Challenge Virus in Nasal Secretions: Isolation and Confirmation

Presence of the challenge virus in nasal secretions is an unequivocal indicator of infection. Detection of virus in nasal wash samples is a two-step process involving both isolation of the virus and confirmation of the specific viral strain. Isolation procedures across studies were similar, whereas confirmation procedures differed between and within studies depending on the virus. Protocols for each study are described below.

In all studies, nasal wash samples were collected for viral isolation on Quarantine Day 0 before viral challenge, and on all subsequent post-challenge days.  To ensure that participants had not become infected with a naturally occurring (wild) virus prior to viral challenge, nasal secretions from both Quarantine Day 0 and Day 1 were screened for non-challenge strains of rhinovirus (Pittsburgh studies only; see Screening for Pre-Challenge Infections with Nonspecific 'Other' Rhinoviruses. Recovery of challenge virus from a participant’s Day 0 nasal secretions (or of a non-challenge rhinovirus strain on Day 0 or Day 1) resulted in all subsequent viral challenge data collected on that participant being excluded from analyses. ¹

BCS

Isolation 

Nasal secretion specimens were mixed with viral collecting broth and stored in aliquots at -70°C. Rhinoviruses (RV2, RV9, & RV14) were detected in O-HeLa cells, respiratory syncytial virus (RSV) in HEp-2 cells, and coronavirus (CV229E) in the C-16 strain of continuous human fibroblast cells. ²  When a characteristic cytopathic effect was observed in the tissue culture, specimens were transferred to further cultures and tests were performed to identify the virus.

Confirmation

RVs and CV229E were confirmed by neutralization tests with specific rabbit immune serum;³ and RSV was confirmed by immunofluorescent staining of culture cells. ⁴

PCS1, PCS2, PCS3, and PMBC-rhinovirus

Isolation

Frozen nasal lavage (NL) samples that had been stored at -70°C were quickly thawed in a 37°C water bath and then vortexed and spun at 800 x g (1800 rpm) for 10 minutes in a refrigerated centrifuge. Each of two tubes of human embryonic lung fibroblast cells (MRC-5 and WI-38 cell lines) was then inoculated with 0.2 ml of NL/VCB mixture. A titration of a viral pool of known titer also was included in each batch for assessment of cell sensitivity to rhinovirus. All tubes were incubated at 33°C in a revolving drum which rotated the tubes one full revolution every 3-5 minutes. Fibroblasts were read every other day for 14 days before they were discarded as negative.

Rhinovirus was identified by the appearance of a characteristic cytopathic effect. When 3+ to 4+ cytopathic effect (CPE) was achieved, the cell monolayer was scraped using the tip of a serologic pipette and cell debris and media were frozen at -70°C. If a weak positive did not progress or the amount of CPE diminished, 0.4 to 0.8 ml of scraped cell inoculum was passed to new monolayers of MRC-5, and the remnant frozen at -70^oC. ⁵

Confirmation

Isolated rhinoviruses were confirmed using a neutralization assay. Enough 96-well plates for all samples were prepared using MRC-5 cells. Cell culture harvests from one positive specimen per participant were thawed in a 36^oC water bath and placed on ice. Serial 10-fold dilutions from 100 to 103 for each specimen were prepared by adding 180 μl of 10% Eagle’s minimal essential medium (EMEM) to each well, and an initial 20 μl of sample nasal lavage to 4 wells across the 100 position. A control specimen of rhinovirus of the desired serotype was included in all experiments to confirm the ability of the specific serum to neutralize growth.

The type-specific antiserum and corresponding pre-immune serum was diluted (10% EMEM) to give 20 antibody (Ab) units/l00 μl. 50 μl of pre-immune serum was then added to wells in columns 1, 2, 5, 6, 9, and 10; and 50 μl of type-specific antiserum was added to wells in columns 3, 4, 7, 8, 11, and 12.

After incubating for 1 hour at 33°C, 5% CO₂, the virus mixture was transferred into 96-well plates containing MRC-5 cells with media removed. Plates were incubated at 33°C, 5% CO₂ for 7 days and checked every other day for cytopathic effect. The rhinovirus isolate was confirmed as corresponding to the type of the specific antiserum if the virus titer in the wells with pre-immune serum was at least 2 logs greater than the titer in the wells with the specific antiserum. ⁵

PMBC-influenza

Isolation

Specimens were inoculated into Madin-Darby canine kidney (MDCK) cells and incubated at 34°C. After 30 minutes, 0.5 ml of fluid maintenance medium containing 20 µg/ml of trypsin was added and the cultures were then incubated at 34°C in 5% CO₂. Cytopathic effect was scored microscopically every day. Aliquots of culture fluid from samples showing cytopathic effect were then inoculated into the allantoic cavities of 11-day fertile eggs and incubated at 35°C for 40 to 48 hours. Harvested allantoic fluids were tested for hemagglutinin (HA) with 0.5% chicken red blood cells. Virus titer was expressed in 50% egg-infecting dose (EID₅₀) per ml. ⁶

Confirmation

Influenza A virus was confirmed using a hemagglutination inhibition (HI) assay with antisera prepared against the viral strain of interest. ⁶

Notes & References

¹ Because up to 2 weeks may be necessary to culture rhinovirus from a biological specimen, whether participants were infected with the challenge virus on Day 0 was not known until after the period of quarantine had ended.  Thus, viral challenge data were collected on all participants, regardless of their Day 0 status, and only later excluded if either virus type had been isolated. 

² Phillpotts, R. J. (1983). Clones of MRC-C cells may be superior to the parent line for the culture of 229E-like strains of human respiratory coronavirus. Journal of Virological Methods, 6, 267-269.

³ Al-Nakib W., Dearden, C. J., & Tyrrell, D. A. J. (1989). Evaluation of a new enzyme-linked immunosorbent assay (ELISA) in the diagnosis of rhinovirus infection. Journal of Medical Virology, 29, 268-272.

⁴ McQuillin, J., & Gardner, P. S. (1968). Rapid diagnosis of respiratory syncytial virus infection by immunofluorescent antibody techniques. British Medical Journal, 1, 602-605.

⁵ Gwaltney, J.M. Jr., Colonno, R.J., Hamparian, V.V., & Turner, R.B. (1989). Rhinovirus. In N.J. Schmidt & R.W. Emmons (Eds). Diagnostic procedures for viral, rickettsial, and chlamydial infections, 6th ed (pp. 579-614). Washington, D.C.: American Public Health Association.

⁶ Tobita, K., Sugiura, A. Enomoto, C., & Furuyama, M. (1975). Plaque assay and primary isolation of influenza A viruses in an established line of canine kidney cells (MDCK) in the Presence of Trypsin. Medical Microbiology & Immunulogy, 162, 9-14.