAIVs) expressing chimeric HAs (cHAs). These vaccines are designed to contain identical hemagglutinin stalk domains from H1N1 virus but antigenically unrelated globular head domains from avian influenza virus subtypes H5, H8 and H9. Mouse experiments demonstrated enhanced cross-protection of cHA-containing LAIVs compared to the relevant vaccine viruses expressing natural HAs, and this enhanced protection was driven by stalk-HA-reactive IgG antibodies. The establishment of fully functional cross-protective immunity after two doses of cHA LAIV vaccination in naïve animals suggests that a similar effect might be expected after a single cHA LAIV dose in primed individuals, or after two to three doses in naïve children.

Establishment of the cross-clade antigen detection system for H5 subtype influenza viruses using peptide monoclonal antibodies specific for influenza virus H5 hemagglutinin

The H5 subtype of highly pathogenic avian influenza (H5 HPAI) viruses is a threat to both animal and human public health and has the potential to cause a serious future pandemic in humans. Thus, specific and rapid detection of H5 HPAI viruses is required for infection control in humans. To develop a simple and rapid diagnostic system to detect H5 HPAI viruses with high specificity and sensitivity, we attempted to prepare monoclonal antibodies (mAbs) that specifically recognize linear epitopes in hemagglutinin (HA) of H5 subtype viruses. Nine mAb clones were obtained from mice immunized with a synthetic partial peptide of H5 HA molecules conserved among various H5 HPAI viruses. The antigen-capture enzyme-linked immunosorbent assay using the most suitable combination of these mAbs, which bound specifically to lysed H5 HA under an optimized detergent condition, was specific for H5 viruses and could broadly detect H5 viruses in multiple different clades. Taken together, these peptide mAbs, which recognize linear epitopes in a highly conserved region of H5 HA, may be useful for specific and highly sensitive detection of H5 HPAI viruses and can help in the rapid diagnosis of human, avian, and animal H5 virus infections.

A Portable Multi-Channel Turbidity System for Rapid Detection of Pathogens by Loop-Mediated Isothermal Amplification.

This study aimed at developing a portable multi-channel turbidity system (21 cm in length, 15.5 cm in width and 11.5 cm in depth) by real-time loop-mediated isothermal amplification (LAMP) method for rapid detection of pathogens. The developed system herein includes temperature control unit, photoelectric detection unit, turbidity calibration unit, power management unit, human machine unit, communication unit and ARM-based microcontroller. The coefficient of variation for eight channels is less than 0.25% in noise analysis. Legionella bacteria (LEG) and H7 subtype virus (H7) were successively detected by the designed and developed system within 60 minutes. Moreover, its specificity for LEG is satisfactory and its sensitivity for H7 is 10 copies/mL. Besides, this system for point-of-care diagnosis allows a rapid, small size, low cost, and automatic detection with the characteristics of high-efficiency, excellent stability and high uniformity.

Au/Fe3O4 core-shell nanoparticles are an efficient immunochromatography test strip performance enhancer-a comparative study with Au and Fe3O4 nanoparticles.

Immunochromatography test strips that use metal particles constructed from Au, Fe3O4, and Au/Fe3O4 nanoparticles were developed for the rapid detection of avian influenza virus subtype H7 (AIV H7). The principle of this immunochromatography test strip was based on a sandwich immunoreaction in which AIV H7 antigens bind specifically to their corresponding antibodies on a nitrocellulose membrane. An antibody–metal (Au, Fe3O4 or Au/Fe3O4) nanoparticle conjugate was used as a label and coated onto a glass fiber membrane, which was used as a conjugate pad. To create a test and a control zone, an anti-H7 polyclonal antibody and an anti-IgG antibody were immobilized onto the nitrocellulose membrane, respectively. Positive samples displayed brown/red lines in the test and control zones of the nitrocellulose membrane, whereas negative samples resulted in a brown/red line only in the control zone. The limit of detection (LOD) of the Au/Fe3O4 nanoparticle-based immunochromatography test strips was found to be 103.5 EID50 (EID50: 50% Egg Infective Dose), which could be visually detected by the naked eye within 15 min. In addition, 200 clinical samples were tested using the Au/Fe3O4 nanoparticle-based immunochromatography test strip to estimate its performance, and seven were positive for AIV H7. In summary, the Au/Fe3O4 nanoparticle-based immunochromatography test strip offers a simple and cost-effective tool for the rapid detection of AIV H7.

Serological evidence of H5-subtype influenza A virus infection in indigenous avian and mammalian species in Korea.

In Korea, H5-subtype highly pathogenic avian influenza (HPAI) has caused huge economic losses in poultry farms through outbreaks of H5N1 since 2003, H5N8 since 2013 and H5N6 since 2016. Although it was reported that long-distance migratory birds may play a major role in the global spread of avian influenza viruses (AIVs), transmission from such birds to poultry has not been confirmed. Intermediate hosts in the wild also may be a potential factor in viral transmission. Therefore, a total of 367 serum samples from wild animals were collected near major migratory bird habitats from 2011 to 2016 and tested by AIV-specific blocking ELISA and hemagglutination inhibition (HI) test. Two mammalian and eight avian species were seropositive according to the ELISA test. Among these, two mammalian (Hydropotes inermis and Prionailurus bengalensis) and three avian (Aegypius monachus, Cygnus cygnus, and Bubo bubo) species showed high HI titres (>1,280) against one or two H5-subtype AIVs. As H. inermis (water deer), P. bengalensis (leopard cat), and B. bubo (Eurasian eagle owl) are indigenous animals in Korea, evidence of H5-subtype AIV in these animals implies that continuous monitoring of indigenous animals should be followed to understand interspecies transmission ecology of H5-subtype influenza viruses.

Single Dose of Consensus Hemagglutinin-Based Virus-Like Particles Vaccine Protects Chickens against Divergent H5 Subtype Influenza Viruses.

The H5 subtype highly pathogenic avian influenza (HPAI) virus is one of the greatest threats to global poultry industry. To develop broadly protective H5 subunit vaccine, a recombinant consensus HA sequence (rHA) was constructed and expressed in virus-like particles (rHA VLPs) in the baculovirus-insect cell system. The efficacy of the rHA VLPs vaccine with or without immunopotentiator (CVCVA5) was assessed in chickens. Compared to the commercial Re6 or Re6-CVCVA5 vaccines, single dose immunization of chickens with rHA VLPs or rHA-CVCVA5 vaccines induced higher levels of serum hemagglutinin inhibition titers and neutralization titers, mucosal antibodies, IFN-γ and IL-4 cytokines in sera, and cytotoxic T lymphocyte responses. The rHA VLPs vaccine was superior to the commercial Re6 vaccine in conferring cross-protection against different clades of H5 subtype viruses. This study reports that H5 subtype consensus HA VLP single dose vaccination provides broad protection against HPAI virus in chickens.

Universal influenza virus vaccines: what can we learn from the human immune response following exposure to H7 subtype viruses?

Several universal influenza virus vaccine candidates based on eliciting antibodies against the hemagglutinin stalk domain are in development. Typically, these vaccines induce responses that target group 1 or group 2 hemagglutinins with little to no cross-group reactivity and protection. Similarly, the majority of human anti-stalk monoclonal antibodies that have been isolated are directed against group 1 or group 2 hemagglutinins with very few that bind to hemagglutinins of both groups. Here we review what is known about the human humoral immune response to vaccination and infection with H7 subtype influenza viruses on a polyclonal and monoclonal level. It seems that unlike vaccination with H5 hemagglutinin, which induces antibody responses mostly restricted to the group 1 stalk domain, H7 exposure induces both group 2 and cross-group antibody responses. A better understanding of this phenomenon and the underlying mechanisms might help to develop future universal influenza virus vaccine candidates.

Development and application of a triplex real-time PCR assay for the simultaneous detection of avian influenza virus subtype H5, H7 and H9

Avian influenza virus (AIV), especially subtypes H5, H7 and H9, has contributed to enormous economic losses and poses a potential pandemic threat to global human public health. Early screening of suspected cases is key to controlling the spread of AIVs. In this study, an accurate, rapid, and triplex real-time polymerase chain reaction (PCR) assay was developed for the simultaneous detection of AIV subtypes H5, H7 and H9. The sensitivity of the real-time PCR was at least 100 times higher than that of the conventional PCR, with a detection limit of 50 copies and an EID50 of 1 (50% egg infections dose) for the H5, H7, and H9 subtypes. The lack of cross-reaction with other avian respiratory viruses suggested that the real-time PCR assay was highly specific. The reproducibility of the assay was confirmed using plasmids containing targets genes. Furthermore, 362 clinical field samples were evaluated. Subtypes H5, H7 and H9 were detected in 102 (28.18%) samples by real-time PCR and in 35 (9.67%) samples by conventional virus isolation. These results indicate that the triplex real-time PCR assay has good sensitivity, specificity and reproducibility and that it might be useful for laboratory surveillance and rapid diagnosis of the H5, H7 and H9 subtypes of influenza A viruses.

Pathogenicity and immunosuppressive potential of fowl adenovirus in specific pathogen free chickens

To elucidate the effect of fowl adenovirus (FAdV)-C in specific-pathogen-free (SPF) chickens, we investigated the pathogenicity, body weights, enzymatic systems, and immune organs of chickens in response to Newcastle disease virus (NDV) and avian influenza virus subtype H9 (AIV-H9) vaccination. Chickens were divided randomly into four groups, which included injection groups (FAdV-C, vaccination, and FAdV-C plus vaccination) and a negative control group. The results indicated that FAdV-C was highly pathogenic in SPF chickens and led to a 40% mortality rate and growth retardation, compared with the control birds. Significant changes in clinical chemical markers of all infected birds, together with histopathological lesions, indicated impairment of the liver and heart integrity and function. Furthermore, chickens in the FAdV-C plus vaccination group had significantly lower titers of antibodies against NDV and AIV-H9 than the uninfected and vaccinated chickens. The results of this study provide new insights into the pathogenesis of hydropericardium syndrome, a disease that progresses to a metabolic disorder and causes serious growth retardation and immunosuppression.