Chorioallantoic membranes of embryonated chicken eggs as an alternative system for isolation of equine influenza virus

Abstract

BackgroundInfluenza virus isolation in embryonated chicken eggs (ECEs) is not applicable for rapid diagnosis, however it allows the recovery and propagation of the viable virus. A low number of infectious virus particles in the swabs, poor quality of samples or individual strain properties can lead to difficulties during the virus isolation process. We propose to utilize chorioallantoic membranes (CAM) of ECEs with the assistance of real-time RT PCR to facilitate equine influenza virus isolation.MethodsReal-time RT PCR was used to detect influenza virus genetic material in amniotic/allantoic fluids (AF) and CAM of ECEs. Haemagglutination assay was used for AF. We used highly diluted virus as a substitute of clinical specimen for ECEs inoculation.ResultsOur study demonstrated that real-time RT PCR testing of CAM homogenates was more useful than testing of AF for EIV detection in ECEs. Positive results from CAM allowed to select the embryos from those with haemagglutination assay (HA) - and real-time RT PCR-negative AF for further passages. Using homogenates of CAM for subsequent passages, we finally obtained HA-positive AF, which confirmed virus replication.ConclusionWe postulate that real-time RT PCR testing of CAM homogenates and their subsequent passages may facilitate the isolation of equine influenza viruses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-017-0788-3) contains supplementary material, which is available to authorized users.

Equine influenza: a comprehensive review from etiology to treatment

“…Lung auscultation may present altered sounds, such as increased breathing intensity, crackles and wheezing. These changes to auscultation may be present in a horse with secondary bacterial pneumonia (Timoney, 1996;Gross et al, 1998Gross et al, , 2004Morley et al, 2000;Cullinane and Newton, 2013).…”

“…Among the most common clinical signs are nasal discharge, serous initially and subsequently mucopurulent, dry cough, fever, depression, and lack of appetite. The presence of these signs and their intensity may vary according to the age of the animal and the individual susceptibility to the disease (Timoney, 1996;Gross et al, 1998Gross et al, , 2004Morley et al, 2000;Cullinane and Newton, 2013). The occurrence of abortion in pregnant mares is not frequent (Radostits et al, 2003).…”

“…Rapid tests for the diagnosis of infection by EIV should not be considered substitutes for laboratory tests, because the performance of the latter leads to the identification of the virus, which allows the outbreak to be characterized, to carry out vaccine and epidemiological studies, and guarantees, with certainty, that an animal is infected with EIV (Radostits et al, 2003). The most commonly used laboratory test to detect the virus and diagnose the disease is the reverse-transcription PCR of samples collected from nasopharyngeal swab (Chambers et al, 1994;Oxburgh and Hagstrom, 1999;Back et al, 2016;Gora et al, 2017;OIE, 2019c). This technique was used, for example, in the detection of an outbreak in Mongolia in which the horses with EIV presented uncommon clinical signs.…”

“…or pneumonia have a high number of granulocytes, especially neutrophils in this secretion (Gross et al, 1998). In terms of histology, it is possible to observe necrotic lesions in the bronchi and alveoli, infiltration of neutrophils, formation of hyaline membranes, squamous metaplasia and hyperplasia of the airway epithelium (Patterson-Kane et al, 2008).…”

A Comprehensive Review on Equine Influenza Virus: Etiology, Epidemiology, Pathobiology, Advances in Developing Diagnostics, Vaccines, and Control Strategies

“…Isolation of virus is usually done in embryonated chicken eggs by amniotic route. Chorioallantoic membranes can also be used for isolation of the virus and instead of HA, qRT-PCR can be employed for detection of the virus rapidly (Gora et al, 2017 ). Cell lines like Madin Darby canine kidney (MDCK) are suitably used for virus isolation (Easterday et al, 1997 ).…”