Rhinovirus transmission by aerosol and lower respiratory tract disease after inoculation

In the next instalment to answer the question posed in last week's post, we also find that rhinovirus can a lower respiratory tract infection (LRTI), if it is delivered directly to the site; several issues around this topic are contentious in current age of PCR diagnosis of lower respiratory tract disease using specimens from the upper respiratory tract (URT).

From Thomas R. Cate et al, Am J Epidemiol.

Author: Thomas R Cate et al
Journal:  Am J Epidemiol 81(1):95-105
Year: 1965
RV type used: NIH 1734 (RV-A15¹)
RV receptor type: major group; ICAM-I

This study set out to investigate the impact of RV on the lower respiratory tract.

Key features of the study layout..

• 16 healthy adult male inmate volunteers
• Safety-tested preparation of RV-15
• 6 volunteers given 1ml nasopharyngeal serum-inactivated virus via a hand atomizer (coarse droplets expected to mainly deposit in the upper respiratory tract), and 1ml instilled intransally by pipette with subject lying on back
• 8, RV-15-antibody-free volunteers were exposed to 10l of air (16, 20 or 66 TCID₅₀ RV15), via a mask, containing 15-second-old 0.2-3.0um particles generated from a Collison atomizer (see Figure)
• A number of re-inoculations were also performed on each virus-delivery group
• Aerosols was also sampled using a Shipe impinger (this device contained cell culture medium onto which some aerosol was impacted) for virus isolation, after storage at -70°C. These data determined the dose that had been used
• Prior (2-days) to inoculation, nasal, pharyngeal and anal swab specimens and 10ml of nasopharyngeal wash (NPW) were collected, frozen at -20°C for testing to identify pre-existing viruses or bacteria (all culture based). The same specimen types were collected after inoculation (minus the anal swab). RV culture was conducted on human embryonic fibroblast cultures, with rotation at 33°C)

Key results included...

• Only 1 other virus, apart form RV-15, was found in the subjects. Culture may have missed fastidious or unculturable respiratory viruses (like the RV-Cs) however.
• During the 1st week after inoculation, usually starting from day-2..
• NPWs contained culturable virus in at least 1 specimen from 8/8 subjects
• During the 2nd week after inoculation..
• 7/8 subjects gave virus-positive NPWs
• During the 3rd week after inoculation..
• 5/8 subjects gave intermittent virus-positive NPWs
• Maximal virus titre aligned in time with most severe illness
• Nasal and pharyngeal swabs specimens did not yield virus as often as NPWs
• All subjects had a rise in antibody titre of 4-fold or greater, indicating infection, by 3-weeks with a further bump after 4-5-weeks
• Tracheobronchitis was diagnosed in 6/8 antibody-free aerosol-inoculated volunteers. This is a lower respiratory tract disease.
• Signs and symptoms included cough (sometimes in fits), substernal chest pain,, wheezing, tender trachea.
• 3 had a primary diagnosis of tracheobronchitis , the other 3 also had a prominent coryzal illness (nasal obstruction/discharge, sneezing, sore throat, swollen neck lymph nodes). 
• Fever was determined in 5/8, within the 1st 1-2-days.
• Signs and symptoms lasted for 1-4 days, a little longer for a rhinitis-alone
• No tracheobronchitis developed among 31 antibody-free volunteers inoculated through a course spray/drop method into the nasopharynx
• No infection (no suitable rise in antibody) or illness was detected among 6 volunteers inoculated with a preparation of virus that had first been inactivated by incubation with an antibody-positive serum. This identified that there were no other viruses/bacteria in the preparation that could have caused the disease. This had been, infrequently, found in other preparations by the authors so this step was important part of their comprehensive approach.
• 4-weeks later, 2 volunteers from the aerosol infection group, 2 from the inactivated virus group, and 2 new volunteers, were (re-)inoculated
• No infection, illness or virus shedding resulted in the aerosol pair
• No illness but infection and shedding occurred in the pair previously inoculated with inactivated virus
• Infection, illness and shedding were apparent in the new volunteer pair
• Neutrophil counts were significantly raised in aerosol-inoculated volunteers at illness onset and also, but to a lesser extent, in the 6 volunteers given inactivated virus. This explains to me why in those with a predisposition to severe RV outcomes, including those with asthma, a symptomatic RV infection is not necessary to trigger an attack.

The authors concluded...

• The aerosols generated here, which carried relatively small amounts of virus, would likely travel beyond the nasopharynx and tracheobronchial tree and be carried into the lungs, probably with <50% deposited and the remainder exhaled
• No evidence of pneumonia was found
• If RV is suitably aerosolized in sufficiently small particles, inhalation can result in lower respiratory tract disease while site-specific installation into the upper respiratory tract usually results a typical URTI or "common cold"

How do these findings translate to everyday exposures to RV coughs and sneezes and in children? In the general community we are constantly exposed to virus and have a complex, person-specific panoply of antibodies resulting from different infections beginning in childhood. This is probably why we are incapacitated by bad colds and LRTIs all the time! An addendum in the discussion of Cate's paper highlights how symptoms resulting from RV infection are best considered as part of the entire spectrum of possible outcomes. 

Previous symptomatic infection, as shown above, protects from lower respiratory tract disease hence adults are less likely to have LRTIs than children who see these viruses for the first time. Also, there is literature showing that the antibody to some RVs can protect against, or moderate, disease due to infection by other RVs. If you are antibody-free, then disease can potentially be more severe.

Cate's studies are all conducted without knowledge of the 50+ RV-Cs because they could not be grown (detected) using the cells employed by the culture methods of the day. Why is that relevant? Because some consider RV-Cs to be more asthmagenic/pathogenic and because we don't know the receptor or natural tissue tropism/distribution of the RV-Cs in humans. How the RV-Cs perform in human volunteer infections is unknown.

Certainly room remains for some new research building upon excellent studies like this one by Cate et al and highlighting (a) that RV can infect the lungs and cause disease if an aerosol is encountered and (b), that one outcome from RV infection does not fit all.

Further reading and references...

1. First HRV nomenclature assignment publication
   http://www.nature.com/nature/journal/v213/n5078/pdf/213761a0.pdf

Rhinovirus (RV) transmission by aerosol: does it happen or is transmission solely by hand-contact and self-inoculation?

I'll be writing a few posts over the coming weeks based in the papers I've found on this topic of RV transmission. How applicable these study results are to transmission of other respiratory viruses is unknown.

The focus will be on answering the question of "Do rhinoviruses transmit by an aerosol route?" The endpoint is usually the development of a clinical upper respiratory tract infection (URTI) or "common cold". 

A lot of volunteer human infection experiments have been conducted using RVs since their identification 60-years ago. This is likely because RVs were seen to cause only mild illness, reducing the health risk for human volunteers. Less common were influenza studies of this sort (correct me if I'm wrong though). It's also worth noting that adults rather than children were included, so the true spectrum of RV disease was not observed. 

From Elliot Dick et al, the Journal of
Infectious Diseases 

Author: Elliot Dick et al
Journal:  J Infect Dis 156(3):442-448
Year: 1987
RV type used: RV-A16
RV receptor type: major group; ICAM-I

This study set out to see whether RV was transmitted by aerosol, indirect contact, or both.

Key features of the study layout..

• 27-34 males >18-years of age were inoculated intranasally with 56-2,500 TCID₅₀ of safety tested¹ RV-A16 on 2 successive days.
• 8-days after inoculation, the 8 cases with the most severe URTIs played cards with 12 RV-B16 neutralizing antibody-free males for ~12-hours in a room containing 4 tables spaced 1.4m apart.
• Each table seated 2 "donors" and 3 "recipients" and the recipients moved locations each hour. Donors were replaced with fresh donors if their URTIs waned
• Coughs, sneezes, nose blows and hand-to-face movements were monitored
• Acquisition of a separate infection during meal times was eliminated by staggering their egress and entry into the card-playing room and by seating recipients 50ft (15m) apart in a well-ventilated room
• 4 experiments, A-D, were performed.
• Experiment A-C tested aerosol transmission.
• 6/12 males used cloth handkerchiefs; the remaining 6 were restrained from any hand-to-head movements
• In experiment A, a 3ft (1m) plastic collar was worn around the neck
• In experiment B and C, arm restraints were used
• Experiment D utilised Experiment C's contaminated furniture and card playing equipment, moving it all into a 2nd card playing room. 
• 12 new recipients were immediately introduced to the room for 12-hours of poker with exaggerated hand-to-face movements
• Card-playing equipment was exchanged between rooms each hour to keep the contaminant levels high
• All meals were eaten in the experiment room to avoid contact with any donors
• After the 12-hour game, recipients returned to the laboratory each day for 2-weeks to provide nasal washings and record symptoms. If they were symptomatic they were taken to a separate laboratory for sampling.
• Exaggerated exposures of "sentinel" recipients consisted of recipients present during the donor's nasal wash collections or undertaking nasal washing alongside symptomatic recipients
• Nasal washing were collected into Hanks balanced sslt solution (HBSS) medium with 0.5% gelatin and inoculated onto WI-38, Hep-2 and primary rhesus monkey kidney cells within 4-hours after collection

Key results included...

• Experiment A: 11/2 recipients were infected, 5 by aerosol alone
• Experiment B: 6/12 infected, 1 by aerosol alone
• Experiment C: 5/12 infected, 4/5 in the restrained recipients
• 12/18 (67%) control recipients (could be infected by any route) were infected versus 10/18 (56%) restrained recipients
• infected recipients were symptomatic and shed virus for ≥1-day 
• Experiment D: no infections but 5/8 donor hands yielded culturable RV-A16 virus while none of the recipient's hands did
• No sentinel recipients became symptomatic

The authors concluded...

• Aerosol transmission was the most important  mechanism of natural spread of RV in adults in this study
• Aerosol transmission was nearly as efficient as transmission by combined aerosol/direct contact/indirect contact
• RV-A16 load declined rapidly to near zero on the journey between donor and the nose of the recipient.
• Virus shedding in a recipient was usually first detected 3-days after proximity to the donor

The authors raised some interesting points...

• Some previous studies to defining that RV transmission was primarily due to fomite and droplet contact may have failed to detect a small and large aerosol modality because recipient exposure was too short or to too small a viral inoculum
• In a previous study by these authors, the donor had to have a mild to moderate URTI, in which they shed ≥10³ TCID₅₀/ml, before transmission reached the desired endpoint
• Brief, casual exposures to an infected RV case infrequently results in adequate transmission as measured by occurrence of a symptomatic episode
•  Exposure by direct inoculation with fresh nasal secretions is practically unlikely

Further reading and references...

1. Safety testing of RV preparations.
   D'Alessio et al. J Infect Dis. 1976;133:28-36.