H1 Subtypes Research Articles

Arbidol targeting influenza virus A Hemagglutinin; A comparative study

Influenza (flu) is a serious global health threat. The Hemagglutinin (HA) protein binds the flu virus to the sialic acids at the surface of the host cells' membrane which allows the endocytosis of the virus. Therefore, potential inhibitors can attach to the active site of HA and block the virus life-cycle. In this study, the antiviral drug arbidol (ARB) and 16 HA-subtypes were docked and analyzed to represent different approaches in predicting the conformation of protein-ligand, protein-protein, and protein-glycan complex and its binding energy. Our findings show that ARB interacts with all HA subtypes, and H7 possesses the best affinity. The next influenza pandemic could be caused by H4, H5, H6, and H14 subtypes, which prompts further studies in investigating the interaction between these particular HA subtypes and other antiviral drugs to obtain higher efficacy.

SARS-CoV-2 spike and its adaptable furin cleavage site

SARS-CoV-2 spike and its adaptable furin cleavage site

Seroprevalences of specific antibodies against avian pathogens in free-ranging ring-necked pheasants (Phasianus colchicus) in Northwestern Germany.

Infectious diseases in captive pheasants (Phasianus colchicus) are well known, but there is a lack of knowledge about occurrence and distribution of pathogens in free-ranging pheasants in Germany. We investigated 604 sera from hunted pheasants and 152 sera from wild caught pheasants between 2011 to 2015, with the aim to determine the prevalence of specific antibodies against different viruses: Avian influenza virus (AIV) of subtypes H5, H7, H9, paramyxovirus type 1 (PMV-1), avian encephalomyelitis virus (AEV), infectious bursitis disease virus (IBDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), avian metapneumovirus (aMPV) and Salmonella sp., Mycoplasma synoviae (MS) and Mycoplasma gallisepticum (MG). In addition, 178 caeca were investigated for Histomonas meleagridis. The study reveals an ongoing circulation of IBV in the wild pheasant population during the study. Also high seroprevalences of specific antibodies against aMPV depending on the area and a strong increase in prevalence of IBDV antibodies in sera of pheasants in Lower Saxony were detected. ILTV antibody prevalences differed between areas and AEV antibody detection differed between years significantly, whereas specific antibodies against PMV-1 could not be detected and antibodies against AIV-H5, -H7 and -H9 and Mycoplasma spp. were detected in very few cases.

Genetic characteristics of avian influenza A (H9N9) virus in Poyang Lake area, 2014–2019

Objective To understand the molecular characteristics of avian influenza A (H9N9) virus isolated in Poyang Lake area in Jiangxi province between 2014 and 2019. Methods A total of 31854 poultry related environmental samples were collected in Poyang Lake area between 2014 and 2019. The samples were transported to Chinese National Influenza Center (CNIC) at low temperatures for virus isolation. All allantoic fluids were tested by influenza A virus fast diagnostic kits and influenza A virus positive allantoic fluids were harvested, then, the isolates were deeply sequenced. Software CLC, CD-HIT, MAFFT 7.475, MEGA 6.06, FastTree and FigTree v1.4.4 were used for the sequence analysis. Results Between 2014 and 2019, a total of 18 influenza A (H9N9) virus strains were isolated in Poyang Lake area. Phylogenetic analysis of their full genome showed that they were reassortant of H9N2 virus and H7N9 virus. The NA segments of the H9N9 virus isolated in Jiangxi and the H9N9 virus isolated from Eastern China were in different sub-clade. Amino acid sites analysis of these H9 subtype viruses showed that they belonged to low pathogenic avian influenza virus, but some crucial amino acids substitution had been found, such as Q226L in HA protein, 550L in PA protein, N30D, T215A in M1 protein and P189D, F103L and M106I in NS1 genes. Conclusion The influenza A (H9N9) virus strains isolated in our study during 2014–2019 were the reassortant of H9N2 and H7N9 viruses, which might be highly associated with the high incidences of H9N2 virus and H7N9 virus infections during 2013–2017. Although, there were no reported about human infection with influenza A (H9N9) virus, the genetic characteristic analysis results has shown it has potential to infect human. It is necessary to strengthen the continuous surveillance and research on reassortant of avian influenza viruses.

Conserved Sequence Analysis of Influenza A Virus HA Segment and Its Application in Rapid Typing.

The high mutation rate of the influenza A virus hemagglutinin segment poses great challenges to its long-term effective testing and subtyping. Our conserved sequence searching method achieves high-specificity conserved sequences on H1–H9 subtypes. In addition, PCR experiments show that primers based on conserved sequences can be used in influenza A virus HA subtyping. Conserved sequence-based primers are expected to be long-term, effective subtyping tools for influenza A virus HA.

Administration of Multivalent Influenza Virus Recombinant Hemagglutinin Vaccine in Combination-Adjuvant Elicits Broad Reactivity Beyond the Vaccine Components.

Combining variant antigens into a multivalent vaccine is a traditional approach used to provide broad coverage against antigenically variable pathogens, such as polio, human papilloma and influenza viruses. However, strategies for increasing the breadth of antibody coverage beyond the vaccine are not well understood, but may provide more anticipatory protection. Influenza virus hemagglutinin (HA) is a prototypic variant antigen. Vaccines that induce HA-specific neutralizing antibodies lose efficacy as amino acid substitutions accumulate in neutralizing epitopes during influenza virus evolution. Here we studied the effect of a potent combination adjuvant (CpG/MPLA/squalene-in-water emulsion) on the breadth and maturation of the antibody response to a representative variant of HA subtypes H1, H5 and H7. Using HA protein microarrays and antigen-specific B cell labelling, we show when administered individually, each HA elicits a cross-reactive antibody profile for multiple variants within the same subtype and other closely-related subtypes (homosubtypic and heterosubtypic cross-reactivity, respectively). Despite a capacity for each subtype to induce heterosubtypic cross-reactivity, broader coverage was elicited by simply combining the subtypes into a multivalent vaccine. Importantly, multiplexing did not compromise antibody avidity or affinity maturation to the individual HA constituents. The use of adjuvants to increase the breadth of antibody coverage beyond the vaccine antigens may help future-proof vaccines against newly-emerging variants.

Monoclonal antibodies to hemagglutinin of influenza A/H7N3 virus (Orthomyxoviridae: Alphainfluenzavirus: Influenza A virus)

Variants of influenza virus A/H7 have the same high pandemic potential as A/H5. However, the information about the antigenic structure of H7 hemagglutinin (НА) is considerably inferior in quantitative terms to similar data for H5 НА.The aims of the study were development and characterization of the monoclonal antibodies (MAbs) panel for HA subtype H7 of the influenza A virus. Viruses were accumulated in 10-day-old chicken embryos. Purification and concentration of the virus, determination of protein concentration, preparation of MAbs and ascitic fluids, hemagglutination and hemagglutination inhibition (HI) tests, assessment of antibodies' activity in indirect enzyme-linked immunosorbent assay (ELISA), as well as determination of MAbs isotypes and neutralization reaction (NR) were carried out by standard methods. The obtained MAbs to А/mallard/Netherlands/12/2000 (H7N3) strain were studied in HI test with a set of strains of different years of isolation belonging to different evolutionary groups. MAbs had a reduced reactivity compared to the immunogen-virus for all the studied strains. Cross-interaction of MAbs 9E11 and 9G12 in HI test with influenza A/H15 virus has been observed. Influenza A agent with H7 HA variant could serve as a potential cause of a future pandemic. Development of the MAbs panel for subtype H7 HA is an urgent task for both veterinary medicine and public health. The obtained MAbs can be used not only for epitope mapping of the H7 HA molecule (currently insufficiently studied) and as reagents for diagnostic assays, but also for determining common («universal») epitopes in HA of different strains of this subtype.

Changes of avian influenza virus subtypes before and after vaccination in live poultry in Nanchang, China from 2016 to 2019

We investigated fluctuations in the detection rates of avian influenza virus (AIV) subtypes H5, H7, and H9 in live poultry in Nanchang city, Chinese province Jiangxi, before and after the Chinese nationwide AIV vaccination campaign against highly pathogenic (HP) AIV subtype H5 and H7. Samples were tested for nucleic acid of type A avian influenza virus by real-time reverse transcription polymerase chain reaction technology. The H5, H7 and H9 subtypes of influenza viruses were further classified for the positive results. Based on the analysis of 2119 samples collected from February 2016 to December 2019, we found that AIV subtypes H5, H7, H9 showed a seasonal pattern, and the positive rate of avian influenza tended to reach its peak in the colder season. The detection rate of AIV subtypes H5, H7, H9 of chickens (39.26%) was significantly higher than that of ducks (5.78%) and pigeons (4.31%). After vaccination, the positive rates of the H5 subtype (0.27%) and the H7 subtype (0.00%) decreased significantly, while the positive rate of the H9 subtype (29.95%) increased significantly. The H9 subtype has become the dominant subtype detected in live poultry and occupies a dominant position in the live bird market. This study showed that the government of China should establish measures for the long-term control of avian influenza.

Antigenic evolution of contemporary clade 2.3.4.4 HPAI H5 influenza A viruses and impact on vaccine use for mitigation and control

Since 2003, highly pathogenic avian influenza (HPAI) viruses of the H5 subtype have been maintained in poultry, periodically spilling back into wild migratory birds and spread to other geographic regions, with re-introduction to domestic birds causing severe impacts for poultry health, production and food sustainability. Successive waves of infection have also resulted in substantial genetic evolution and reassortment, enabling the emergence of multiple clades and subtypes within the H5 2.3.4.4 HPAI viruses. Control of AI is principally through either culling or through vaccination using conventional vaccines. Here, we antigenically and genetically characterise the emerging 2020/21 H5NX clade 2.3.4.4 strains and assess cross-reactivity to putative vaccine strains using chicken antisera. We demonstrate significant antigenic differences between commercially available poultry vaccines and currently circulating viruses suggesting that vaccination options might be suboptimal in the current outbreaks.

Genetic characterization of an H5N6 avian influenza virus with multiple origins from a chicken in southern China, October 2019

BackgroundHighly pathogenic avian influenza viruses (HPAIVs) of H5 subtype pose a great threat to the poultry industry and human health. In recent years, H5N6 subtype has rapidly replaced H5N1 as the most predominate HPAIV subtype circulating in domestic poultry in China. In this study, we describe the genetic and phylogenetic characteristics of a prevalent H5N6 strain in Guangdong, China.ResultsNucleotide sequencing identified a H5N6 subtype HPAIV, designated as A/chicken/Dongguan/1101/2019 (DG/19), with a multibasic cleavage site in the hemagglutinin (HA). Phylogenetic analysis revealed DG/19 was a reassortant of H5N1, H5N2, H5N8, and H6N6 subtypes of avian influenza viruses. A number of mammalian adaptive markers such as D36N in the HA were identified.ConclusionsOur results showed that HPAIV H5N6 strains still emerge in well-managed groups of chicken farms. Considering the increasing prevalence of H5N6 HPAIV, and the fact that H5N6 HPAIVs are well adapted to migratory birds, an enhanced surveillance for the East Asian-Australasian flyway should be undertaken to prevent potential threats to the poultry industry and human health.

Quaternary ammonium salts based on (-)-borneol as effective inhibitors of influenza virus.

A series of compounds containing a 1,7,7-trimethylbicyclo[2.2.1]heptane fragment were evaluated for their antiviral activity against influenza A virus strain A/Puerto Rico/8/34 (H1N1) in vitro. The most potent antiviral compound proved to be a quaternary ammonium salt based on (-)-borneol, 10a. In in vitro experiments, compound 10a inhibited influenza A viruses (H1, H1pdm09, and H3 subtypes), with an IC50 value of 2.4-16.8 µM (depending on the virus), and demonstrated low toxicity (CC50 = 1311 µM). Mechanism-of-action studies for compound 10a revealed it to be most effective when added at the early stages of the viral life cycle. In direct haemolysis inhibition tests, compound 10a was shown to decrease the membrane-disrupting activity of influenza A virus strain A/Puerto Rico/8/34. According to molecular modelling results, the lead compound 10a can bind to different sites in the stem region of the viral hemagglutinin.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00705-021-05102-1.

Distribution of avian influenza viruses according to environmental surveillance during 2014\u20132018, China

BackgroundRecurrent infections of animal hosts with avian influenza viruses (AIVs) have posted a persistent threat. It is very important to understand the avian influenza virus distribution and characteristics in environment associated with poultry and wild bird. The aim of this study was to analyze the geographic and seasonal distributions of AIVs in the 31 provinces, municipalities and autonomous region (PMA) of China, compare the AIVs prevalence in different collecting sites and sampling types, analyze the diversity of AIVs subtypes in environment.MethodsA total of 742 005 environmental samples were collected from environmental samples related to poultry and wild birds in different locations in the mainland of China during 2014–2018. Viral RNA was extracted from the environmental samples. Real-time RT-PCR assays for influenza A, H5, H7 and H9 subtypes were performed on all the samples to identify subtypes of influenza virus. The nucleic acid of influenza A-positive samples were inoculated into embryonated chicken eggs for virus isolation. Whole-genome sequencing was then performed on Illumina platform. SPSS software was used to paired t test for the statistical analysis. ArcGIS was used for drawing map. Graphpad Prism was used to make graph.ResultsThe nucleic acid positivity rate of influenza A, H5, H7 and H9 subtypes displayed the different characteristics of geographic distribution. The nucleic acid positivity rates of influenza A were particularly high (25.96%–45.51%) in eleven provinces covered the Central, Eastern, Southern, Southwest and Northwest of China. The nucleic acid positivity rates of H5 were significantly high (11.42%–13.79%) in two provinces and one municipality covered the Southwest and Central of China. The nucleic acid positivity rates of H7 were up to 4% in five provinces covered the Eastern and Central of China. The nucleic acid positivity rates of H9 were higher (13.07%–2.07%) in eleven PMA covered the Southern, Eastern, Central, Southwest and Northwest of China. The nucleic acid positivity rate of influenza A, H5, H7 and H9 showed the same seasonality. The highest nucleic acid positivity rates of influenza A, H5, H7, H9 subtypes were detected in December and January and lowest from May to September. Significant higher nucleic acid positivity rate of influenza A, H5, H7 and H9 were detected in samples collected from live poultry markets (LPM) (30.42%, 5.59%, 4.26%, 17.78%) and poultry slaughterhouses (22.96%, 4.2%, 2.08%, 12.63%). Environmental samples that were collected from sewage and chopping boards had significantly higher nucleic acid positivity rates for influenza A (36.58% and 33.1%), H5 (10.22% and 7.29%), H7(4.24% and 5.69%)and H9(21.62% and 18.75%). Multiple subtypes of AIVs including nine hemagglutinin (HA) and seven neuraminidase (NA) subtypes were isolated form the environmental samples. The H5, H7, and H9 subtypes accounted for the majority of AIVs in environment.ConclusionsIn this study, we found the avian influenza viruses characteristics of geographic distribution, seasonality, location, samples types, proved that multiple subtypes of AIVs continuously coexisted in the environment associated with poultry and wild bird, highlighted the need for environmental surveillance in China.Graphic

Genomic surveillance: Brief summary of the Influenza virological activity in Ecuador reported by National Influenza Centre of Ecuador from INSPI

For H1N1pdm were grouped within clade 6B.1, and related to the strain of vaccine reference for that year, regarding the H3 subtype were grouped within genotype 3C.2a, which are related to the strain of vaccine reference for the H3N2 subtype recommended by WHO in the formulation of the 2016 Southern Hemisphere and 2017-18 Northern Hemisphere (A/Hong Kong/ 4801/2014), but other group of Ecuadorian samples from 2017, were genetically related to clade 3C.3a, this cluster is more related to Northern Hemisphere Vaccine strain 2015-2016 and Southern Hemisphere 2015 (A/Switzerland/9715293/2013), so we could observe different genetic lineages of H3N2 subtype co-circulate in Ecuador during 2017. In the case of Influenza B we also had a co-circulation of Yamagata and Victoria lineages but the trivalent formulation only had Victoria lineages.

Sero-Detection of Avian Influenza A/H7 in Nigerian Live-Bird Markets in Plateau State

Avian influenza has been reported in domestic birds in Nigeria since 2006 and subtype H5 of the Gs/Gg lineage has continued to be detected up till date. It has been suggested that waterfowls and local birds sold in live-bird markets may be natural reservoir and source of reinfection of different subtype of avian influenza in poultry farms. This study aims at serodetection of avian influenza virus in waterfowls and local birds at live-bird markets in Plateau State, Nigeria. A total of three hundred and nine (309) blood samples were collected over a period of three months and two hundred and ninety-two (292) sera were analysed by c-ELISA for influenza A nucleoprotein using standard protocols. Haemagglutination Inhibition (HI) specific for subtypes H5, H9, and H7 was also carried out using standard protocols on ELISA positive samples. The results showed seroprevalence of 5.14% (n=15) for influenza A. Serotype H7 was thereafter detected by HI in 5 of the 15 influenza A positive samples. The H7 positive sera also reacted with H7N3, H7N4, H7N1 and H7N7 virus strains with HI titre ranging between 1:32 to 1:512. This investigation for the first time showed serological evidence of influenza A subtype H7 in local birds and waterfowls sold at the live bird market in Nigeria. Further virological surveillance to isolate the virus is important in order to better understand influenza virus epidemiology in Nigeria and the potential risk that other subtypesof influenza poses to poultry production and public health.
 Keywords: Influenza A, subtype H7, serological detection, live bird market, Nigeria.

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.

Biological characteristics of influenza virus subtype H6N8 isolated from wild birds in the south of Western Siberia

Aim. The aim is to explore the molecular and biological characteristics of influenza virus subtype H6N8.Material and Methods. Regular collecting of biological samples from wild birds was carried out in 2016 in the south of Western Siberia. Isolation of avian influenza virus strains from birds’ cloacal swabs was performed using three passages in embryonated chicken eggs. Hemagglutination assay was used to detect viruses in the allantoic fluid. Type and subtype of influenza viruses were identified using reverse transcription PCR (RT‐PCR). Results. This is the first time the avian influenza virus H6N8 subtype has been isolated in Russia. Phylogenetic analysis revealed that hemagglutinin (HA) and neuraminidase (NA) segments of A/gadwall/Chany/97/2016(H6N8) strain genome belonged to the Eurasian lineages of avian influenza viruses. Phylogenetic analysis showed that the HA sequence of the strain was closely related to the strain isolated from a duck in Bangladesh in 2015 (A/duck/Bangladesh/25767/2015(H6N1).Conclusion. Avian influenza viruses subtype H6N8 have the ability to circulate among wild waterfowl and poultry, increasing the chance of reassortment and transmission of H6 virus strains among birds. For this reason, it is of crucial importance to strengthen surveillance for avian influenza H6 subtype among wild birds in the south of Western Siberia.

Development of an immunochromatographic strip for rapid detection of H7 subtype avian influenza viruses

BackgroundH7N9 avian influenza virus (AIV) including highly and low pathogenic viruses have been detected in China since 2013. H7N9 AIV has a high mortality rate after infection in humans, and most human cases have close contacted with poultry in the live poultry market. Therefore, it is necessary to develop a rapid point-of-care testing (POCT) technique for H7N9 AIV detection.MethodsThe H7N9 AIV was inactivated and purified, and was used as the antigen to immunize BALB/c. Twelve H7-HA specific monoclonal antibodies (McAbs) were produced through the hybridoma technique. The McAb 10A8 was conjugated with colloid gold as detecting antibody; McAb 9B6 was dispensed on the nitrocellulose membran as the capture test line and the Goat-anti mouse IgG antibody was dispensed as control line respectively. The immunochromatographic strip was prepared.ResultsThe analysis of ELISA and virus neutralization test showed that the obtained McAbs specifically recognized H7 HA. Based on the prepared strip, the detection of H7 AIV was achieved within 10 min. No cross-reaction occurred between H7 AIVs and other tested viruses. The detection limit of the strip for H7 was 2.4 log10EID50/0.1 mL for chicken swab samples.ConclusionThe McAbs were specific for H7 and the immunochromatographic strip developed in this study was convenient, rapid and reliable for the detection of H7 AIV. The strip could provide an effective method for the rapid and early detection of H7 AIV.

Serosurvey of Avian Influenza Viruses (H5, H7, and H9) and Associated Risk Factors in Backyard Poultry Flocks of Lahore District, Pakistan.

Rural poultry constitutes 56% of the total poultry population in Pakistan; however, epidemiological information about avian influenza viruses (AIVs) in backyard poultry flocks is lacking. A cross-sectional survey of villages of Lahore district was conducted from July 2009 to August 2009 using two-stage cluster sampling and probability proportional to size (PPS) sampling to estimate seroprevalence and its associated risk factors. A random selection of 35 clusters from 308 villages of Lahore were considered, and from each cluster, six chickens aged >2 months were selected. A total of 210 serum samples were collected and examined by the hemagglutination inhibition (HI) test for specific antibodies against AIV subtypes H5, H7, and H9. Overall weighted seroprevalence for AIVs was 65.2% (95% CI: 55.6–74.8%), and for subtype H5, H7 & H9 was 6.9% (95% CI: 10.8–23.0%), 0% (95% CI: 0–1.7%), and 62.0% (95% CI: 52.2–71.8%) respectively. However, none of the samples were positive for H7. The average flock size was 17.3 birds, and the main purpose of keeping poultry was for eggs/meat (70.6%, 95% CI: 59.7–81.4). A majority of them were reared in a semi-caged system (83%, 95% CI: 74.5–91.3). Backyard birds were received from different sources, that is, purchased from the market or received as a gift from friends or any NGO, and were 5.7 times more likely to become avian influenza (AI) seropositive than those that were not exposed to these sources (CI 95%: 2.0–716.0). Backyard birds which were received from different sources, that is, purchased from the market or received from friends or any NGO, were 5.7 times more likely to become AI seropositive compared to those that were not (CI 95%: 2.5–18.7). To reduce the risk of AIV in Pakistan, continuous surveillance of backyard poultry would be needed.

AS03-adjuvanted H7N9 inactivated split virion vaccines induce cross-reactive and protective responses in ferrets

Human infections with avian H7N9 subtype influenza viruses are a major public health concern and vaccines against H7N9 are urgently needed for pandemic preparedness. In early 2013, novel H7N9 influenza viruses emerged in China that caused about 1600 human cases of infection with a high associated case fatality rate. In this study, two H7N9 split virion vaccines with or without AS03 adjuvant were tested in the naive ferret model. Serological analyses demonstrated that homologous hemagglutination inhibition and microneutralization antibody titers were detectable in the ferrets after the first immunization with the AS03-adjuvanted vaccines that were further boosted by the second immunization. In addition, heterologous antibody titers against older H7 subtype viruses of the North American lineage (H7N7, H7N3) and newer H7 subtype viruses of the Eurasian lineage (H7N9) were detected in the animals receiving the AS03-adjuvanted vaccines. Animals receiving two immunizations of the AS03-adjuvanted vaccines were protected from weight loss and fever in the homologous challenge study and had no detectable virus in throat or lung samples. In addition, microscopic examination post-challenge showed animals immunized with the AS03-adjuvanted vaccines had the least signs of lung injury and inflammation, consistent with the greater relative efficacy of the adjuvanted vaccines. In conclusion, this study demonstrated that the AS03-adjuvanted H7N9 vaccines elicited high levels of homologous and heterologous antibodies and protected against H7N9 virus damage post-challenge.

Discordant evolution of organellar genomes in peas (Pisum L.)

Plastids and mitochondria have their own small genomes, which do not undergo meiotic recombination and may have evolutionary fates different from each other and that of the nuclear genome. For the first time, we sequenced mitochondrial genomes of pea (Pisum L.) from 42 accessions mostly representing diverse wild germplasm from throughout the wild pea geographical range. Six structural types of the pea mitochondrial genome were revealed. From the same accessions, plastid genomes were sequenced. Phylogenetic trees based on the plastid and mitochondrial genomes were compared. The topologies of these trees were highly discordant, implying not less than six events of hybridisation between diverged wild peas in the past, with plastids and mitochondria differently inherited by the descendants. Such discordant inheritance of organelles could have been driven by plastid-nuclear incompatibility, which is known to be widespread in crosses involving wild peas and affects organellar inheritance. The topology of the phylogenetic tree based on nucleotide sequences of a nuclear gene, His5, encoding a histone H1 subtype, corresponded to the current taxonomy and resembled that based on the plastid genome. Wild peas (Pisum sativum subsp. elatius s.l.) inhabiting Southern Europe were shown to be of hybrid origin, resulting from crosses of peas related to those presently inhabiting the eastern Mediterranean in a broad sense. These results highlight the roles of hybridisation and cytonuclear conflict in shaping plant microevolution.