Avian Influenza: Mixed Infections and Missing Viruses

Leann Lindsay,Vivien Dugan,Xudong Lin,Terra Kelly,Seth Schobel,Magdalena Plancarte,David Wentworth,Walter Boyce

doi:10.3390/v5081964

Leann Lindsay, Vivien Dugan + Show 6 more

Open Access

https://doi.org/10.3390/v5081964

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Abstract

A high prevalence and diversity of avian influenza (AI) viruses were detected in a population of wild mallards sampled during summer 2011 in California, providing an opportunity to compare results obtained before and after virus culture. We tested cloacal swab samples prior to culture by matrix real-time PCR, and by amplifying and sequencing a 640bp portion of the hemagglutinin (HA) gene. Each sample was also inoculated into embryonated chicken eggs, and full genome sequences were determined for cultured viruses. While low matrix Ct values were a good predictor of virus isolation from eggs, samples with high or undetectable Ct values also yielded isolates. Furthermore, a single passage in eggs altered the occurrence and detection of viral strains, and mixed infections (different HA subtypes) were detected less frequently after culture. There is no gold standard or perfect reference comparison for surveillance of unknown viruses, and true negatives are difficult to distinguish from false negatives. This study showed that sequencing samples prior to culture increases the detection of mixed infections and enhances the identification of viral strains and sequences that may have changed or even disappeared during culture.

Highlights

IntroductionField and laboratory efforts should yield results that accurately reflect nature—screening tests should have 100% sensitivity and specificity, and virus isolation and sequencing should not alter or bias results
Influenza A surveillance in wild birds is crucial to understanding the origin, evolution and transmission of viruses that pose a potential health risk for humans, domesticated animals, and wildlife [1,2,3,4].Ideally, field and laboratory efforts should yield results that accurately reflect nature—screening tests should have 100% sensitivity and specificity, and virus isolation and sequencing should not alter or bias results
Each sample was inoculated and cultured in embryonated chicken eggs (ECE) to detect live virus, and allantoic fluid (ALF) was tested by matrix real time PCR, HA-subtyped by conventional PCR, and full genomes sequenced by a multisegment-RTPCR and generation sequencing (NGS)

Summary

Introduction

Field and laboratory efforts should yield results that accurately reflect nature—screening tests should have 100% sensitivity and specificity, and virus isolation and sequencing should not alter or bias results. This is rarely the case, and many studies have shown that researchers must carefully consider the limitations and biases associated with different sampling strategies and diagnostic tests [5,6,7,8,9,10]. Duplicate cloacal samples from each bird were screened for virus nucleic acid by matrix real-time (RT) PCR, and these pre-culture samples were subtyped by conventional PCR/sequencing of a 640 bp section of the HA gene. Each sample was inoculated and cultured in embryonated chicken eggs (ECE) to detect live virus, and allantoic fluid (ALF) was tested by matrix real time PCR, HA-subtyped by conventional PCR, and full genomes sequenced by a multisegment-RTPCR and generation sequencing (NGS)

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