Virus Pathotype and Deep Sequencing of the HA Gene of a Low Pathogenicity H7N1 Avian Influenza Virus Causing Mortality in Turkeys

Munir Iqbal,Ian H. Brown,Sharon M. Brookes,Steve C. Essen,Kolli B. Reddy,John W. McCauley,Paul Digard

doi:10.1371/journal.pone.0087076

Munir Iqbal, Ian H. Brown + Show 5 more

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https://doi.org/10.1371/journal.pone.0087076

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Journal: PloS one	Publication Date: Jan 28, 2014
Citations: 40	License type: CC BY 4.0

Affiliation: The Pirbright Institute, Animal and Plant Health Agency

Abstract

Low pathogenicity avian influenza (LPAI) viruses of the H7 subtype generally cause mild disease in poultry. However the evolution of a LPAI virus into highly pathogenic avian influenza (HPAI) virus results in the generation of a virus that can cause severe disease and death. The classification of these two pathotypes is based, in part, on disease signs and death in chickens, as assessed in an intravenous pathogenicity test, but the effect of LPAI viruses in turkeys is less well understood. During an investigation of LPAI virus infection of turkeys, groups of three-week-old birds inoculated with A/chicken/Italy/1279/99 (H7N1) showed severe disease signs and died or were euthanised within seven days of infection. Virus was detected in many internal tissues and organs from culled birds. To examine the possible evolution of the infecting virus to a highly pathogenic form in these turkeys, sequence analysis of the haemagglutinin (HA) gene cleavage site was carried out by analysing multiple cDNA amplicons made from swabs and tissue sample extracts employing Sanger and Next Generation Sequencing. In addition, a RT-PCR assay to detect HPAI virus was developed. There was no evidence of the presence of HPAI virus in either the virus used as inoculum or from swabs taken from infected birds. However, a small proportion (<0.5%) of virus carried in individual tracheal or liver samples did contain a molecular signature typical of a HPAI virus at the HA cleavage site. All the signature sequences were identical and were similar to HPAI viruses collected during the Italian epizootic in 1999/2000. We assume that the detection of HPAI virus in tissue samples following infection with A/chicken/Italy/1279/99 reflected amplification of a virus present at very low levels within the mixed inoculum but, strikingly, we observed no new HPAI virus signatures in the amplified DNA analysed by deep-sequencing.

Highlights

Avian influenza (AI) viruses are divided into subtypes on the bases of the antigenic properties of their two surface glycoproteins, the haemagglutinin (HA) and the neuraminidase (NA)
The results presented in figure 3B clearly demonstrated that the presence of Low pathogenicity avian influenza (LPAI) cDNA in HPAIspecific detection reaction samples did not interfere with the highly pathogenic avian influenza (HPAI) virus cDNA detection specificity or sensitivity, with the slopes and detected by the H7 HA-common probe. (B) The effect of addition of cDNA reverse transcribed from LPAI virus RNA to the cDNA prepared from HPAI virus RNA. (6) A constant amount of LPAI (Italy/1279) virus RNA corresponding to 104.85 EID50 virus titre or (m) water was added to a 10-fold dilution series of RNA extracted from the HPAI virus (A/ostrich/Italy/984/00) infectious allantoic fluid containing 106 EID50 virus/0.1 ml
The aim of the study described here was to investigate whether there was selective pressure that might result in the generation of HPAI viruses during replication of a LPAI virus within an infected host

Summary

Introduction

Avian influenza (AI) viruses are divided into subtypes on the bases of the antigenic properties of their two surface glycoproteins, the haemagglutinin (HA) and the neuraminidase (NA). Viruses belonging to the H5 and the H7 subtypes are known to be able to evolve to a high pathogenicity (HP) form by acquiring a series of multiple basic amino acids (arginine and lysine residues) at the HA cleavage site [7]. HP forms of AI viruses are differentiated from their LP counterparts by acquiring an ability to replicate in the internal body organs and tissues leading to death due to organ failure [8].the differentiation of pathotypes (LP and HP forms) is performed using a combination of intravenous infection of chickens, to assess the clinical disease and define the intravenous pathogenicity index (IVPI) of a virus, and by molecular analysis for presence or absence of a series of basic amino acids at the cleavage site of HA molecule [7,9]. Whilst LPAI viruses do not cause severe disease in chickens infected experimentally, they are able to cause variable disease signs in other galliforme species [10,11]

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