Susceptibility and transmissibility of pigeons to Asian lineage highly pathogenic avian influenza virus subtype H5N1.

To determine whether or not pigeons are susceptible to infection with Asian lineage highly pathogenic (HP) avian influenza virus (AIV) subtype H5N1 and can serve as a transmission host for H5N1 HPAIV, we experimentally infected 187 young and adult pigeons with five different isolates of H5N1 HPAIV and co-habited some experimentally infected pigeons with susceptible specific pathogen free chickens. Results showed that all infected pigeons remained clinically healthy during the observation period. No gross lesions or histopathological changes were observed in the infected pigeons, and haemagglutination inhibition antibodies were not detected in serum samples of the infected pigeons. Additionally, all chickens placed in contact with AIV H5N1 infected pigeons remained healthy, and no virus or haemagglutination inhibition antibodies were detected in samples from the chickens. Our data suggest that pigeons are not susceptible to Asian lineage H5N1 HPAIV and do not transmit the virus to chickens.     

Partial protection against challenge with the highly pathogenic H5N1 influenza virus isolated in Japan in chickens infected with the H9N2 influenza virus

Summary The protective effect of the A/Ck/Yoko/aq55/01 (H9N2) avian influenza virus against the highly pathogenic H5N1 virus, i.e., A/Ck/Yama/7/04 (genotype V), was examined. Three 5-week-old chickens were inoculated intranasally with the H9N2 virus (10^8.6 EID₅₀/head) and were kept with two contact chickens. All of the infected chickens were reinoculated with the same virus at 20 weeks of age, and 10 days later, they were challenged intranasally with the H5N1 virus (10^4.0 EID₅₀/head). Five chickens simultaneously challenged with only the H5N1 virus (challenge control) died within 4 days postchallenge (d.p.c.). In contrast, four out of the five challenged, immune chickens died from 5 to 8 d.p.c. The median time to death in the immune chickens (6.3 days) was significantly longer than that in the challenge controls (3.4 days) (P < 0.01). No H5N1 virus shedding into the tracheae and feces of the challenged, immune chickens were detected for 3 d.p.c., but H5 genes were detectable in only one chicken by a loop-mediated isothermal amplification method. The H5N1 viruses were detected in the tracheae and/or feces of the dead immune chickens at death or 1 to 2 days before death. Only one out of the five challenged, immune chickens survived the H5N1 challenge without any signs for 14 d.p.c., but the virus and H5 gene were sporadically detected in the trachea only 7 and 14 d.p.c., respectively. This study shows that the H9N2 viruses may have the potential to induce cross-protection to the challenge with a recent lethal H5N1 virus (genotype V).     

Co-infection of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> or <Emphasis Type="Italic">Haemophilus paragallinarum</Emphasis> exacerbates H9N2 influenza A virus infection in chickens

Summary H9N2 influenza viruses are frequently isolated from chicken meat and bone marrow imported from China to Japan since 2001. These isolates were experimentally inoculated into specific pathogen-free chickens intranasally. Viruses were recovered from the meat and bone marrow of birds showing no overt signs. On the other hand, chickens co-infected with H9N2 virus and either Staphylococcus aureus or Haemophilus paragallinarum showed clinical signs severer than those shown by birds infected only with the virus alone or each of the bacteria alone. In addition, H9N2 viruses were more efficiently recovered from the chickens co-infected with S. aureus or H. paragallinarum than those from the birds infected with only the virus. The present results indicate that co-infection of H9N2 influenza virus with S. aureus or H. paragallinarum enhances the replication of the virus in chickens, resulting in exacerbation of the H9N2 virus infection.     

从我国人群中再次分离到H9N2亚型流感病毒

目的 了解分离流感病毒毒株表面抗原亚型和特性及其来源。方法 病毒通过ＭＤＣＫ细胞分离,用红细胞凝集抑制（ＨＩ）和神经氨酸酶抑制（ＮＩ）测定对病毒株表面抗原进行鉴定和特性分析,人血清中抗体测定采用ＨＩ和中和实验。对患者进行个」案调查。结果 分离物为甲型流感病毒Ｈ９Ｎ２亚型,属Ｇ９类似毒株,它的ＨＡ抗原特性与已经从人、鸡和鸽分离到的Ｈ９Ｎ２亚型毒株均有差异。患者恢复期血清对分离物的ＨＩ抗体滴度为４００     

Development of an antigen-capture ELISA for detection of H7 subtype avian influenza from experimentally infected chickens

Emergence of highly pathogenic avian influenza H7N1 was due to mutation of low pathogenic avian influenza H7N1 strain, which caused outbreaks in Italy between 1999 and 2000, and resulted in complete mortality of infected poultry. This outbreak places increased importance on the early detection of H7N1 AIV. Here we describe the development of a detection method for H7N1 virus from infected chickens using a specific antigen-capture-ELISA (AC-ELISA). A panel of mAbs was developed against the surface antigen HA of H7N1 AIV strain A/chicken/Singapore/94. The mAbs were screened by immunofluorescence assays, ELISA and immunoblotting. Selected mAbs 5E5 and 8F10 were of isotypes IgM and IgG and were conformation- or linear epitope-specific, respectively. These mAbs were used as capture antibodies for AC-ELISA development. The detection limit was as little as 10(2)-10(3) TCID(50) units of virus derived from tissue culture supernatants. Virus from the tracheal swab samples of experimentally infected chickens was detected from days 3 to 7 post-infection using the AC-ELISA, with results being confirmed by RT-PCR. AIV subtypes H4N1, H5N3 H9N2 and H10N5 did not react in the AC-ELISA but were RT-PCR positive, indicating that this AC-ELISA is specific for H7N1 strains.     

Experimental assessment of the pathogenicity of eight avian influenza A viruses of H5 subtype for chickens, turkeys, ducks and quail

Clinical signs, death, virus excretion and immune response were measured in 2-week-old chickens, turkeys, quail and ducks infected by intramuscular, intranasal and contact routes with eight influenza viruses of H5 subtype. Six of the viruses: A/chicken/Scotland/59 (H5N1), ck/Scot; A/tern/South Africa/61 (H5N3), tern/SA; A/turkey/Ontario/ 7732/66 (H5N9); ty/Ont; A/chicken/Pennsylvania/1370/83 (H5N2); Pa/1370; A/turkey/Ireland/83 (H5N8); ty/Ireland, and A/duck/Ireland/ 113/84 (HSN8); dk/Ireland, were highly pathogenic for chickens and turkeys. Two viruses, A/chicken/Pennsylvania/1/83 (H5N2), Pa/1 and A/turkey/Italy/ZA/80 (H5N2), ty/Italy, were of low pathogenicity. Ck/Scot was more pathogenic for chickens than turkeys while ty/Ont was more pathogenic for turkeys than chickens. Other viruses showed little difference in their pathogenicity for these two hosts. No clinical signs or deaths were seen in any of the infected ducks. Only two viruses, dk/Ireland and ty/Ireland, produced consistent serological responses in ducks, although intramuscular infection with tern/SA and ty/Italy resulted in some ducks with positive HI titres. These four were the only viruses reisolated from ducks. Quail showed some resistance to viruses which were highly pathogenic for chickens and turkeys, most notably to ck/Scot and ty/Ont and to a lesser extent tern/SA and Pa/1370. Transmission of virus from intranasally infected birds to birds placed in contact varied considerably with both host and infecting virus and the various combinations of these.     

Isolation and preliminary characterisation of a paramyxovirus from collared doves (Streptopelia decaocto)

A haemagglutinating virus was isolated from the caecal tonsils of dead collared doves (Streptopelia decaocto). The virus was identified as an avian paramyxovirus showing some antigenic relationships with PMV-7 /dove/Tennessee/4/75 and PMV-?/pigeon/Otaru/76. Of 15 collared doves and 24 feral pigeons trapped and sampled at the site at which dead doves were found, only two pigeons had significant antibody litres to the isolate. The virus was apathogenic for experimentally infected pigeons and chickens and stimulated only a weak antibody response.     

Role of aflatoxin toxicity on transmissibility and pathogenicity of H9N2 avian influenza virus in turkeys

The study was conducted to investigate the role of aflatoxin on the infectivity and transmissibility of H9N2 AI virus. The experiment was performed on 80 non-vaccinated turkeys, divided into 4 groups of 20 birds each. Group A was kept as non-infected and a non-treated negative control; Group B was inoculated intratracheally with H9N2 AI virus (1 × 10⁷ EID₅₀) at 4 weeks of age; Group C was fed on a diet containing 0.5 ppm aflatoxin from Day 1 through the entire experiment period and Group D was fed on diet containing 0.5 ppm aflatoxin as for Group C but inoculated intratracheally with H9N2 AI virus (1 × 10⁷ EID₅₀) at the fourth week of age and then mixed with naïve birds. Infected and contact birds showed clinical signs of different severity, showing the most prominent disease signs in birds of the aflatoxin + H9N2 group. All infected birds showed virus shedding, however, the pattern of virus shedding was different for birds of the aflatoxin + H9N2 group showing pronounced virus secretion. Similarly, efficient transmission of virus was observed between infected and contact birds, but more prominent virus transmission was seen in those birds inoculated and fed aflatoxin-treated diet. Moreover, significantly lower antibody titres against H9N2 AIV were observed in birds fed aflatoxin-treated diet, indicating an immunotoxic nature of aflatoxin as the reason for poor seroconversion. Similarly, decreased IFN_γ mRNA expression and higher mortality (35%) suggest an immunotoxic and immunosuppressive effect of aflatoxin leading to enhanced pathogenesis of H9N2 viruses in aflatoxin-fed birds. The immunosuppressive nature of aflatoxin might delay influenza virus clearance and this may be one of the reasons for increased pathogenicity of H9N2 LPAI viruses in turkeys under field conditions.     

Characterization of two pigeon paramyxovirus type 1 isolates in China

For over three decades, there has been a continuing panzootic caused by a virulent variant avian paramyxovirus type 1 strain, the so-called pigeon paramyxovirus type 1. It is found primarily in racing pigeons, but it has also spread to wild birds and poultry. In this study, two pigeon paramyxovirus type 1 strains, SD12 and BJ13, obtained from diseased pigeons in China, were characterized. Phylogenetic analysis based on complete sequences allowed characterization of both strains as genotype VI, class II. Further phylogenetic analysis of a 374-nucleotide section of the fusion gene showed that SD12 fell into lineage VIbii-d and BJ13 into VIbii-f. The deduced amino acid sequence of the cleavage site of the fusion protein confirmed that both isolates contained the virulent motif ¹¹²K/RRQKR↓F¹¹⁷ at the cleavage site. Nevertheless, the values of intracerebral pathogenicity indices showed the SD12 isolate to be a velogenic strain and BJ13 isolate to be a mesogenic strain. The SD12 isolate was further investigated via clinical observation, RNA detection, histopathology and viral serology in experimentally infected 3-week-old chickens. It showed a mild pathological phenotype in chickens, with viral replication restricted to a few tissues. The molecular mechanism for the SD12 isolate to have a virulent motif but low levels of virulence for chickens requires further study.     

Molecular analysis of the hemagglutinin genes of Australian H7N7 influenza viruses: Role of passerine birds in maintenance or transmission?

In 1985 a fowl plague-like disease occurred in chickens in Lockwood, Victoria, Australia and caused high mortality. An H7N7 influenza virus was isolated from the chickens (A/Chicken/Victoria/1/85); additionally, an antigenically similar virus was isolated from starlings (A/Starling/Victoria/5156/85) and serological evidence of H7N7 virus infection was found in sparrows. Antigenic analysis with monoclonal antibodies to H7, oligonucleotide mapping of total vRNA, and sequence analysis of the HA genes established that the chicken and starling influenza viruses were closely related and probably came from the same source. There was high nucleotide sequence homology (95.3%) between the HA genes of A/Chick/Vic/85 and a fowl plague-like virus isolated from chickens in Victoria 9 years earlier [A/Fowl/Vic/76 (H7N7)]. The sequence homologies indicated that the A/Chick/Vic/85 and A/Fowl/Vic/76 were derived from a common recent ancestor, while another recent H7N7 virus, Seal/Mass/1/80 originated from a different evolutionary lineage. Experimental infection of chickens and starlings with A/Chick/Vic/1/85 (H7N7) was associated with high mortality (100%), transmission to contact birds of the same species, and virus in all organs. In sparrows one-third of the birds died after infection and virus was isolated from most organs; transmission to contact sparrows did not occur. In contrast, the H7N7 virus replicated in ducks and spread to contact ducks but caused no mortality. These studies establish that the host species plays a role in determining the virulence of avian influenza viruses, and provide the first evidence for transmission of virulent influenza viruses between domestic poultry and passerine birds. They support the hypothesis that potentially virulent H7N7 influenza viruses could be maintained in ducks where they cause no apparent disease and may sometimes spread to other wild birds and domestic poultry.     

Production and diagnostic application of monoclonal antibodies against influenza virus H5

Nine monoclonal antibodies (mAbs) against avian influenza virus (AI) H5 subtype from mice immunized with inactivated virus H5N1 (A/Turkey/ON/6213/66) were produced. Upon testing, the results indicated that the binding epitopes of eight out of the nine mAbs were conformational, while one mAb (#7) reacted with denatured H5N1 only. Two mAbs #10 and #11 reacted with all of the thirteen H5 strains tested indicating that the binding epitopes of these mAbs were conserved among these H5 subtypes.Possible applications of these mAbs in rapid tests for H5 antigen were explored. Double antibody sandwich (DAS) ELISAs were developed using two selected mAbs #10 and #11. This DAS ELISA detects specific H5 viruses and is able to identify all thirteen H5 strains tested. Three mAbs showed reactivity with AI H5 antigen for both immunofluorescence (IF) and immunohistochemistry. A cELISA used to screen chickens that had been infected with an H5 virus was developed with mAb #9 and recombinant H5 antigen. The sera from chickens that have been infected with an H5N1 virus were examined using the cELISA. 80% of the sera from H5 infected chickens showed a positive H5 specific antibody response at 7 days post-infection (dpi) and remained positive until the end of the experiment on day 30 (>40% inhibition). This panel of the AI H5 specific mAbs is valuable for the development of various immunoassays.     

The influenza virus gene pool in a poultry market in South central china

We surveyed influenza activity in a live poultry market in Central China for 16 months, isolating viruses from 1% of 6360 fecal samples. We obtained multiple H3N6, H9N2, H2N9, H3N3, and H4N6 isolates and single H1N1 and H3N2 isolates. Two distinct H3 molecules were identified; other hemagglutinin subtypes were phylogenetically homogeneous. The H3N6 viruses (9 genotypes) and H9N2 viruses (4 genotypes) were genetically heterogeneous, whereas the H2N9, H3N3 and H4N6 viruses had single genotypes. Thirteen representative viruses were tested for their ability to replicate in quail and chickens. All tested viruses replicated in the respiratory tract of quail. Only nine of the viruses were shed in detectable levels in infected chickens, and four of these were detected in less than 50% of infected birds. A single H4N6 isolate caused disease and systemic spread in chickens. These findings show that quail are broadly susceptible to different subtypes of influenza A virus.     

Generation and characterisation of monoclonal antibodies specific to avian influenza H7N9 haemagglutinin protein

Introduction: Emerging virulent strains of influenza virus pose a serious public health threat with potential pandemic consequences. A novel avian influenza virus, H7N9, breached the species barrier from infected domestic poultry to humans in 2013 in China. Since then, it has caused numerous infections in humans with a close contact to poultry. Materials and Methods: In this study, we describe the preliminary characterisation of five murine monoclonal antibodies (MAbs) developed against recombinant haemagglutinin (rHA) protein of avian H7N9 A/Anhui/1/2013 virus by their Western blot and enzyme-linked immunosorbent assay (ELISA) reactivity and binding affinity. Results: Of the five MAbs, four were highly specific to H7N9 HA and did not show any cross-reactivity in ELISA with rHA protein from pandemic as well as seasonal H1N1, H2N2, H3N2, H5N1 and influenza virus B (B/Brisbane/60/2008). However, one of the MAbs, MA-24, in addition to HA protein of H7N9 also reacted strongly with HA protein of H3N2 and weakly with HA of pandemic and seasonal H1N1 and H2N2. All the five MAbs also reacted with H7N9 rHA in Western blot. The MAbs bound H7N9 rHA with an equilibrium dissociation constant (K_D) ranging between 0.14 and 25.20 nM, indicating their high affinity to HA. Conclusions: These antibodies may be useful in developing diagnostic tools for the detection of influenza H7N9 virus infections.     

Evaluation of vaccination against infectious laryngotracheitis (ILT) with recombinant herpesvirus of turkey (rHVT-LT) and chicken embryo origin (CEO) vaccines applied alone or in combination

ABSTRACT

The chicken embryo origin (CEO) infectious laryngotracheitis (ILT) live attenuated vaccines, although capable of protecting against disease and reducing challenge virus replication, can regain virulence. Recombinant ILT vaccines do not regain virulence but are partially successful at blocking challenge virus replication. The objective of this study was to evaluate the effect of rHVT-LT vaccination on CEO replication and how this vaccination strategy enhances protection and limits challenge virus transmission to naïve contact chickens. The rHVT-LT vaccine was administered at 1 day of age subcutaneously and the CEO vaccine was administered at 6 weeks of age via eye-drop or drinking water. CEO vaccine replication post vaccination, challenge virus replication and transmission post challenge were evaluated. After vaccination, only the group that received the CEO via eye-drop developed transient conjunctivitis. A significant decrease in CEO replication was detected for the rHVT-LT?+?CEO groups as compared to groups that received CEO alone. After challenge, reduction in clinical signs and challenge virus replication were observed in all vaccinated groups. However, among the vaccinated groups, the rHVT-LT group presented higher clinical signs and challenge virus replication. Transmission of the challenge virus to naïve contact chickens was only observed in the rHVT-LT vaccinated group of chickens. Overall, this study found that priming with rHVT-LT reduced CEO virus replication and the addition of a CEO vaccination provided a more robust protection than rHVT alone. Therefore, rHVT-LT?+?CEO vaccination strategy constitutes an alternative approach to gain better control of the disease.     

Genetic and phenotypic characterization of a low-pathogenicity avian influenza H11N9 virus

An H11N9 low-pathogenicity avian influenza virus, A/duck/WA/663/97, was isolated from a sick Mandarin duck kept in an outdoor bird exhibit. Genetic and phenotypic characterization of the virus suggested that it originated from free-flying birds, a concept supported by genetic similarity with waterfowl isolates from the same geographic area and time period. This duck-origin virus had genetic features typical of H11 and N9 viruses, including no neuraminidase stalk deletion, no differences in putative glycosylation sites in either surface protein, and no addition of basic amino acid residues at the hemagglutinin cleavage site compared to published sequences. It replicated in both avian and mammalian cells in vitro, and experimentally challenged chickens developed mild acute upper respiratory lesions but no clinical signs of disease. It elicited immune responses in chickens, resulting in seroconversion in all infected birds, although antibody titers remained low over the experimental period.     

Characterization of avian influenza viruses isolated from domestic ducks in Vietnam in 2009 and 2010

In the surveillance of avian influenza in Vietnam, 26 H9N2, 1 H3N2, 1 H3N8, 7 H4N6, 3 H11N3, and 1 H11N9 viruses were isolated from tracheal and cloacal swab samples of 300 domestic ducks in April 2009, and 1 H9N6 virus from 300 bird samples in March 2010. Out of the 27 H9 virus isolates, the hemagglutinins of 18 strains were genetically classified as belonging to the sublineage G1, and the other nine belonged to the Korean sublineage. Phylogenetic analysis revealed that one of the 27 H9 viruses was a reassortant in which the PB2 gene belonged to the Korean sublineage and the other seven genes belonged to the G1 sublineage. Three representative H9N2 viruses were intranasally inoculated into ducks, chickens, pigs, and mice. On the basis of experimental infection studies, it was found that each of the three viruses readily infected pigs and replicated in their upper respiratory tracts, and they infected chickens with slight replication. Viruses were recovered from the lungs of mice inoculated with two of the three isolates. The present results reveal that H9 avian influenza viruses are prevailing and genetic reassortment occurs among domestic ducks in Vietnam. It is recommended that careful surveillance of swine influenza with H9 viruses should be performed to prepare for pandemic influenza.     

Low infectivity of a novel avian-origin H7N9 influenza virus in pigs

We studied the pathogenesis and transmissibility of a novel avian-origin H7N9 influenza virus in pigs. When pigs were infected with H7N9 influenza virus, they did not show any clear clinical signs (such as sneezing, fever and loss of body weight), and they shed viruses through their noses for 2 days after infection. No transmission occurred between infected and naïve pigs. Pigs suffered from mild pneumonia, which was accompanied by the induction of inflammatory cytokines and chemokines such as IL-8 and CCL1. Taken together, our results suggest that pigs may not play an active role in transmitting H7N9 influenza virus to mammals.