Pathobiology of avian influenza virus infection in minor gallinaceous species: a review

Susceptibility to avian influenza viruses (AIVs) can vary greatly among bird species. Chickens and turkeys are major avian species that, like ducks, have been extensively studied for avian influenza. To a lesser extent, minor avian species such as quail, partridges, and pheasants have also been investigated for avian influenza. Usually, such game fowl species are highly susceptible to highly pathogenic AIVs and may consistently spread both highly pathogenic AIVs and low-pathogenic AIVs. These findings, together with the fact that game birds are considered bridge species in the poultry–wildlife interface, highlight their interest from the transmission and biosecurity points of view. Here, the general pathobiological features of low-pathogenic AIV and highly pathogenic AIV infections in this group of avian species have been covered.     

Pathogenesis of pancreatic atrophy by avian influenza a virus infection

Specific-pathogen-free (SPF), 2-day-old chicks were inoculated with type A influenza virus (A/whistling swan/Shimane/499/83/(H5N3)) into their caudal thoracic air sac. The original isolate of the virus was of low virulence (ICPI 0. 20 to 0.40), and was passaged 10 times through the respiratory organs of SPF chicks. Most of the chicks inoculated with the passaged virus (strain 499) showed respiratory and alimentary signs. Three of 30 chicks died on days 2, 6 and 7 post-inoculation (p.i.). Almost half of the infected chicks showed poor growth, and the variation of body size in the flock became prominent from day 10 p.i. Infected chicks consistently had pathological changes in the pancreas, liver, kidneys and respiratory tracts, and occasionally in the brain, duodenum and bone marrow. Positive immunoreaction to avian influenza virus (AIV) antigen and recovery of the virus persisted for longer period in the pancreas than in other organs. The pancreatic lesions were caused by a direct, lytic virus infection of the acinar cells and contributed to poor growth of the chicks.     

Detection by microneutralization of antibodies against avian influenza virus in an endemic avian influenza region

Detection by microneutralization of low-titre antibodies (anti-H5 micro-NT titre ≤1 : 80) against avian influenza virus (H5N1) is usually taken to be a false-positive result. In this prospective study of 242 intensive-care unit patients admitted for severe community-acquired pneumonia, the prevalence of low-titre anti-H5 micro-NT was 2.4%. Prior exposure to poultry was the sole independent risk factor for these low-titre antibodies (adjusted OR 42.41; 95% CI 22.45–64.51; p <0.001). We suggest that low anti-H5 micro-NT titres be interpreted in conjunction with plausible poultry, environmental and human exposure to H5N1.     

Prototype single step lateral flow technology for detection of avian influenza virus and chicken antibody to avian influenza virus

A rapid and effective lateral flow assay (LFA) for detection of avian influenza virus (AIV) was developed. For antigen capture, the assay used monoclonal antibody specific for a conserved nuclear protein (NP) epitope, immobilized on a cellulose acetate matrix, in conjunction with a second NP monoclonal antibody chemically linked to either coloured latex beads or colloidal gold particles contained in a sample pad for detection. Virus sample added to the sample pad flowed into the trapping antibody to form a visible band as well as a second, control band further along the acetate strip. The control band consisted of recombinant protein A/G, also immobilized on the matrix. A second LFA for detection of chicken antibody to AIV was developed where NP antigen was immobilized on the matrix with recombinant protein A/G immobilized as a control band. Latex beads or colloidal gold particles to which monoclonal anti-chicken antibody was attached, were used as the indicator system.     

Incomplete avian influenza A virus displays anomalous interference

Summary The interfering activity of incomplete avian influenza A (fowl plague) virus was titrated using an infectious centre reduction assay. It was found that the interference property had poissonian kinetics only at low multiplicities of infection.With 2 Figures     

Cytometric microsphere array for subtyping avian influenza virus

Avian influenza is a highly contagious disease, and different subtypes of avian influenza virus (AIV) have different levels of pathogenicity. A microsphere-based fluorescent assay was initially established for subtyping AIV. DNA fragments were amplified with biotinylated primers. AIV subtype-specific DNA probes with an amino-linker at the 5' end were covalently bound with carboxy-modified encoded beads. The modified beads and the denatured DNA fragments were mixed together for hybridization. Then, quantum dots-streptavidin (QDs-streptavidin) was added to conjugated biotinylated PCR products. The reaction products were screened by flow cytometry. AIV strains (such as H5N1 and H9N2) could be determined and subtyped according to their combination of encoded beads and fluorescent QDs. The method's combined sensitivity of the nucleic acids of H5N1 and H9N2 avian influenza virus at a threshold of 74 pg and 1 pg could be detected. This is a powerful method for detecting many pathogens or many types of a pathogen simultaneously.     

Attenuation of a human H9N2 influenza virus in mammalian host by reassortment with an avian influenza virus

Summary. In order to develop a surrogate virus strain for production of an inactivated influenza vaccine against a human H9N2 virus, A/Hong Kong/1073/99 (HK1073: H9N2) was co-infected in embryonated chicken eggs with an apathogenic avian influenza virus, A/Duck/Czechoslovakia/56 (Dk/Cz: H4N6), for gene segment reassortment. Multiple-gene reassortants obtained were examined for replication in mammalian hosts in vitro and in vivo by infecting MDCK cells and by intranasal administration to hamsters, respectively. A 2–6 gene reassortant with both surface glycoproteins of HK1073 origin and the rest of Dk/Cz origin, HK/CZ-13, was shown to replicate poorly in the mammalian hosts both in vivo and in vitro comparing with HK1073, although this reassortant replicated as efficiently as each parental strain in embryonated eggs. No sequence difference was observed in the HA1 region between HK1073 and HK/CZ-13, indicating that the reassortant would be equivalent in its immunogenicity to the parental HK1073 strain when it is used as an inactivated vaccine. A virus strain with attenuation in mammalian hosts is preferable for production of an H9 vaccine, since it should reduce the risk of manufacturing-related infections of employees during the vaccine production. HK/CZ-13 can therefore be a surrogate strain for production of an inactivated vaccine as well as diagnostic antigens in case of a possible future pandemic caused by an HK1073-like H9 influenza virus.     

An avian influenza A virus killing a mammalian species — the mink

Summary During October of 1984 an influenza epidemic occurred on mink farms in the coastal region of South Sweden. Six strains of an influenza A virus were isolated. All six isolates were of the H10 subtype in combination with N4. The H10 subtype in combination with various N subtypes was hitherto only known to occur in avian strains, the prototype being the A/chicken/Germany/N/49 (H10N7) virus.With 1 Figure     

Incomplete avian influenza virus contains A defective non-interfering component

Summary Incomplete avian influenza (fowl plague) virus derived by undiluted egg passage, displayed an increased capacity to promote the synthesis of intracellular virus-specific proteins when compared with standard virus. Thein vitro virionbound RNA polymerase activity of incomplete virus was also greater than could be explained by the presence of residual infectious virus.When the titres of infectious and interfering virus species were determined directly, they did not account for all the virus present. The existence of defective non-interfering (DNI) virus, even in standard virus preparations, was inferred. DNI virus is capable of initiating infection, synthesis of mRNA and proteins but cannot complete a productive replication cycle, and does not interfere with multiplication of standard virus. Such DNI virus could exaggerate the true extent of DI virus formation by lowering the PFU:HAU ratio and so account for the failure to correlate infectivity with RNA composition or RNA polymerase activity.With 3 Figures     

Growth characteristics of influenza virus type C in avian hosts

Summary Influenza virus type C could be propagated to high yield in primary chick embryo kidney cell culture (PCEK) provided that trypsin (2 µg/ml) was used as a medium supplement. The virus could also be titrated by plaque assay using PCEK host cells and influenza C virus that had been plaque-purified in PCEK cells could then be serially passaged to high titer using the allantoic route of 10–11-day-old embryonated eggs.With 1 Figure     

Genes involved in the virulence of an avian influenza virus

A/chicken/Japan/24 (Japan) (HavlNegl) isolated from an outbreak of fowl plague-like illness among chickens is comparable to fowl plague viruses in its virulence, causing paralysis and death of chickens within 3 days. It also kills chick embryos after allantoic infection of embryonated eggs. Another avian influenza virus, A/duck/Ukraine/1/63 (Ukraine) (Hav7Neg2), in contrast, causes no illness or death in either chickens or chick embryos. In an attempt to determine the genes involved in the observed difference in virulence, we prepared a series of recombinants between Japan and Ukraine viruses. Parental derivation of genes in the recombinants was determined by urea-polyacrylamide gel electrophoresis of viral RNA. The virulence was assessed by three criteria: (1) lethality for chickens after intracerebral inoculation, (2) lethality for embryos after allantoic inoculation into 15-day-old embryonated eggs, and (3) lethality for embryos after allantoic inoculation into 10-day-old embryonated eggs. The recombinants in which genes coding for hemagglutinin (HA), neuraminidase (NA), and matrix (M) proteins had been derived from Japan virus and five other genes from Ukraine virus were fully virulent by all three criteria. The recombinants which had received HA and NA genes from Japan virus showed a slightly but definitely reduced virulence in chickens and 15-day-old embryos but retained the virulence in 10-day-old embryos unchanged. The recombinants which had received only HA gene from Japan virus had a markedly diminished virulence in chickens and 15-day-old embryos but were as lethal for 10-day-old embryos as Japan virus. It was concluded that HA gene was the key determinant of virulence of Japan virus, but, in addition, NA and M genes were required for the full expression of virulence. Requirement of HA and NA genes was confirmed by reconstitution of virulence after reuniting the two genes.     

Protection afforded by avian influenza vaccination programmes consisting of a novel RNA particle and an inactivated avian influenza vaccine against a highly pathogenic avian influenza virus challenge in layer chickens up to 18 weeks post-vaccination

The efficacies of an oil adjuvanted-inactivated reverse genetics-derived H5 avian influenza virus (AIV) vaccine and an alphavirus replicon RNA particle (RP) AIV vaccine were evaluated in commercial Leghorn chickens. Challenge utilized A/turkey/MN/12582/2015, an isolate representing the U.S. H5N2 Clade 2.3.4.4 responsible for the 2015 highly pathogenic avian influenza (HPAI) epornitic in commercial poultry the United States. As part of a long-term, 36-week study, chickens were challenged at seven weeks of age after receiving a single vaccination, at 18 weeks of age following a vaccine prime-single boost, and at 36 weeks of age after a prime- double-boost. All vaccine programmes reduced virus oropharyngeal and cloacal shedding and mortality compared to the non-vaccinated control birds; however, chickens receiving at least one administration of the RP vaccine generally had diminished viral shedding especially from the cloacal swabbings. A detectable serum antibody response and protection were observed through 18 weeks post-vaccination. Our data suggest that, in conjunction with a comprehensive eradication, enhanced biosecurity and controlled marketing plan, vaccination programmes of commercial layer chickens with novel RP vaccines may represent an important tool for preventing HPAI-related mortalities and decreasing viral load during a catastrophic influenza outbreak.

RESEARCH HIGHLIGHTS

• Immunization of poultry following a vaccination schedule consisting of inactivated and RNA particle vaccines offered significant protection against lethal disease following HPAIV challenge.

• Virus shedding was significantly (P?<?0.05) reduced in chickens vaccinated with either inactivated and/or recombinant vaccines.

• Serum antibody titres were not a reliable indicator of protection.

• An inactivated vaccine containing 384 HAU of the homologous antigen was unable to induce complete protection.

     

Co-infection of mallards with low-virulence Newcastle disease virus and low-pathogenic avian influenza virus

Waterfowl are considered the natural reservoir of low-virulence Newcastle disease viruses (loNDVs) and low-pathogenic avian influenza viruses (LPAIVs). The objective of this study was to investigate the effect of co-infections with loNDV and LPAIV on the infectivity and excretion of these viruses in mallards. One-month-old mallards were inoculated intranasally with 10⁶ median embryo infectious doses of a wild-bird-origin loNDV and A/Mallard/MN/199106/99 (H3N8) LPAIV on the same day or received the LPAIV 2 or 5 days after loNDV inoculation. All mallards became infected with both viruses based on detection of seroconversion and viral shedding. Co-infection resulted in a higher number of cloacal swabs detected positive for LPAIV and a lower number of cloacal swabs detected positive for loNDV in some groups, although differences between groups were not statistically significant. Co-infection did not affect replication of LPAIV in epithelial cells of the lower intestine and bursa of Fabricius. In summary, the results of this study indicate that co-infection with LPAIV and loNDV does not affect the ability of mallards to be infected with either virus although it may have minimal effects on patterns (source and timing) of viral shedding.     

Reassortment and gene interactions in the crossing of low-pathogenic avian influenza H5 virus with human influenza virus

The reassortants obtained via the crossing of highly productive influenza virus A/Puerto Rico/8/34 (H1N1) strain and the low pathogenic avian influenza virus A/Duck/Primorie/2621/2001 (H5N2) strain were genotyped and characterized. The H5N2 reassortant having 6 genes from A/Puerto Rico/8/34 virus has the high level of reproduction in chick embryos, while slightly more moderate than in the parent A/Puerto Rico/8/34 strain. The reproduction of the H5N1 reassortant that had 7 genes from A/Puerto Rico/8134 virus was very low. The serial passage selection allowed the investigators to obtain the H5N1 strain that was reproductively close to the H5N2 reassortant. This variant had one amino acid substitution in hemagglutinin (N244D, H3 numbering) and a lower affinity for fetuin. By the level of virulence to mice, the H5N1 and H5N2 reassortants were close to A/Puerto Rico/8/34 virus and greatly differed in this respect from low virulent A/Duck/Primorie/2621/2001 (H5N2). The results are discussed in connection with the problem of vaccination when there is a threat for H5N1 virus subtype-caused pandemic.     

Experimental data on avian influenza virus detection using magnetic immunosorbents

To increase the sensitivity and specificity of detecting high-pathogenic avian influenza variant (HSN1), laboratory studies were conducted at low virus concentrations in water samples, by using magnetic immunosorbent (MIS) test systems and selective avian influenza virus concentrating units. MIS-based selective virus concentrating, followed by rapid assays (ELIZA and RT-PCR), detect the low concentrations (as low as 10 nm in 5,000 ml of water) of avian influenza A/H5N1 antigen and RNA. The method developed opens new avenues for indication of an avian influenza pathogen in different environmental objects, including the water of surface water reservoirs of unlimited volume, with varying pollutions and low virus concentrations.     

Contact infection of mink with 5 subtypes of avian influenza virus

Avian influenza viruses of H3N8, H11N4, H7N7, H8N4, and H5N3 infected mink by contact.     

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.     

Development and application of monoclonal antibodies against avian influenza virus nucleoprotein

Rapid and accurate diagnosis of avian influenza (AI) infection is important for an understanding epidemiology. In order to develop rapid tests for AI antigen and antibody detection, two monoclonal antibodies (mAbs) against influenza nucleoprotein (NP) were produced. These mAbs are designated as F26-9 and F28-73 and able to recognize whole AI virus particles as well as the recombinant NP. Both of the mAbs were tested in a slot blot for their reactivity against 15 subtypes of influenza virus; F28-73 reacted with all tested 15 subtypes, while F26-9 failed to react with H13N6 and H15N8. The mAb binding epitopes were identified using truncated NP recombinant proteins and peptide array techniques. The mAb F26-9 reacted with NP-full, NP-1 (638bp), NP-2 (315bp), NP-4 (488bp), and NP-5 (400bp) in the Western blot. The peptide array results demonstrated that the mAb F26-9 reacted with NP peptides 15-17 corresponding to amino acids 71-96. The mAb F28-73 recognized the NP-full, -1 and -4 fragments, but failed bind to NP-2, -3, -5, and any peptides. This antibody-binding site is expected to be contained within 1-162 amino acids of AI NP, although the exact binding epitope could not be determined. The two mAbs showed reactivity with AI antigen in immunofluorescence, immunohistochemistry and immune plaque assays. Immune response of AI infected animals was determined using the mAb F28-73 in a cELISA. All tested chickens were positive at 11 days post-infection and remained positive until the end of the experiment on day 28 (>50% inhibition). The two mAbs with different specificities are appropriate for developing various tests for diagnosis of AI infection.