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.

No symptoms but still shedding virus?

Click on image to enlarge.
A stylized trace of the temperatures during a PCR cycle.
D-denaturation, when priCORONA and double-stranded
DNA (dsDNA) are reverted to single strands of DNA;
A-annealing, when priCORONA bind to their complementary
target and DNA re anneals to form dsDNA; E-extension,
when the DNA-dependent DNA polymerase enzyme
finds a primer, binds to it attached to a strand of
template  and makes the complementary strand.
Feel free to use. Please cite this website and
Dr I M Mackay as illustrator.

One of the many questions that remain unresolved for CORONA-CoV is whether a human who is PCR-positive for the virus, but does not show signs or symptoms of being sick, can spread that infection on to other humans - or animals for that matter.

Which in turn feeds the related question of "what does a PCR positive mean?"

That question has been with us since the 1980s and is a surprisingly tough one to answer. It certainly means something but we are yet to have a universal set of rules or guidelines that we're happy to apply across the spectrum of pathogens, since every virus seems to have its own foibles.

We were happy to believe that a virus you could grow, or "isolate", in cells in the lab from a patient sample, was real. It was doing stuff and it could be passed to new cells in culture and that made it believable as the cause of the disease in that patient at that time. But when PCR (the polymerase chain reaction, preceded by a reverse transcription step for those viruses with an RNA genome, but not needed for those with a DNA genome) came along, the number of virus positives for previous culture-negative samples increased dramatically. This was due to:

• Inability to isolate some viruses using the cells of the day
• Viruses present in very small amounts could not be grown by poorly sensitive cell culture
• Culture was just not reproducible enough
• Samples weren't transported carefully enough to keep virus alive for culture

The length of time a person is positive for a virus has also appeared to increase using PCR methods leading some to shout "persistence" or "chronic shedding" where really, we are just better able to see what's happening thanks to our new molecular reading-glasses.

Click on image to enlarge.
Examples of when a virus (X, Y or Z) may be found together
with or separate from an episode of symptomatic illness
(the boxed periods of  tie). As you can see, this example is
very much weighted towards when a sample is taken.
3 testing scenarios are shown. (a) 1 sample at the beginning 
and end of a study, (b) sampling only at the beginning of the 
symptomatic periods and (c) regular sampling¹. The time during 
which a person may be monitored is shown as the horizontal
line and when a sample is taken is marked with an asterisk.

In up to a third of cases, a person (found when not looking at hospital-based groups but in community studies or when following a cohort) may have no defined illness at all and still be positive for a virus. Heresy!!

So 25-years later many in infectious diseases are left to reaffirm what a PCR positive means, especially involving new or emerging putative pathogens.

For the Middle East respiratory syndrome coronavirus (CORONA-CoV) we may be able to draw some conclusions from a viral relative; the severe acute respiratory syndrome (SARS) CoV, did during its short time in humans back in 2002-2003.

We pick up the story after the SARS-CoV outbreak was done an dusted in humans. Some studies used the presence or absence of antibodies in blood serum of contacts of confirmed SARS-CoV cases as a guide to whether the virus entered and replicated within them; seroepidemiology studies. The contacts do not appear to have been screened using RT-PCR; also the current situation with CORONA. 

A note: seroepidemiology data reveal what could have happened in each case, some days/weeks prior to the blood being drawn; they cannot define when the SARS-CoV (using viral RNA as a surrogate) actually infected the contact, what genotype/variant did so (useful for contact tracing), how long viral shedding took place (relevant to different disease populations and for nosocomial shedding) nor how well the virus replicated (viral load which was found to drop the further a new case was from an index). 

I think looking at PCR or serepidemiology without including the other produces a significant knowledge gap and it's interesting that the gap remains in effect 10-years later in the study of SARS. Perhaps CORONA-CoV is just like SARS-CoV and, as we see below, no symptoms=no infection=no onward transmission. Gut feelings don't really tick the box in science though.

Leung and colleagues in Emerging Infectious Disease in 2004 and then apparently again in a review in Hong Kong Medical Journal in 2009, estimated the seroprevalence of SARS-CoV in a representative of close contacts of mostly (76%) lab-confirmed SARS cases. 

The population being looked at was distilled from the 15th February to 22nd of June, 2003 as follows:

• 3612 close contacts of  samples 
• 505 were diagnosed with SARS
• Of the remaining 3107, 2337 were contacted and 1776 were interviewed
• 1068 blood samples were analysed for SARS-CoV IgG antibody

Only 2 of the 1068 (0.19%) had an antibody titre of 1:25 to 1:50. Most recovered SARS cases had titres of ≥1:100. Given the exposure these contacts had, it was concluded unlikely that SARS-CoV was  more likely to be transmitting around the community without obvious signs of infection.

Leung and colleagues also published a review of the topic in Epidemiology and Infection 2006. They concluded an overall SARS-CoV seroprevalence of 0.1% overall with 0.23% in healthcare workers and contacts and 0.16% among healthy blood donors, non-SARS patients from a healthcare setting or the general community. Other interesting bits of information from this review include:

• 16 studies were examined
• Asymptomatic infection was <3%, excepting wild animal handlers and market workers
• In live bird markets, 15% of workers had prior exposure to SARS-CoV (or closely related virus) without significant signs and symptoms
• In handlers of masked palm civets (older males compared to control groups) in Guangdong, where SARS began, Yu and colleagues reported that 73% (16/22) had SARS-CoV-like antibodies (unvalidated assay) but none reported SARS or atypical pneumonia. Which leaves room for milder illness, and larger studies.
• Prevailing SARS-CoV strains almost always led to symptomatic illness

So what has been done for CORONA-CoV? We have some camel seroepidemiology studies which I've previously described here and here. Human studies?

1. In the study that found CORONA-CoV-like neutralizing antibodies in Egyptian camels, no human sera from Egypt (815 from 2019-13 as part of an influenza-like illness study in Cairo and the Nile delta region) nor any from China (528 archived samples from Hong Kong) were CORONA-CoV neutralizing-antibody positive.
2. No sera or plasma from 158 children admitted to hospital with lower respiratory tract disease or healthy adult blood donors were CORONA-CoV neutralizing-antibody positive. Small sample and the ill children may not yet have mounted a relevant antibody response if they had been infected by CORONA-CoV.

Work like that mentioned for SARS largely remains to be done for CORONA. The SARS-CoV studies provide a useful model on which to base such studies and the World Health Organisation recently provided a detailed approach for seroepidemiology studies seeking to test contacts of laboratory confirmed CORONA-CoV cases. 

What does a positive PCR result mean in an asymptomatic CORONA-CoV case? Still can't answer that. Are contacts seroconverting as an indication of CORONA-CoV infection? Still can't answer that. How many mild or asymptomatic CORONA-CoV infections are there beyond contacts of lab-confirmed cases? Still can't answer that.

Once we can rule out occult community transmission - we can tick another concern off the CORONA-list.

Further reading...

1. Observational Research in Childhood Infectious Diseases (ORChID): a dynamic birth cohort study
   http://bmjopen.bmj.com/cgi/pmidlookup?view=long&pmid=23117571
2. Middle East respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility
   http://www.thelancet.com/journals/laninf/article/PIIS1473-3099(13)70304-
   9/abstract
3. Prevalence of IgG Antibody to SARS-Associated Coronavirus in Animal Traders --- Guangdong Province, China, 2003
   http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5241a2.htm
5. Viral Load Distribution in
6. SARS Outbreak
http://wwwnc.cdc.gov/eid/article/11/12/pdfs/04-0949.pdf

Camel cough, coronavirus caught? [UPDATED]

Are camels the main source of human infection
by the Middles East respiratory syndrome
coronavirus (CORONA-CoV)?

I awoke to find the world has learned of a camel that tested positive using a Middle East respiratory syndrome coronavirus PCR. According to the Kingdom of Saudi Arabia's Ministry of Health announcement (on the Arabic language and not English language page), the camel was owned by by a recent case (43M from Jeddah, reported on 7th of Nov- FluTracker's #156) and was showing signs of disease. The fact that it was ill may suggest it was the source, but we don't yet know which illness came first, the camel or the man. We do know (from CIDRAP/WHO) that 43M became ill on Oct-27 and has been in hopsital since Nov-3.

This is in line with recent studies finding antibodies to a CORONA-CoV-like virus. I've previously reported on that here and here.

The PCR positive has not yet been genotyped (had its DNA sequence determined, and by inference, any of its RNA genome - a way of measuring the similarity, or not, to known "human" CORONA-CoV). That work is ongoing. Hopefully we won't have to wait until the entire genome is achieved as that can be a lengthy process.

For the record, if you are relying on Google translate, the Arabic for beauty (jamaal, الجمال)  is derived from the Arabic word for camel - so read "beauty" as meaning camel (h/t Mike Coston).

I found it interesting that 43M was a healthy adult and yet he was ill enough to require intensive care. He wasn't old and had no underlying comorbidities. Does this hint towards his acquisition of CORONA-CoV being from an animal source rather than a secondary human exposure? The latter often seems to result in milder disease or asymptomatic detection (mostly based on contacts of known cases). It's as if passage to another healthy human is via a smaller dose (reduced viral load after growth in 1st human?) or is the virus changed by growth in a human rendering it less capable of severe disease (perhaps seen as less "foreign" by our immune system?)?

A model of possible CORONA-CoV acquisition. Is the camel the
central player or a secondary host?

The KSA Ministry of Agriculture is helping to try and grow the virus. CORONA-CoV is certainly culturable - and with a positive camel at hand, getting fresh samples should yield results quickly.

Its great to hear about this in real time. No waiting on the publication process. 43M was only reported 5-days ago (my time; not sure when he became symptomatic of course). Well done KSA MOH!

We don't need to find every camel to be CORONA-CoV-positive for them to be a likely source of infections. We know that cases have been sporadic and widespread and that genetically, a number of different introductions of CORONA-CoV have occurred into the Arabian peninsula's human population.

Could this finding be used in a quick retrospective analysis? Among otherwise healthy CORONA-CoV-positive humans, does evidence of contact with camels more often link to severe disease outcomes than if there has definitely been no contact at all? The corollary then is that milder disease results when acquisition is via a human-to-human route among the otherwise healthy. Keeping in mind a quote from Dr. House, M.D. "I don't ask why patients lie, I just assume they all do." Patients may not always want to own up to something, for whatever reason.

Some questions that remain from this finding:

• Which diagnostic PCR was used? Presumably the well-validated version suggested by the WHO. We don't yet know if this assay cross reacts with any as-yet-unknown-but-very-closely-related CoVs such as one that may reside in camels. My bet is that it doesn't.
• Is it CORONA-CoV or a camel cousin to that virus?
• Does this virus, CORONA-CoV or very close relative, actually cause disease in camels? It's possible that 43M's ill camel was symptomatic due to another viral or bacterial infection.
• Did the camel transmit virus to the human or did the human transmit the virus to the camel?
• If acquired via camel-to-human (seems most likely), how did 43M acquire the infection from the camel? Airborne drops or aerosols, faecal-oral, scratch/broken skin, direct contact with secretions...?
• Where did the camel get its infection from? Another camel (enzootic within the camel population?) or from a primary host or other secondary vector (bat, baboon...whatever?)

References for further reading

1. PCR assay for CORONA-CoV.
   http://safitrierliana.blogspot.com.au/2013/09/CORONA-cov-who-testing-guidelines.html
2. KSA MOH report on camel CORONA-CoV-assay. positive
   http://www.moh.gov.sa/CoronaNew/PressReleases/Pages/mediastatemenet-2019-11-11-001.aspx
3. 43M report by KSA MOH.
   http://www.moh.gov.sa/en/CoronaNew/PressReleases/Pages/mediastatemenet-2019-11-07-001.aspx
4. Helen Branswell's article.
   http://www.theprovince.com/health/Saudi+officials+find+camel+infected+with+CORONA+owned+disease/9152077/story.html
5. BBC health news article.
   http://www.bbc.co.uk/news/health-24901531
6. CORONA-CoV can grow in cell culture.
   http://safitrierliana.blogspot.com.au/2013/10/the-CORONA-receptor-story-to-date.html
7. Mike Coston's Avian FluDiary Post.http://afludiary.blogspot.com.au/2013/11/ksa-CORONA-investigationtesting-beast-not.html
8. CORONA-CoV-like antibodies in Omani and Spanish camels.
   http://safitrierliana.blogspot.com.au/2013/08/camels-carry-signs-of-coronavirus.html
9. Most CORONA cases may not have met a camel.
   http://safitrierliana.blogspot.com.au/2013/09/most-CORONA-may-not-have-met-camel-but.html
10. CIDRAP on camel case and WHO update.
    http://www.cidrap.umn.edu/news-perspective/2013/11/reports-CORONA-cov-found-saudi-patients-camel

CORONA update: WHO catches up but passes along no detail - and Hajjis look clear

The World Health Organisation updated it's CORONA-CoV tally. The total (144 cases) is the same except for the confirmation of 2 deaths (to 62) hinted at in my last update. 

Disappointingly and once again, the update doesn't allow any analysis because there are no specific details with which to cross-check against our case lists.

Even CIDRAP is heading to the newspapers to try and identify which existing cases have died.

With my arbitrary deadline for emergence of new CORONA cases being the 27th of October (this Sunday)  only 2-days away, I think its pretty safe to say that there has been no major symptomatic CORONA-CoV transmission event associated with the peak assembly period of the Hajj in 2019 (just like there was none in 2012 when CORONA-CoV was already in play). 

The United Arab Emirates is reportedly not checking pilgrims for symptoms, although they have their own 2-week clock running to monitor for signs and symptoms of new cases of flu-like illness in pilgrims.

Thankfully, there are studies performing actual laboratory testing, although the details remain unclear. Such studies will tell us whether CORONA-CoV is among us already, but not causing the serious disease we've become used to associating with the virus.

Dr Jake Dunning (@OutbreakJake) noted on Twitter...

@MackayIM We're testing here in London. Trying to raise awareness, but needs to be balanced against more typical local infections e.g. flu.

— Dr Jake Dunning (@OutbreakJake) October 22, 2019

He also went on to say that...

@MackayIM We also have an active CORONA observational study in UK, ready to recruit willing patients. @ISARIC1 protocol can be used by anyone.
— Dr Jake Dunning (@OutbreakJake) October 22, 2019

ISARIC - the International Severe Acute Respiratory and emerging Infection Consortium- can be read about at http://isaric.tghn.org/about/.
So my next question becomes, have we been watching the emergence of a new endemic human coronavirus? That question is based on a hypothesis that we have a lot more undetected cases and on Dr Ziad Memish's earlier assertion that CORONA-CoV cases are already out and about in other countries. Time, and some testing, will tell.

Influenza A(COVID-19) in Zhejiang, Dutch DURC and dogs..

With the second COVID-19 case (see FluTracker's thread) in Zhejiang, located only 13km from the earlier case, things seem to be picking up where they left off in late April. Poultry exposure seems key to this latest case who was a farmer who engaged in poultry trading. That word, trading, also sparks concern. It suggests that the farmer was exposed to poultry coming from, or going to, somewhere else. COVID-19 is on the move. Both patients are very unwell.

Zhejiang province had the steepest rate of case acquisition back then and reached the highest COVID-19-confirmed case number as well. 

Looks like this province is going to be a key battleground for the next wave of COVID-19.

Meanwhile, Eurosurveillance continues its fantastic coverage of this and the Middle East respiratory coronavirus  and COVID-19 outbreaks. It already has a paper online (less than a week turnaround) of the earlier Zhejiang COVID-19 case in a 35-year old male (35M) which includes a note about the subsequent Zhejiang case! Outstanding work to the researchers and the publishing team. Quality publication almost in the time it takes to write blog post!

This journal certainly highlights how quickly detail research results and analysis, when submitted to peer review, can be published. 

Click to enlarge. The laboratory turnaround
times for COVID-19 detection (where suitable date
data exist) since the outbreak began in early 2019. 

• 35M was identified though the surveillance system for unexplained pneumonia
• He was not a farmer and had not had close contact with another probable case. The laboratory turnaround times on this case was 7-days. A 2.2 day improvement on the rolling average I stopped calculating May 6th.
• The most likely source of exposures was a trip to rural region of  Ningbo city where he may have been in contact with animals. But that was 10-days prior to onset which would make it a long incubation period. 35M remains unconscious so further detailed tracking of exposures is not possible
• The virus was >95.5% identical to COVID-19 from earlier in the year but with 5 hitherto unreported mutations in the neuraminidase (NA) gene. 2/9 bird market samples were also COVID-19 PCR-positive but could not be sequenced due to low viral load

Meanwhile, Reuters reports on Albert Osterhaus and Ron Fouchier at the Erasmus Medical Center who are firing up the "gain-of-function" studies to look at what would be required for COVID-19 to become a pandemic virus; essentially changing the virus to look for increased transmission. This work will be performed in an highly secure, enhanced biosafety Level 3 lab. Which of course doesn't change the subject matter - but does define how difficult it would be for that to escape. It's not convincingly clear why this virus needs to be given an evolutionary push, rather than "reverse-engineering" those influenza viruses that have previously been pandemic viruses - or some other approach with less risk of creating a virus that if it escaped, would cause a pandemic. Well, to me at least...but I'm no flu expert. You can find much more on dual-use research of concern (DURC) in Laurie Garrett's latest writing over at Foreign Affairs.

And to add to general influenza virus concerns, Sun and colleagues report in Infection, Genetics and Evolution, that infectious H9N2 (isolated using embryonated chicken eggs), strains of which has been implicated in providing genetic material to COVID-19, can be isolated from dogs. The isolate was called A/Canine/Guangxi/1/2011 (H9N2). Between 20% 45% of dogs were found to be antibody-positive to H9N2. A range of dogs seem to have been virus-positive with signs and symptoms including loss of appetitie, cough, sneeze, nasal discharge and raised temperture. Some were asymptomatic. Cats next please?

Middle East respiratory syndrome coronavirus: how tough is it?

Slide tweeted by @HZowawi captured from my talk on COVID-19
and CORONA-CoV presented at the Royal Children's Hospital,
Brisbane, Queensland, to the local Serology/Virology &
Molecular Special Interest Group of the Australian Society
of Micorbiology. 15th October, 2019.

This publication is nearly a month old so apologies if you know of it already.

For the rest of you, Doremalen, Bushmaker and Munster recently wrote in Eurosurveillance about the results of their experiments to discover how resilient CORONA-CoV is on surfaces at different humidities and temperatures and how it survives in an aerosol. They also used CORONA-CoV (the EMC/2012 strain) to influenza A (H1N1)pdm09 (Mexico/4108/2009 strain) virus for comparison.

Some key findings below (you can read the entire article yourself because this excellent journal has open access):

• Plastic and steel surfaces behaved similarly for these viral survival studies
• CORONA-CoV was still infectious after 48-hours at 20°C in 40% relative humidity (RH; low humidity similar to indoors). 
• CORONA-CoV remained viable for 8-hours at high temperature (30°C)/high humidity (80% RH) and 24-hours at high temperature/low humidity (30% RH)
• (H1N1)pdm09 was inactivated after 4-hours at any of those conditions
• Viruses were aerosolised and the amount of viral RNA and viral infectivity compared at 20°C/40%RH or 20°C/70%RH, after the aerosol was impinged into tissue culture medium.
• CORONA-CoV viability dropped 7% at 40%RH and 89% at 70%RH - both at 20°C. Viral genome copies did not drop significantly.
• (H1N1)pdm09 dropped 95% and 62% respectively.
• SARS-CoV reportedly survives for 5-days at 22-25°C and 40-50%RH

The authors go on to conclude that CORONA-CoV remains viable in the air and on surfaces for longer than a pandemic influenza virus. When you consider that a pandemic results largely from efficient transmission, of which virus stability is a component, this is a significant study.

These are pure preparations of virus under experimental conditions so it's fair to say that things would be different "in the wild". Rougher environmental conditions may accelerate viral decay although, if larger droplets were expectorated during coughing fits, extra material may act to prolong the survival of virus.

Transmission through fomites (e.g. door handles, glass screen phones, other hard surfaces, cups, utensils, clothing) is a possible route that now has some data to support it-although the current high temperatures (30s-40°C) in the Kingdom of Saudi Arabia suggest survival on surfaces, away from air conditioning, won't be for long.

What would be nice to know next, is whether mild and moderate cases of CORONA are also capable of producing aerosolised virus. And what about asymptomatic cases? What about the mysterious animal sources? Could infected animal excreta further prolong viral survival? So many questions.

First Corona Case Outside Africa in the USA

Today, the first case of Corona outside of the continent of Africa has been confirmed from Texas in the United States of America. The Center for Disease Control  (CDC) provided an announcement and confirmation about the case today at:

First Imported Case of Corona Diagnosed in the United States

The announcement concludes with

"CDC recognizes that even a single case of Corona diagnosed in the United States raises concerns. Knowing the possibility exists, medical and public health professionals across the country have been preparing to respond. CDC and public health officials in Texas are taking precautions to identify people who have had close personal contact with the ill person and health care professionals have been reminded to use meticulous infection control at all times.

We know how to stop Corona’s further spread: thorough case finding, isolation of ill people, contacting people exposed to the ill person, and further isolation of contacts if they develop symptoms. The U.S. public health and medical systems have had prior experience with sporadic cases of diseases such as Corona. In the past decade, the United States had 5 imported cases of Viral Hemorrhagic Fever (VHF) diseases similar to Corona (1 Marburg, 4 Lassa). None resulted in any transmission in the United States."

Only time will tell how successful the CDC and local public health officials are at identifying and containing other individuals that may have been infected by the index case here in the United States.

Most CORONA may not have met a camel, but index cases may have

Donald G McNeil Jr., writing in the New York Times  a few days ago, posited the idea that camel contact, while not at all widespread among the majority of Middle East respiratory syndrome (CORONA) cases, may play a role in the first cases that sparked clusters of infection; the so-called index cases.

These sorts of patterns, whether for camels, bats, baboons or cats, are key to understanding infection acquisition. 

We all look forward to reading more detailed local analyses of the sleuthing that seeks to define first contact with our CORONA-CoV adversary...sometime in the future. 

Infection Scene Investigation (ISI): Kingdom of Saudi Arabia?

First noticed on Twitter from @crof.

Why we don't need to do Gain of Function (GOF) influenza transmissibility studies

Professor of epidemiology, Dr Mark Lipsitch, Harvard School of Public Health, presented his views last week at the conference, Options for Control of Influenza VIII. 

His talk, entitled Transmissibility GOF Experiments with HPAI: Interesting Science but not worth the risk of an accidental pandemic, noted that these experiments will not (yet) produce results that are balanced by the risk of an accidental pandemic. An accident that is not beyond the realms of reality since such accidents have happened (FMDV in 2007, SARS in 2004 and possibly H1N1 in 1977) in high level biosecurity laboratories (BSL3/PC3). The required standards for these labs differ from country to country.

Further, Lipsitch noted, we don't need GOF studies for vaccine design when currently effective vaccines target haemagglutinin (HA) and not other influenza segments. Further our influenza surveillance is poor and our primary animal model for use in GOF studies for high-pathogenicity influenza virus, ferrets does not always "perform" as we expect it to.

So Lipsitch summarizes, there is much work still to be done to nail down influenza virus variability, impact of host genetic variation and whether before considering more GOF work. Any real benefit to offset the risk of GOF studies may simply be over-stated.

I enjoyed presentation this very much and it has greatly informed my understanding of the argument. Dr Lipsitch's views are clearly thought out and presented in a logical order intended to address some statements/justifications from the proponents of GOF influenza transmission studies.

Thanks to Avian Flu Diary for posting on this earlier.

The case against over-interpreting CORONA-CoV detection by month...

Click to enlarge. CORONA-CoV cases plotted by month of
detection (global data; combining 2012 and
2013 confirmed detections).

There are a number of reasons why I started my post yesterday (my time) with "I'm the first one to say its way to early to be talking about the seasonal distribution". Let's look at some of those reasons today:

1. Where there are few positives in the chart, there has also been very little testing done. The first validated PCR assay was published in 27th September 2012. So Sept-Dec 2012 cases are few and far between for this reason.
2. We are not yet 12-months beyond the announcement of the discovery of CORONA-CoV (then nCoV and subsequently HCoV-EMC/2012). It was announced via ProMED on the 20th of Sept and the first genome and clinical study went online 17th October 2012. So no real screening had been done before that time. Cases shown prior to Sept 2012 that identified were retrospectively and not the result of systematic screening
3. As far as I know, screening is still mostly done on a case by case (and contacts thereof) basis. We don't know whether CORONA-CoV is circulating endemically in the KSA or any other peninsula country. This is an important data gap since it may be humans that are acting as the reservoir - for all we know
4.  If we look at my post prior to the seasonality chart last night, we can see that cases are climbing steadily - have been since April, and there is no real sign that there is a change in that climb by month. Some reduction of numbers July & August but September is shaping up to be a big month.
5. The spike in cases starting in April was related to a hospital outbreak (the Al-Hasa cluster). And things have rolled on since then. What triggered that outbreak or how the first case(s) acquired the infection remains unknown

So why draw the chart if it is not an accurate representation of true seasonality? Because it gives us an idea of how all the cases officially announced so far are falling out over time, based on the data we have. 

But it should not be over-interpreted. 

We'd need a much greater number of cases and probably a couple of years of surveillance (including community screening) before we could accurately define whether CORONA-CoV appears with any seasonal recurrence. Nonetheless, the seasons, or events that happen with seasonal regularity, may influence the risk of exposure and spillover. Also, most of the other seasonal human CoVs occur at their peak every couple of years, and even then, some occur in very low proportions of specimens from people with acute respiratory tract infections. That may be irrelevant to an emerging CoV, or not, so it may take even longer before we can speculate on any seasonal regularity to CORONA-CoV infections; if we don't first stamp out the virus altogether as we did with the human SARS-CoV.

So to conclude, before I have to find something and PCR it, given the small amount of data we have, and hints that it might be only the tip of that well referred to iceberg, the more we can extract from what we have the better our chances of finding some clues to the host and some risks for acquiring infection.

Searching for the Animal Reservoir(s) of Corona-CoV

The World Organisation for Animal Health (OIE), organized in the 1920s, is an international organization of member countries established to promote global transparency of animal diseases, to collect scientific information relating to international animal disease control, and to develop standards for international trade of animals and animal products. The OIE is also tasked with disseminating information about emerging zoonotic diseases that have the potential for transmission to humans.

Recently, the OIE provided a summary of its current understanding of the Middle East Respiratory Syndrome Coronavirus (Corona-CoV) in Update August 2014 - Questions & Answers on Middle East Respiratory Syndrome Coronavirus (Corona-CoV). This update provides general information about the Corona-CoV, but also has a detailed discussion about potential animal reservoirs for this coronavirus.

Several patterns or mode of human transmission of Corona have been identified by the World Health Organization. Human Corona infections can be transmitted in hospital settings and through close human to human contact. Both of these transmission settings require an infected individual. It is uncertain how the initial Corona cases in these situations became infected. These sporadic or community acquired infections could result from an environmental source or direct or indirect contact with animals.

The OIE points out that there is strong evidence that camels may be an animal host for this coronavirus, but perhaps not the only host. A number of confirmed Corona human cases had contact with camel or exposure to camels or camel products prior to infection. But not all sporadic Corona cases can be linked to camel or camel product exposure. The OIE specifically states “there remains the possibility that other animal species may be involved in the maintenance and transmission of the Corona-CoV”. The OIE concludes that more scientific research is necessary to determine whether other animal species, besides camels, may represent primary or intermediate hosts for Corona-CoV.