An avian H5N1 influenza A virus (A/Hong Kong/156/97) was isolated from a tracheal aspirate obtained from a 3-year-old child in Hong Kong with a fatal illness consistent with influenza. Serologic analysis indicated the presence of an H5 hemagglutinin. All eight RNA segments were derived from an avian influenza A virus. The hemagglutinin contained multiple basic amino acids adjacent to the cleavage site, a feature characteristic of highly pathogenic avian influenza A viruses. The virus caused 87.5 to 100 percent mortality in experimentally inoculated White Plymouth Rock and White Leghorn chickens. These results may have implications for global influenza surveillance and planning for pandemic influenza.

https://science.sciencemag.org/content/sci/279/5349/393.full.pdf

Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness.

Recent outbreaks of highly pathogenic avian influenza A virus infections (H5 and H7 subtypes) in poultry and in humans (through direct contact with infected birds) have had important economic repercussions and have raised concerns that a new influenza pandemic will occur in the near future. The eradication of pathogenic avian influenza viruses seems to be the most effective way to prevent influenza pandemics, although this strategy has not proven successful so far. Here, we review the molecular factors that contribute to the emergence of pandemic strains.

Influenza: lessons from past pandemics, warnings from current incidents

Highly pathogenic (HP) avian influenza (AI) (HPAI) is an extremely contagious, multi-organ systemic disease of poultry leading to high mortality, and caused by some H5 and H7 subtypes of type A influenza virus, family Orthomyxoviridae. However, most AI virus strains are mildly pathogenic (MP) and produce either subclinical infections or respiratory and/or reproductive diseases in a variety of domestic and wild bird species. Highly pathogenic avian influenza is a List A disease of the Office International des Epizooties, while MPAI is neither a List A nor List B disease. Eighteen outbreaks of HPAI have been documented since the identification of AI virus as the cause of fowl plague in 1955. Mildly pathogenic avian influenza viruses are maintained in wild aquatic bird reservoirs, occasionally crossing over to domestic poultry and causing outbreaks of mild disease. Highly pathogenic avian influenza viruses do not have a recognised wild bird reservoir, but can occasionally be isolated from wild birds during outbreaks in domestic poultry. Highly pathogenic avian influenza viruses have been documented to arise from MPAI viruses through mutations in the haemagglutinin surface protein. Prevention of exposure to the virus and eradication are the accepted methods for dealing with HPAI. Control programmes, which imply allowing a low incidence of infection, are not an acceptable method for managing HPAI, but have been used during some outbreaks of MPAI. The components of a strategy to deal with MPAI or HPAI include surveillance and diagnosis, biosecurity, education, quarantine and depopulation. Vaccination has been used in some control and eradication programmes for AI.

Highly pathogenic avian influenza.

Role of hemagglutinin cleavage for the pathogenicity of influenza virus.

Mammalian influenza viruses are descendants of avian strains that crossed the species barrier and underwent further adaptation. Since 1997 in southeast Asia, H5N1 highly pathogenic avian influenza viruses have been causing severe, even fatal disease in humans. Although no lineages of this subtype have been established until now, such repeated events may initiate a new pandemic. As a model of species transmission, we used the highly pathogenic avian influenza virus SC35 (H7N7), which is low-pathogenic for mice, and its lethal mouse-adapted descendant SC35M. Specific mutations in SC35M polymerase considerably increase its activity in mammalian cells, correlating with high virulence in mice. Some of these mutations are prevalent in chicken and mammalian isolates, especially in the highly pathogenic H5N1 viruses from southeast Asia. These activity-enhancing mutations of the viral polymerase complex demonstrate convergent evolution in nature and, therefore, may be a prerequisite for adaptation to a new host paving the way for new pandemic viruses.

https://www.pnas.org/content/pnas/102/51/18590.full.pdf

The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host

Pathologic changes and distribution of viral antigen as determined by immunohistochemistry were compared among 4-wk-old specific-pathogen-free chickens inoculated intratracheally with avian influenza virus (AIV) isolates of either low or high pathogenicity. Viruses of low pathogenicity, previously characterized as mildly pathogenic (MP), included A/chicken/Pennsylvania/21525/83 (H5N2) (MP-Penn) and A/chicken/Alabama/7395/75 (H4N8) (MP-Alab). Viruses of high pathogenicity included A/chicken/Pennsylvania/1370/83 (H5N2), A/chicken/Victoria/A185/85 (H7N7), and A/turkey/Ontario/7732/66 (H5N9). Extremely variable clinical signs ranging from mild respiratory distress to high mortality were present among chickens inoculated with these viruses. Chickens inoculated with highly pathogenic (HP) virus had histologic lesions of necrosis and inflammation in cloacal bursa, thymus, spleen, heart, pancreas, kidney, brain, trachea, lung, and skeletal muscle, whereas chickens inoculated with MP virus had histologic lesions most frequently in lung and trachea or lacked histologic lesions. Immunospecific staining for avian influenza viral proteins was most common in cells within heart, lung, kidney, brain, and pancreas of chicken inoculated with HP viruses, but immunospecific staining was present only and infrequently in trachea and lung of chickens inoculated with MP-Penn AIV. MP-Alab did not produce lesions nor have viral antigen in inoculated chickens but did produce serologic evidence of infection. The pattern of organ involvement and viral antigen distribution in chickens intratracheally inoculated with HP AIV isolates indicates a common capability to spread beyond the respiratory tract and confirms the pantrophic replicative, pathobiologic, and lethal nature of the viruses. However, variability in severity and lesion distribution exists between different HP AIVs. By contrast, MP viruses had the ability to replicate in respiratory or enteric tracts or both and produce lesions within the respiratory tract. These MP viruses exhibited a restricted ability to replicate or produce lesions or both in nonrespiratory or nonenteric tissues; such effects were associated with only sporadic deaths.

Comparative pathology of chickens experimentally inoculated with avian influenza viruses of low and high pathogenicity.

SUMMARY Titration endpoints from any 2-fold dilution series represent either true or modified base-2 logarithms and can be recorded conveniently by tube or dilution number. The simple arithmetic mean of titers thus expressed can be easily converted to geometric mean titers (GMT) by use of a table of antilogarithms, which is presented. This approach not only facilitates the handling of large volumes of data but also simplifies GMT calculation. RESEARCH NOTE

A simple method for recording and analyzing serological data.

The influence of the composition of water-in-oil emulsions on their physical characteristics was determined by preparing experimental emulsions with various water-to-oil ratios and various emulsifiers. Emulsions containing Tween 80 in the aqueous phase and Arlacel A or Arlacel 80 in the oil phase were lower in viscosity than emulsions containing only an oil-phase emulsifier. Viscosity decreased as the concentration of oil increased. Oil-emulsion vaccines prepared with aqueous- and oil-phase emulsifiers had low viscosity, were stable for more than 12 weeks at 37 C, and induced a marked primary antibody response in chickens.

https://www.jstor.org/stable/pdfplus/1589643.pdf

Preparation of inactivated oil-emulsion vaccines with avian viral or Mycoplasma antigens.

Inactivated oil-emulsion (OE) Newcastle disease (ND) and avian influenza (AI) vaccines were injected into 18-day-old white rock (WR) and white leghorn (WL) chicken embryos to evaluate their immunologic efficacy and their effects on hatchability. Embryonating eggs were inoculated at 1.5 inches depth with various vaccine volumes and antigen concentrations. Serum hemagglutination-inhibition (HI) titers were first detected in chickens at 2 wk posthatch. Protection against morbidity and mortality was demonstrated in all of 10 chickens vaccinated as embryos and challenged with viscerotropic velogenic ND virus at 53 days of age and also in all of eight in ovo- vaccinated chickens challenged with highly pathogenic AI virus at 34 days of age. All of five unvaccinated control chickens for each respective ND- and AI-vaccinated group died. In pooled groups from successive hatches, the hatchability of WR or WL embryos injected with 100 microliters of vaccine was not significantly different (P > 0.05) from unvaccinated hatchmate controls when needle gauges of 22, 20, and 18 were used. Seroconversion rates of chickens vaccinated as embryos ranged from 27% to 100% with ND vaccination and 85% to 100% for AI vaccination. For ND, geometric mean HI titers of chickens per vaccine group ranged from 11 to 733, and in pooled groups, the range was 49 to 531. Titers for AI vaccine groups ranged from 156 to 1178. This study demonstrated that acceptable hatchability, seroconversion rates, and protective immunity can be attained with in ovo inoculation of ND or AI OE vaccines if the vaccines are prepared with sufficient antigen and administered properly.

In ovo vaccination of chicken embryos with experimental newcastle disease and avian influenza oil-emulsion vaccines

Replicate samples of serum from chickens immune to Newcastle disease were titrated to determine the influence of certain test conditions on hemagglutination-inhibition (hi) titers. The test conditions studied were those most likely to vary in normal laboratory operations. The most marked effect on magnitude of HI titers was incubation time of twofold serum dilutions in antigen-saline; the average titer increase after incubation of the serum-antigen mixture for 1 hr at 37 C was log2 2.3 (fivefold). Twofold increases in virus concentration of the antigen-saline diluent caused an average titer reduction of log2 0.8. Shifts in HI titers were only minor with twofold changes in erythrocyte concentration (log2 0.3), with variations of test reading times from 0.5 to 2.0 hr (log2 0.1), and with variations in the period between preparation of the initial 1:10 serum dilution in antigen-saline and the subsequent serum dilutions (log2 0.3).

Max Brugh

Papers

Comparative pathology of chickens experimentally inoculated with avian influenza viruses of low and high pathogenicity.

A simple method for recording and analyzing serological data.

Preparation of inactivated oil-emulsion vaccines with avian viral or Mycoplasma antigens.

In ovo vaccination of chicken embryos with experimental newcastle disease and avian influenza oil-emulsion vaccines

The influence of test conditions on Newcastle disease hemagglutination-inhibition titers.