A real-time reverse transcriptase PCR (RRT-PCR) assay based on the avian influenza virus matrix gene was developed for the rapid detection of type A influenza virus. Additionally, H5 and H7 hemagglutinin subtype-specific probe sets were developed based on North American avian influenza virus sequences. The RRT-PCR assay utilizes a one-step RT-PCR protocol and fluorogenic hydrolysis type probes. The matrix gene RRT-PCR assay has a detection limit of 10 fg or approximately 1,000 copies of target RNA and can detect 0.1 50% egg infective dose of virus. The H5- and H7-specific probe sets each have a detection limit of 100 fg of target RNA or approximately 103 to 104 gene copies. The sensitivity and specificity of the real-time PCR assay were directly compared with those of the current standard for detection of influenza virus: virus isolation (VI) in embryonated chicken eggs and hemagglutinin subtyping by hemagglutination inhibition (HI) assay. The comparison was performed with 1,550 tracheal and cloacal swabs from various avian species and environmental swabs obtained from live-bird markets in New York and New Jersey. Influenza virus-specific RRT-PCR results correlated with VI results for 89% of the samples. The remaining samples were positive with only one detection method. Overall the sensitivity and specificity of the H7- and H5-specific RRT-PCR were similar to those of VI and HI.

Development of a Real-Time Reverse Transcriptase PCR Assay for Type A Influenza Virus and the Avian H5 and H7 Hemagglutinin Subtypes

https://www.thelancet.com/pdfs/journals/lancet/PIIS0140-6736(97)11212-0.pdf

Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus

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.

VERY few veterinary surgeons have thought fit to write a book on diseases of poultry. Mr. Ernest Gray has done justice to the subject and is to be congratulated on his effort. A book of this size, written by one with specialized knowledge, will add to the value of any library or private bookshelf. Diseases of Poultry Their Aetiology, Diagnosis, Treatment and Control; with a Section on the Normal Anatomy and Physiology of the Fowl. By Ernest Gray. (Lockwood's Agricultural and Horticultural Handbooks.) Pp. x + 198 + 16 plates. (London: Crosby Lockwood and Son, Ltd., 1940.) 9s. 6d. net.

Diseases of Poultry

A review of avian influenza in different bird species

Molecular changes in the haemagglutinin (HA)-coding regions and proteolytic cleavage sites from multiple H5N2 subtype viruses isolated during a recent outbreak of avian influenza (AI) in central Mexico have been characterized. Eighteen isolates, collected during a 15 month period (October 1993 to January 1995) from six central states, were sequenced. None of the 18 predicted HA1 amino acid sequences were identical and changes were not restricted to a specific region of the sequence. Phylogenetic analyses of the HA1 sequences demonstrated two virus lineages, designated Puebla and Jalisco, with sequence variation as high as 10.5% for amino acid and 6.2% for nucleotide sequences. During the latter months of the surveillance period, highly pathogenic (HP) strains of Al emerged causing lethal disease in commercial poultry flocks. In each of the HP strains isolated, the HA protein was cleaved in chicken embryo fibroblast cells in the absence of trypsin, and two alterations not found in earlier non-HP isolates were detected. In the HA protein, HP strains all had a glutamic acid → lysine substitution at amino acid position 324 and an insertion of arginine and lysine as new residues 325 and 326. The insertion appears to be due to a duplication of the nucleotide sequence AAAGAA at nucleotide positions 965–970 of the HA1-coding region. Computer-assisted secondary structure analyses place the target for the insertion in a predicted RNA stem-loop structure. A mechanism is suggested by which the polymerase duplicates the sequence.

Heterogeneity in the haemagglutinin gene and emergence of the highly pathogenic phenotype among recent H5N2 avian influenza viruses from Mexico

The presence of low-pathogenic H7 avian influenza virus (AIV), which is associated with live-bird markets (LBM) in the Northeast United States, was first detected in 1994 and, despite efforts to eradicate the virus, surveillance of these markets has resulted in numerous isolations of H7 AIVs from several states from 1994 through 1998. The hemagglutinin, nonstructural, and matrix genes from representative H7 isolates from the LBM and elsewhere were sequenced, and the sequences were compared phylogenetically. The hemagglutinin gene of most LBM isolates examined appeared to have been the result of a single introduction of the hemagglutinin gene. Evidence for evolutionary changes were observed with three definable steps. The first isolate from 1994 had the amino acid threonine at the -2 position of the hemagglutinin cleavage site, which is the most commonly observed amino acid at this site for North American H7 AIVs. In January 1995 a new genotype with a proline at the -2 position was detected, and this genotype eventually became the predominant virus isolate. A third viral genotype, detected in November 1996, had an eight-amino-acid deletion within the putative receptor binding site. This viral genotype appeared to be the predominant isolate, although isolates with proline at the -2 position without the deletion were still observed in viruses from the last sampling date. Evidence for reassortment of multiple viral genes was evident. The combination of possible adaptive evolution of the virus and reassortment with different influenza virus genes makes it difficult to determine the risk of pathogenesis of this group of H7 AIVs.

Phylogenetic Analysis of H7 Avian Influenza Viruses Isolated from the Live Bird Markets of the Northeast United States

A Novel Carbohydrate Addition Site on the Hemagglutinin Protein of a Highly Pathogenic H7 Subtype Avian Influenza Virus

Consistent occurrence of hemagglutinin variants among avian influenza virus isolates of the H7 subtype

A study was conducted to examine the effect of avian influenza virus (AIV) on chicken lymphocyte activation. Unprimed or Brucella abortus antigen (Ag)-primed lymphocytes were incubated with various doses of the T-cell mitogen concanavalin A (Con A) or Ag, respectively, plus serial dilutions of inactivated AIV for 72 hr, and cell proliferation was measured via uptake of tritiated thymidine. AIV enhanced the proliferative response to Con A or Ag by 150% or better, and the enhancement decreased in a viral dose-dependent manner. The effects were more readily observed in cells that had not been maximally activated by the Con A or Ag. The enhanced response was observed in lymphocytes from both white rock and white leghorn breeds of chicken and in mature peripheral blood lymphocytes or immature thymocytes. The viral activity could be abrogated by pre-treatment of the viral preparation with AIV-specific antisera or prior adsorption of the AIV with chicken erythrocytes. These results indicate that AIV can interact with and modify the in vitro activity of chicken lymphocytes and may exert modulatory effects on the avian immune system.

https://www.jstor.org/stable/info/1591154

John W. Latimer

Papers

Heterogeneity in the haemagglutinin gene and emergence of the highly pathogenic phenotype among recent H5N2 avian influenza viruses from Mexico

Phylogenetic Analysis of H7 Avian Influenza Viruses Isolated from the Live Bird Markets of the Northeast United States

A Novel Carbohydrate Addition Site on the Hemagglutinin Protein of a Highly Pathogenic H7 Subtype Avian Influenza Virus

Consistent occurrence of hemagglutinin variants among avian influenza virus isolates of the H7 subtype

Stimulatory effect of avian influenza virus on chicken lymphocytes.