key: cord-301577-3qc56c7d
authors: Lamson, Daryl M.; Ramani, Rama; Kleabonas, Matthew; Metcalfe, Maureen; Humphrey, Charles; St. George, Kirsten
title: An unusual case of influenza-like illness after yellow fever vaccination
date: 2014-05-31
journal: Journal of Clinical Virology
DOI: 10.1016/j.jcv.2014.01.020
sha: 
doc_id: 301577
cord_uid: 3qc56c7d

Abstract Yellow fever (YF) is an important public health concern in areas where the disease is endemic. For more than 60 years a highly effective live attenuated vaccine has been available, its widespread use resulting in a dramatic decrease in the number of cases. On rare occasions, YF vaccine can cause mild to severe disease and rare adverse vaccine-associated events have been reported. Additionally, an average viremia of 3–5 days after administration of the YF vaccine has been published. Here we present a case where YF vaccine was isolated in cell culture from a respiratory swab collected from a patient presenting with influenza-like illness. To the best of our knowledge, this is the first report finding replicating YF vaccine in the respiratory sample of a post inoculated individual.

Yellow fever (YF) is an important public health concern in areas where the disease is endemic. For more than 60 years a highly effective live attenuated vaccine has been available, its widespread use resulting in a dramatic decrease in the number of cases. On rare occasions, YF vaccine can cause mild to severe disease and rare adverse vaccine-associated events have been reported. Additionally, an average viremia of 3-5 days after administration of the YF vaccine has been published. Here we present a case where YF vaccine was isolated in cell culture from a respiratory swab collected from a patient presenting with influenza-like illness. To the best of our knowledge, this is the first report finding replicating YF vaccine in the respiratory sample of a post inoculated individual.

© 2014 Elsevier B.V. All rights reserved.

Influenza-like illness (ILI) is a major health issue during winter months in temperate climates and can be caused by a large range of pathogens including influenza, rhinovirus, human metapneumovirus, parainfluenza viruses and some bacteria [1] . The use of culture to detect respiratory pathogens became increasingly common in clinical virology laboratories during the last 50 years with the commercial availability of cell cultures. However, with the introduction of commercially manufactured and FDA-approved molecular assays, including multiplexed PCR assays, the preference for testing viral pathogens using cell culture systems is diminishing [2] . In this case study we describe the utility of cell culture followed by electron microscopy (EM) to identify an unknown pathogen causing ILI in a healthy 19 year old woman. Moreover, the case highlights the importance of providing relevant case history in order to avoid unnecessary testing.

A 19 year old female was seen in a college student health clinic, presenting with fever of 103 • F (39 • C) for 3 days, nausea, vomiting, cough and myalgia. The patient history did not note any recent travel, vaccination, animal contact or insect bites. ILI was the initial diagnosis and a respiratory swab was sent for influenza testing. 

The testing algorithm included both molecular assays and conventional cell culture. The respiratory swab was inoculated into primary Rhesus monkey kidney cells (Diagnostic Hybrids, Athens, OH), human lung adenocarcinoma cells (A549), human embryonic lung cells (HEL) and epithelial colorectal adenocarcinoma (Caco-2) cells. All inoculated cell lines were examined for cytopathic effect (CPE) three times a week for 2 weeks. In parallel, the primary sample was extracted using the bioMérieux easyMAG, tested for influenza A and B using the Centers for Disease Control and Prevention (CDC) Human Influenza Virus Real-time RT-PCR Diagnostic Panel (unpublished protocol, details are available from the CDC on request), and for Influenza A and B, respiratory syncytial virus A and B, human metapneumovirus, rhinovirus, enterovirus, parainfluenza viruses 1-4, and human coronaviruses OC43, 229E, NL63 and HKU1 with the Qiagen ResPlex II kit (Qiagen, Germantown, MD). Multiple real-time (RT)-PCR assays were used for the detection of cytomegalovirus, Epstein-Barr virus, human herpesvirus 6, adenovirus, enterovirus, herpes simplex virus 1, herpes simplex virus 2, varicella zoster virus [3] , rhinovirus [4] , Mycoplasma pneumonia [5] , Legionella pneumophila [6] , and Chlamydia pneumonia (unpublished assay provided by K. Musser).

CPE was observed at 9 days in the Caco-2 cells and all other cell lines exhibited no viral changes after a total of 14 days incubation. Further, the Qiagen ResPlex II assay and all of the real-time (RT)-PCR assays were negative. To further investigate the agent that had grown in the Caco-2 cells, the isolate was extracted using the Qiagen Viral RNA mini kit and tested with the same panel of molecular assays as those used for the primary sample; all results were negative. At this time the submitting laboratory was contacted to obtain additional patient information that may elucidate what could be propagating in cell culture. The clinician reported that there was nothing relevant of note in the history. The virus cultures were then prepared for shipment to the CDC for EM.

Briefly, 1:1 mixtures of infected scraped cells and 5% glutaraldehyde were centrifuged at 1000 × g for 10 min. Supernatant of the centrifugate was adsorbed to formvar-carbon coated grids and negatively stained with 5% ammonium molybdate-1% trehalose, pH 6.9. Subsequently, thin-section electron microscopy was performed on the centrifuged cell culture precipitate using routine methods, similar to those described previously [7] [8] [9] . Specimens were viewed within a Tecnai BioTwin electron microscope operating at 80-120 kV (FEI Company, Hillsboro, OR). Images were captured digitally by using a 2K × 2K CCD camera (Advanced Microscopy Techniques Corp., Woburn, MA).

The images observed on EM were consistent with either a togavirus or a flavivirus (Fig. 1A and B) . Consequent to this observation, nucleic acid from the Caco-2 isolate was amplified using a pan-flavivirus conventional RT-PCR assay that targets a portion of the NS5 region [10] . PCR products of the expected size were sequenced using the PCR primers. Resulting sequences were combined and BLAST analyzed, demonstrating the highest sequence similarity (99%) to YF virus. The student health clinic was contacted for further discussion on the patient history. They provided additional information, including their administration of the YF vaccine to the patient 8 days prior to the collection of the original respiratory specimen, and also that the patient had recovered.

To further characterize this YF virus, published primers [11] and primers designed in this study using Geneious Pro 5.6 (Biomatters, San Francisco, CA), were used to further sequence the Membrane (M), and envelope (E) gene regions (Table 1 ). These targets further identified the virus as having the highest similarity to YF vaccine.

Multiple papers have been published on adverse events associated with the YF vaccine; however, the literature focuses on viscerotropic disease and multisystem organ failure that mimic wild-type disease progression [12] [13] [14] . Here we present a case suffering ILI with fever and cough, eight days after receiving the YF vaccine. YF vaccine was isolated in Caco-2 cells from a respiratory swab. The virus was further prepared, stained and imaged by EM before it was identified by sequence analysis of the NS5 gene. Additional sequence analysis of the E and M gene verified highest identity to YF vaccine (strain 17D), reversions to wild-type YF virus (Asibi strain) were not present [15] .

YF vaccine is a highly effective vaccine that has the ability to greatly decrease the incidence of YF in people traveling to endemic areas. Despite the possibility of an adverse event, the risk of a severe event post-vaccination is low (http://www.cdc.gov/vaccines/pubs/ vis/downloads/vis-yf.pdf). Since replicating YF vaccine was isolated and propagated in cell culture, and in the absence of detection of other known infectious agents, the potential cause of the ILI was the YF vaccine the patient received 8 days previously. Although a known viremia occurs 3-5 days after inoculation of the YF vaccine [16] , this is the first report, to our knowledge, on isolation of YF vaccine from the respiratory sample of a recently vaccinated individual.

This case highlights the importance of the collection and submission of an appropriate case history, and the ongoing availability of cell culture and electron microscopy to assist in the identification of unusual or unexpected agents. EM and cell culture isolation have been utilized to identify many previously unknown and unsuspecting viruses associated with disease outbreaks [17] ; such examples include Zaire ebolavirus, Reston ebolavirus, Hendra virus, Monkeypox virus, and SARS coronavirus. In this case, like the other viruses mentioned, the pathogen was detected by cell culture and was subsequently identified by EM. Without the assistance of EM the relevant molecular assay would not have been chosen to further identify the virus.

Funding was provided from within the Wadsworth Center to perform this study.

MassTag polymerase-chain-reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza-like illness in New York state during

Utilization of nucleic acid amplification assays for the detection of respiratory viruses

Molecular detection of viral causes of encephalitis and meningitis in New York state

Real-time reverse transcription-PCR assay for comprehensive detection of human rhinoviruses

Development of a genomics-based PCR assay for detection of Mycoplasma pneumoniae in a large outbreak in New York state

Design and implementation of a protocol for the detection of Legionella in clinical and environmental samples

Principles and techniques of electron microscopy: biological applications

Same-day diagnostic electron microscopy of infected cell cultures by a modified protocol for conventional and microwave processing

Identification of enterically transmitted hepatitis virus particles by solid phase immune electron microscopy

Comparison of flavivirus universal primer pairs and development of a rapid, highly sensitive heminested reverse transcription-PCR assay for detection of flaviviruses targeted to a conserved region of the NS5 gene sequences

Yellow fever outbreak, Imatong, southern Sudan

Fatal multiorgan failure due to yellow fever vaccine-associated viscerotropic disease

Viscerotropic disease following yellow fever vaccination in Peru

Yellow fever vaccine -how does it work and why do rare cases of serious adverse events take place?

Comparison of the virulent Asibi strain of yellow fever virus with the 17D vaccine strain derived from it

Development of viremia and humoral and cellular parameters of immune activation after vaccination with yellow fever virus strain 17D: a model of human flavivirus infection

Cell culture and electron microscopy for identifying viruses in diseases of unknown cause

The authors would like to thank the Wadsworth Center Applied Genomics Technology Core for performing sequencing and the staff of the Wadsworth Center Virology Laboratory for testing of samples. Special thanks to Alan Dupuis, Dr. Laura Kramer and Dr. Paul Masters for helpful scientific discussion and to Dr. Gino Battaglioli for editorial comments.

None declared.

Ethical approval was given and covered under the New York State Department of Health Institutional Review Board study #07-022 and #02-054.