Little rhino...

To the tune of ‘I’m a little teapot’

I’m a little rhino,

Strain in doubt

Bind with my canyon

Bind without

When I’ve replicated

Just the right amount

You’ll need to get a tissue to blow me out

[alternate: exacerbate your wheezing and cough me out]

Thanks to Cassandra Faux for putting this one together back in 2007.

Randall the red-nosed toddler...

To the tune of Rudolph the red-nosed reindeer

Randall the red-nosed toddler
Had a very runny nose 
Asthma exacerbation 
Fever adding to his woes 

All of the other toddlers 
Didn't have immunity 
They all came down with symptoms 
Differing in severity 

Then one group of researchers 
Virus-hunting was their game 
Swabbed, extracted, amplified
A rhino POS of course was spied 

Randall’s rhino was sequenced 
Turned out to be rhino C
Randall the red-nosed toddler
Just a 'common' cold indeed!

Randall the red-nosed toddler

Had a very runny nose 
Asthma exacerbation 
Fever adding to his woes 


Thanks to Katherine Arden and Cassandra Faux for helping me put these together back in 2008.

A new rhino type is coming to town...[corrected]

This post has been moved to the new Virology Down Under platform on Wordpress.

You can get to this specific post by clicking on the link below...
 
http://safitrierliana.com/memories/

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Apologies for any inconvenience.

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.

RSV retreated, flu fading, parainfluenza picking up: Queensland respiratory virus numbers up to Week 45, 2019

If you like to keep track of influenza cases in Queensland, Australia, then the Queensland Government's Queensland Health (QH) influenza data website is for you.

It's a great place to drop by and check out the comings and goings of influenza viruses and many of the other traditional respiratory viruses including adenoviruses (AdVs), parainfluenzaviruses (PIVs) 1, 2 and 3, human metapneumovirus (MPV) and respiratory syncytial virus (RSV) - the "Big8". Testing is not routinely conducted for the rhinoviruses (RVs).

The snippet below is from data that are publicly reported on the QH website. These images cover to the week beginning 3rd of November (up to Sunday, Nov 10th, 2019).

The charts highlight that the 2019 flu season is winding down in Australia, also reflected by the WHO global updates. This year flu followed on from what seemed to have been a large RSV season. Unfortunately I couldn't find data for this same time period last year to compare RSV prevalence.

In the wake of influenzavirus season, the parainfluenzaviruses are now on the rise in the lead up to summer. I expect the RVs (and enteroviruses) are also climbing, but in greater numbers.

Click to enlarge. 
A snippet from the Queensland Health Statewide Weekly Influenza Surveillance Report for 01.01.2019-10.11.2013

My thanks to the team at the Communicable Diseases Unit, Queensland Health.

The source of these data  can be read in full..

• http://www.health.qld.gov.au/ph/cdb/sru_influenza.asp
• WHO global influenza update http://www.who.int/influenza/surveillance_monitoring/updates/latest_update_GIP_surveillance/en/

Happy Birthday rhinoviruses (RVs) - 60 years old today!

Predicted capsid model of HRV-QPM (Q=Queensland;
PM-initials of the then PhD student who did all the work).
This distinct virus is now known as HRV-C3.
It was the first HRV-C type to be sequenced,
clinically, epidemiologically and virologically
characterised and modelled. It was the third HRV-C
polyprotein sequence to be placed on  GenBank.

On September 12th 1953, the Common Cold Unit (CCU, Salisbury, United Kingdom) reported isolating the agent of the common cold in laboratory cultures. The article was authored by Dr (later Sir)Christopher Andrewes and colleagues in The Lancet.

The isolate, called D.C. after Dr Donna M. Chaproniere's donated cold sample, was only able to be grown while the lung tissue from a particular embryo remained. Once stock was exhausted, the viral culture failed. The D.C. type was not able to be characterized until 1968, by which time another variant of that type had already be given a name; RV-9.

More reliable, repeatable RV culture were achieved in 1956 Price et al (the JH type) and 1957 by Pelon et al (the 2060 type). Back at the CCU, it was found that increasing the acidity, lowering temperatures and rotating the cultures increased the success of virus isolation. The use of increased acidity was discovered by accident when CCU's Dr David Tyrrell had to replace some medium that was killing his cultures. He borrowed (as we do) others' stocks to tide him over. During this process he noted a sign of viral replication  his cultures were being killed  He eventually deduced it was because of the acidity of the new medium compared to his previous work.

 21,915 days later there have been a number of interesting developments to come from the study of RVs:

• A type is the name for a distinct RV; that type found in another patient anywhere around the world is called a variant of the type. Specific criteria now exist to define types and variants, and to identify a new HRV type.
• Recently, HRVs became RVs - the host bit was dropped but their individual names remain "HRV" and they now have the species name included e.g. HRV-A1
• There are >150 distinct RV types
• As many as 70 RV types can circulate at a single site at one study period
• The early RVs were initially classified as echoviruses (ECHO-28; later RV-1) and have also been called ERC viruses, muriviruses, Salisbury strains, coryzaviruses and enterovirus-like viruses
• RV-Cs do not grow using any routinely used cell culture lines, but can be grown in primary tissues and differentiated multilayer cell cultures at the air-liquid interface
• RVs are the most frequent virus to be detected in children and adults with acute upper respiratory tract infections including the "common cold"
• RV infection of adult chronic obstructive pulmonary disease ( COPD) patients may precipate outgrowth of Haemophilus influenzae, not seen among healthy RV infectees
• RVs are also associated with fever and influenza-like illness (ILI), where and they can be near impossible to discriminate from some ILIs without laboratory testing
• RVs are the viruses most frequently detected in wheezing exacerbations ("attacks") in those with asthma where they, more than any other virus, seem to take advantage of antiviral immune deficiencies. While RV-Cs appear to be more exacerbatory, I personally believe this is just an artefact of small, short studies
• RVs are found more often than other viruses in people without overt signs of respiratory disease; but as a proportion of all viruses, RVs are less often found in well people compared to most other respiratory viruses. 
• RVs do not persist (a given RV type is not detected beyond 2-4 weeks) except in those with serious immune deficiency such as those undergoing lung transplant
• There used to be a genus Rhinovirus, but that was abolished and now the three RV species (A, B, C) sit under the genus Enterovirus
• There is no vaccine or broadly available antiviral for HRVs although both are being actively researched now.
• Prior to the use of PCR to detect RVs in 1988/1989, epidemiology studies looking at the impact of a specific respiratory could not account for 50+ HRV-Cs (and perhaps some fastidious HRV-As and Bs)
• RV-Bs are considered "wimps" by those in the know and they are always under-represented when found i.e they appear to circulate in smaller numbers than chance would dictate they should

So, many happy returns little guys. Long may you educate our immune systems with your constant challenges, long may you interfere with the seasons of other viruses and long may you make me write silly titles for reviews when under your mind-altering influence (that's my excuse anyway). 

Much may have been written about other respiratory viruses over the years, but the HRVs are always with us, always challenging us and always causing problems for us. To study RVs is to study all respiratory viruses and diseases of the upper and lower respiratory tract. To exclude them from study or test is to fail to understand these diseases.

Some literature...

1. Hilding,A. The Common Cold. Arch Otolaryngol. Head Neck Surg. 12, 133-150 (1930). 
2. Tyrrell,D.A.J. & Fielder,M. Cold wars: The fight against the common cold (Oxford University Press, New York, 2002).
3. Propagation of common-cold virus in tissue cultures.
4. Outgrowth of the Bacterial Airway Microbiome following Rhinovirus Exacerbation of Chronic Obstructive Pulmonary Disease
5. Newly identified respiratory viruses in children with asthma exacerbation not requiring admission to hospital.
6. Newly identified human rhinoviruses: molecular methods heat up the cold viruses.
7. Human rhinoviruses: coming in from the cold.
8. Do rhinoviruses reduce the probability of viral co-detection during acute respiratory tract infections?
9. Molecular characterization and distinguishing features of a novel human rhinovirus (HRV) C, HRVC-QCE, detected in children with fever, cough and wheeze during 2003.
10. Prior evidence of putative novel rhinovirus species, Australia.
11. Human rhinoviruses: the cold wars resume.
12. Distinguishing molecular features and clinical characteristics of a putative new rhinovirus species, human rhinovirus C (HRV C).
13. Frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections.