Can the Covid-19 tracking app be trusted?

A research team at King's College London have been attempting to track UK and USA Covid19 cases via a self-reporting app, available for iOS and Android at https://covid.joinzoe.com/ and would like to encourage as many people as possible to download the app and self-report every day so we have a better data-set. Some 2 million people have already joined the programme, and results are already being published.

But the ultra-cautious among us might be asking: since the app's source code is not available, how can we be sure it's really doing what it says it is, and not for example spying on our phones?

The development of the app was outsourced to a commercial startup company, Zoe Global Ltd, who unfortunately have been unable to "open-source" the app, probably because, like many commercial companies, they are afraid that disclosing their source code will somehow harm their future business—although in fact there are many good examples of successful companies that do disclose their source code, such as the Android platform itself, and the Jitsi alternative to Zoom, but not everyone has yet fully grasped how to harness such "open-source" business models, and Zoe Global Ltd seems to be one of the companies that cannot do it yet. Nevertheless they were able to quickly produce the app in a crisis, and the research team felt that good positive press coverage by The Guardian, the BBC, King's College London and Professor Tim Spector should be enough assurance of the app's good behaviour without also having to disclose its source code.

Although we were unable to be given the source code when we asked for it, we were nevertheless able to perform a limited independent investigation into how the app works by downloading the Android APK file for the app (package name com.joinzoe.covid-zoe) from a third-party mirror site called APKFab, and looking inside it.

The download from APKFab is a file called "COVID Symptom Tracker_v0.9_apkfab.com.xapk" which can be unpacked using the "unzip" command on GNU/Linux. It contains a "manifest.json" file that gives it the following permissions:

• Receive messages from the Android Cloud to Device Messaging (C2DM) service. This lets the research team send you messages (which they'll probably do only if they want to clarify an exceptional case).
• Access the Internet. This is obviously required to send the results. The app also requests permission to read the state of the Internet and Wi-Fi connections, which could be used for example to check that the Internet connection is good before trying to use it.
   
• Display a window over the top of another app. It's not clear why they want this permission: most likely the requst was left in the code by mistake. This permission (which is requested by 10% of apps on the Google Play store) can in theory be abused by "overlay malware" to pop up a fake login screen on another app, so you think you have to enter login details into the other app but you are actually giving them to the malware author.
   
• Modify audio settings. This allows the app to change the volume, and to change from headphones to speaker or vice versa. Again, there is no clear reason why the Covid19 app should have this permission (unless they want to be able to shout at you in an emergency)—most likely it was left in by mistake.
   
• Run a "service" and start automatically after the phone is restarted. This is used to give you a notification reminding you to report if you haven't done so for a while.
   
• Prompt to install new packages (apps). This permission is usually requested if an app wants to bundle its own update mechanism. It won't be able to actually install other apps unless you say "yes" when asked.
   
• Access the phone's storage, i.e. shared files. This is not strictly necessary unless the app wants to store its data in a place that will persist even after it has been uninstalled and reinstalled, or if it wants to use the external storage card. Apps that request this permission can read files you leave in the shared filespace, so we normally want the app to have a good reason to ask for it.
   
• A permission called "bind get install referrer service" which is automatically added by Google Play Services unless the developer overrides it; it's believed to be harmless analytics on where the app came from.

Looking further into the app, the "config.*" files mostly contain graphics and messages in various languages, although the file "config.arm64_v8a.apk" also contains various library files, including one whose purpose is to detect faces in images. Hopefully this was left in by mistake, since there's no reason for this app to go looking through your album picking out faces (it doesn't have permission to use the camera, but apps with the Storage permission could in theory look at pictures you've already taken). Given that the company had to write the app so quickly, they probably copy/pasted a lot of code from another project and this app ended up with large chunks of unused code which is what we are seeing here; if they'd had more time to develop it properly they'd have hopefully taken this out.

The main part of the app is in a file called "com.joinzoe.covid_zoe.apk" which contains more face-detection resources (in the "assets/models" directory, hopefully left in by mistake), supporting files for Google Play Services and crash-analytics libraries (they want to know what happens if their app crashes), a Soundex library (for matching mis-spelled words), and 16 megabytes of compiled Kotlin code.

It's hard to analyse the code without source code, but there are some tools that can try to back-translate ("decompile") it into code that is partly readable by a developer. We used the "d2j-dex2jar" utility to back-translate the code, although it did fail on some of it, most notably on a couple of libraries from Facebook, one of which is responsible for managing Facebook advertisements. Again we hope this was left in the app by mistake and not actually used.

After "d2j-dex2jar" has done its work, the resulting "jar" files can be inspected using a tool called "jd-gui". We browsed through it and found various user-interface support libraries (not all of which are used), the Amplitude library for tracking what users do in the app, the Bumptech Glide image loading library, and libraries from both Facebook and Google that include advertising functions. The app also bundled a library called Expo that includes a barcode scanner, camera handler (although the app does not have permission to use this), SMS, speech and printing functions\u2014obviously much of this is unused and they didn't have time to take it out (that's how apps end up taking more space on your phone than they should). It also contains an open-source cryptography library which can help send data to their servers securely.

What we were really looking for, though, was the main part of the program—not all libraries are used, so it would be nice to start at the entry point and see which of them actually are used. In future it would help if unused libraries were completely removed, so we wouldn't need to have conversations like “yes we have code in our app that tracks faces and Facebook, but it's switched off and we forgot to take it out”, but obviously the developers had to rush and their method does seem to be “copy everything from another project and use just some of it”.

Unfortunately the all-important main-program classes under "com.joinzoe" do not seem to have been listed as such by the "dex2jar" utility, so finding them would require reading through the best part of a million lines of support code, which would take quite some time. (Perhaps some companies have a deliberate practice of dumping lots of extra unused code in their work just to confuse anyone who tries to analyse it.)

So all we can say at this stage is: we haven't confirmed the app won't contact Facebook, mess with your volume control, pop up misleading boxes over other apps, or look for faces in your photo album, but at least it can't use your camera or microphone, can't read your precise location and can't access the phone, contacts or messages. And the researchers who founded the startup do seem to have a track record of not being particularly devious, so let's apply Hanlon's Razor (“never attribute to malice what is sufficiently explained by stupidity”) and assume the suspect functions were left in by mistake and are most likely turned off. Moreover, we need this data, so do please use the app and self-report every day if you can.

One final note that may be of interest: The app requires at least version 5 of Android to run. Android 5 was released in 2014, and Google figures say 90% of Android users are on Android 5 or above (this is a global figure; they don't say how it's distributed by country). If you are in the 10% who are still using Android phones more than 6 years old, or if you have a non-Android non-iOS phone, then you cannot use the app, which may give the results a certain selection bias. The app asks for your age, so they could in principle correct for the over-representation of younger people (the elderly are less likely to be smartphone users), but it's harder to correct for differences in income: a low-income person stuck on a 7-year-old phone (and therefore excluded from the study) is more likely to have to catch a bus to work in a supermarket every day instead of staying safe at home doing office work. If this bus user with a very old phone is therefore more likely to encounter the virus, then the data is not completely representative if we're not counting them. But some low-income families do in fact have up-to-date smartphones because they've saved up for them and made it a priority, for example because they wanted to use religious or learning apps that required smartphones, and these will now be able to participate in the Covid19 study if they wish.

(written with help from Silas S. Brown)

Coronavirus (6) Animals tested positive for SARS-CoV-2

Three days ago, there was a report saying a Malayan tiger at the Bronx Zoo in New York city has tested positive for the SARS-CoV-2.¹ It is suggested she and the other big cats (two other tigers and three African lions) were infected by an asymptomatic zoo keeper. They started to show COVID-19 symptoms like dry cough and decrease of appetite since late March this year, but they are all expected to make a full recovery soon.

That was the first report of humans transmitting the disease to animals and causing sickness to the animals. Other than that, there had been a couple of instances of pet dogs testing positive for the coronavirus elsewhere in the world.^2,3 It is believed that they were being transmitted by their owners, but none of the dogs got sick or transmitted the disease to other species.

In my first article about coronavirus, I had mentioned that there is a chance animals can be infected, as ACE2, the receptor for SARS-CoV-2, is also present in dogs and cats. This indicates the potential of animals contracting the disease. However, we can expect that the number of coronavirus transmissions from humans to animals is not high, as transfer of a virus to a new host species is not a simple random process. For a virus to replicate and spread, it must be able to 1) bind to a cell-surface molecule; 2) carry out membrane fusion; 3) deliver critical components into the cytoplasm of the host; 4) avoid triggering apoptosis and highly suppressive type I interferon response; 5) interact successfully with cellular cofactors to replicate its genome and structural proteins; and 6) carry out virion assembly and exit the cells.

The infected animals in the Zoo have so far developed very mild symptoms and they are expected to recover soon. It is believed that they are infected with only a "fairly low dose" of the virus as the animals "did not have continual close contact with the asymptomatic zoo keeper", according to Dr Sarah Caddy, Veterinarian and Clinical Research Fellow at the University of Cambridge. However, as I said in my article written earlier, the other reason for very mild to no symptoms developed in the infected animals is their innate defences to suppress the infection. Additionally, their adaptive responses may eliminate the illness even if the infection from the virus overcomes their innate defenses. Therefore, the most common result of the virus transmission from humans to animals is the complete absence of disease or very mild symptoms.*

Although there is no need to fear the transmission of the virus back to humans from the infected animals, there is a possibility that the virus can linger on cats' fur and be "transmitted through touch in the same way it can be picked up from surfaces like tables and doorknobs." Therefore, according to Daniella Dos Santos, president of the British Veterinary Association (BVA), "cats should be kept inside if they live in a household where someone is suffering with the new coronavirus".⁴ Accordingly, the same principle should also apply to all households with animal pets.

*Although the first dog tested positive died few days after being released from mandatory quarantine, it did not show clinical symptoms when it was tested, and it was 17 years old, quite old by the breed's standards. Moreover, it was suffering from other underlying illnesses even before its 'weak positive' test. "It is very unlikely the virus had any contribution to the death of the dog."⁵

References

1. "Coronavirus: Tiger at Bronx Zoo tests positive for Covid-19" BBC News, 6th April, 2020. https://www.bbc.co.uk/news/world-us-canada-52177586

2. "Coronavirus: pet dog belonging to Covid-19 patient infected, Hong Kong health authorities confirm" South China Morning Post, 4thMarch,2020. https://www.scmp.com/news/hong-kong/health-environment/article/3065016/coronavirus-pet-dog-belonging-covid-19-patient

3. "Second Dog in Hong Kong Tests Positive for Covid-19 Virus" Bloomberg, 19th March,2020. https://www.bloomberg.com/news/articles/2020-03-19/second-dog-in-hong-kong-tests-positive-for-covid-19-virus

4. "Cats should be kept inside if owners show coronavirus symptoms, veterinary scientists say" CNBC, 8th April, 2020. https://www.cnbc.com/2020/04/08/cats-should-stay-inside-if-owners-show-coronavirus-symptoms-vets-say.html

5. "Coronavirus: 'very unlikely' Hong Kong dog that tested positive died from Covid-19, source says, citing old age, underlying illnesses" South China Morning Post, 4thMarch, 2020. https://www.scmp.com/news/hong-kong/health-environment/article/3075770/coronavirus-very-unlikely-hong-kong-dog-tested

Coronavirus (5) What is RNA test? Antigen test? Or antibody test?

This summary is not available. Please click here to view the post.

Coronavirus (4) RNA tests using RT-PCR vs using CRISPR

As described in the previous article, detection of SARS-CoV-2 genetic material can be done by both RT-PCR and CRISPR. Although the detection system using CRISPR to detect the SARS-CoV-2 is not yet available in the market, a comparison between the two CRISPR platforms (DETECTR from Mammoth Biosciences and SHERLOCK from Sherlock Biosciences) and the RT-PCR (represented by a protocol developed by CDC from the US), made by the Mammoth Biosciences, let us know more about the advantages of using CRISPR in virus detection.¹

Basically, when comparing the CRISPR platform and RT-PCR, CRISPR has the advantages of shorter running time, no expensive/ special equipment needed, no highly skilled technical staff needed, and thus lower costs in general to run the test. However, the sensitivity of the CRISPR platform is not as good as that of RT-PCR. The lowest limit for detection by CRISPR is in the range of 10 to 70 copies/ul of virus in the sample, while only 3.6 to 10 copies/ul of virus in the sample is already enough to be detected by RT-PCR.

Neither of the CRISPR platform for virus RNA detection have yet been approved by the FDA in the US or by any other country, however I believe that this detection system will become more widely used than the RT-PCR in the near future. The CRISPR detection system would be particularly useful in countries whose resources are very limited, which lack instruments and technical staff to run RT-PCR.

However, one thing that we do need to be aware of is the possibility of the off-target effect generated from the two exonucleases, Cas12a and Cas13a, used in the CRISPR detection system. No study on this has been published so far. The off-target effect of Cas9 in the gene editing system generates unexpected functions of a gene and may result in genomic instability.^2,3 The off-target effect generated from the activity of Cas12a/Cas13a in the CRISPR detection system can result in an incorrect recognition of the nucleotide target, and generates a false positive result. A thorough study of the incident rate of the off-target effect in Cas12a/Cas13a system, and finding out a mechanism to decrease the off-target effect, would give us more assurance of the usability of CRISPR in genetic material detection.

References

1. “A protocol for rapid detection of the 2019 novel coronavirus SARS-CoV-2 using CRISPR diagnostics: SARS-CoV-2 DETECTR” 2nd March, 2020. https://mammoth.bio/wp-content/uploads/2020/03/Mammoth-Biosciences-A-protocol-for-rapid-detection-of-SARS-CoV-2-using-CRISPR-diagnostics-DETECTR.pdf

2. Yanfang Fu, Jennifer A Foden, Cyd Khayter, et al. “High frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells” Nat Biotechnol. 2013 Sep; 31(9):822-826.

3. Seung Woo Cho, Sojung Kim, Yongsub Kim, et al. “Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases” Genome Res. 2014 Jan; 24(1): 132–141.

Coronavirus (3) CRISPR as an alternative to PCR technology in detecting SARS-CoV-2

Coronavirus (3): CRISPR as an alternative to PCR technology in detecting SARS-CoV-2
Until now, RT-PCR is the most commonly used method to detect the nucleic acid strand of SARS-CoV-2. With the use of an automatic machine with a robot arm which pipettes the solution and mixes the liquids on a number of tests simultaneously, in general a real time RT-PCR test can be done in 2 hours.

However, new assays based on CRISPR (clustered regularly interspaced short palindromic repeats) technology, which reduces the assay reaction time to 30 to 60 minutes for nucleic acid detection, are being developed.^1,2 Two biotech companies, Sherlock Biosciences (based in Cambridge, Massachusetts) and Mammoth Biosciences (based in California), that exploit CRISPR as diagnostic tools have simplified the tests so that each test can be done in one tube without any specialized or expansive equipment. Only test kit, two simple thermometer heat blocks, the sample, and basic laboratory equipment such as pipettes and pipette tips are needed for a test process.

The CRISPR nucleic acid detection tests developed by the two companies also made use of three other technologies: isothermal nucleic acid chain reaction expansion system, quenched fluorescent RNA reporter system, and the lateral flow assay. Isothermal nucleic acid chain reaction amplifies nucleic acid sequence from the sample for detection, and thus enhances sensitivity of the test to the attomolar level, that is, only around 100,000 copies are needed in the original sample for reliable detection (18 orders of magnitude smaller than a mole). CRISPR-Cas is used to identify a target sequence and to initiate the reporter system, which in turn indicates the presence of target SARS-CoV-2 sequences. Lateral flow assay is used for developing and showing the result on a strip of paper.

As the whole test does not involve extensive sample manipulation and expensive machinery, this makes the test field-deployable and available for point-of-care.

CRISPR-based lateral flow test kit.#

How CRISPR technology works
CRISPR technology was first applied in gene editing to fix genetic defects, to inactivate undesired genes, to insert new genes, or to study gene functions. For gene editing, CRISPR technology involves two components: Cas9 (CRISPR-associated 9) enzyme, which acts as molecular scissors (called endonuclease), and a custom-designed single-strand guide RNA, which has a sequence matching the target sequence of a gene to be edited. Guide RNA directs Cas9 to a target gene. By changing the guide RNA to match the target of interest, Cas9 can be programmed to efficiently identify and cut at a precise site on genomic DNA.

If the broken ends are left joined by the cell’s cellular repair process, a non homologous end joining (NHEJ) repair will take place. However, the NHEJ repair system is prone to mistakes and can lead to extra or missing bases in the joins. This often results in the inactivation of a gene. On the other hand, by introducing a separate sequence of a template DNA to the CRISPR cocktail, a cell can be induced to perform a different cellular DNA repair process called homology directed repair. The repair system use template DNA as blueprint to direct the rebuilding process. By addition of a template sequence, a defective gene is repaired, or a completely new gene is inserted.

Due to the ability of CRISPR to fix DNA errors, the technology has been used in the treatment of diseases due to genetic defects, such as Turner syndrome and sickle-cell anaemia. The following video provides clear explanation on how the CRISPR works and in which fields the CRISPR gene editing have been applied.

How CRISPR lets you edit DNA. By Andrea M. Henle

How can a CRISPR technology be used in the field of diagnosis
The CRISPR system is not only to be harnessed for gene editing, it can also be used in the field of diagnosis. With the efforts made by researchers working on identifying Cas molecules in bacterial science, different Cas endocucleases with different molecular properties have been identified in the past few years. Both Cas12a (Cpf1) and Cas13a (C2c2), which are found to possess both nucleic acid sequence recognition ability and dual cleavage activities, are being used in the detection of viral RNA or bacterial DNA genomes that cause disease. Cas12a recognizes a DNA sequence, while Cas13a recognizes an RNA sequence.

Cas12a and Cas13a perform specific binding and cleavage with the aid of guide RNA, which is complementary to the target sequence. This mechanism is similar to that used by Cas9. However, the two endonucleases have trans- or collateral cutting activity, which is activated upon target binding. This property is not possessed by Cas9. Once being activated by finding the target, the endocucleases will cut the target sequence and also indiscriminately cut other single-stranded nucleic acid sequences in the vicinity. The two distinct cleavage (cutting) activities of the endonucleases are used to leverage nucleic acid detection, as every endonuclease activation can lead to cleavage of thousands of reporter nucleic acids. This results in potent signal amplification.

Both biotech companies have published in detail how tests based on CRISPR technology were developed, and their detection protocols for SARS-CoV-2 have been released to the public. DETECTR (from Mammoth Biosciences) detects N gene and E gene from the SARS-CoV-2 genome, while SHERLOCKv2 (form Sherlock Biosciences) detects both S gene and Orf1ab. Basically, the detection systems from the two biotech companies involve 4 steps:
1. Extraction of nucleic acid from sample.
2. Amplification of nucleic acids from sample using isothermal amplification. Since isothermal amplification uses a single temperature, no expensive specialized instrumentation is needed to adjust temperatures over time, as would be needed if conventional PCR were in use.
3. Cas12a/Cas13a activation upon target recognition, and this mediates indiscriminate cleavage (random cutting) of reporter RNA: if the target sequence is present in the pool of amplified nucleotides, the non-specific cleavage (random cutting) activity of the Cas becomes activated. The nucleic acid reporters with quenched fluorescent molecules will be cleaved (cut), resulting in activation of the fluorophore. The fluorescent signal is thus an indicator to signal whether the target sequence is present in the test sample.
4. Visual colour readout using paper lateral flow strip, which captures the cleaved reporter RNA with labelled ends on specific antibody bands.

DETECTR have a separate step to extract the nucleic acid from a sample, and combined reactions in steps 2 and 3 into single tube. On the other hand, SHERLOCKv2 have steps 1 to 3 done in one tube by using the HUDSON method (Heating Unextracted Diagnostic Samples to Obliterate Nuclease), to release viral or bacterial nucleic acid from clinical specimens and to protect it from degradation. This bypasses the need for nucleic acid extraction.

The DETECTR platform enables detection that is 30 minutes faster than SHARLOCKv2. This is because the time spent on the additional in vitro transcription step, which is required for SHARLOCK platform, is saved.

Sherlock Biosciences have used synthetic SARS-CoV-2 virus RNA fragment for validation of the test. Currently, the company is collaborating with scientists from the Harvard School of Public Health in trialling the SARS-CoV-2 diagnostic test on patients.



#Picture adopted from "Point-of-care CRISPR/Cas nucleic acid detection: Recent advances, challenges, and opportunities. Biosensors and Bioelectronics, Volume 166, 15 October 2020, 112445"

References
1. “Coronavirus detection using CRISPR diagnostics” https://www.synthego.com/blog/crispr-coronavirus-detection
2. “How SARS-CoV-2 tests work and what’s next in COVID-19 Diagnostics. The Scientist, 3 March, 2020. https://www.the-scientist.com/news-opinion/how-sars-cov-2-tests-work-and-whats-next-in-covid-19-diagnostics-67210

Coronavirus (2) Test kits

Since the outbreak of COVID-19 in Wuhan, China last December, the disease has now spread to more than 70 countries worldwide. While much effort has been put into containing the disease by speeding up the testing for the severe acute respiratory syndrome-coronavirus-2 ( SARS-CoV-2), the news last week about the first test kits distributed by the United States CDC being found unable to produce consistent conclusive results from the negative control, is a bit bizarre .¹

COVID-19 real time RT-PCR test kit by CDC of the United States.

This may arise your interest in knowing what test methods have been used by your own country for the newly emerged coronavirus. How do the tests work? How accurate are the test results from the test kits, and how the test results usually been validated and verified?

RT-PCR test for SARS-CoV-2

Until now, real time RT-PCR (reverse transcription polymerase chain reaction) technology has been the only method to detect the virus globally. PCR is an amplification method by which a targeted nucleotide sequence of an organism can be multiplied exponentially. By first converting the RNA sequence of the virus genome into complementary DNA and subsequently amplifying the target sequence using the complementary DNA as template, even a tiny amount of the virus genome in collection sample can be detected.

Overview of Reverse Transcription-Polymerase Chain Reaction

(From Wikimedia Commons photo: licensed under the Creative Commons Attribution-Share Alike 4.0 International license.)

In early January, a genetic sequence of the newly emerged coronavirus was first released by China.² The analysis of the genome structure revealed that SARS-CoV-2 has 79% identity with the SARS-CoV.³ The whole viral genome contains genes encoding non-structural polyprotein, S (Spike) protein, E (Envelope) protein, M (Membrane) and N (Nucleocapsid) protein (S, E and M proteins together form the viral envelope). Based on the viral genome data, researchers from different countries developed test kits by designing primers, short stretches of DNA, to amplify mainly S, N, and E gene regions of new virus.⁴ The nucleotide regions on the virus which encode the 3 proteins are less prone to mutation and are therefore usually picked for virus detection in RT-PCR test.

3D medical animation still shot showing 2019 novel Coronavirus Structure. From Scientific Animation: www.scientificanimations.com.  Click for full-size image.

How reliable are tests using RT-PCR?

RT-PCR tests have been widely used in diagnosis of other viruses such as mumps, HIV, and influenza, and are normally highly reliable. In order to validate the results of each PCR experiment, negative control and positive control are included in every experiment for sample testing. Simply speaking, the positive signal in positive template control is an indication to show that the experiment works. The clear negative signal in the negative control is an indication of no contamination for the experiment. This is used to validate the positive test result of a sample in the same experiment.

For the new coronavirus, every country uses different criteria to make final diagnosis. These include the clinical observations and epidemiological data. If the RT-PCR result did not match with the clinical observation, in most cases RT-PCR will be repeated. Therefore, the chance of misdiagnosis is lower even the RT-PCR test result is wrong.

What are the possible causes of false-negative from RT-PCR test?

Although controls are used in RT-PCR test experiment to validate the positive results and to prove the experiment is working, false-negative from RT-PCR test is unavoidable. According to experience from China, the false-negative accounts for 3% of RT-PCR tests for COVID-19 patients.⁵

The false-negative from RT-PCR test may come from technical handling errors such as inappropriate specimen collection, storage, and transport. Viral RNA is very much prone to degradation with higher temperature. Therefore, once the sample is collected, it should be placed in a designated collection tube and be kept and transported at 4°C to 8°C. If the test is not going to be done in 24 hours upon sample collection, it should be kept frozen. In addition, the way the tests are being conducted may also cause problems. A dangle or a good rub could mean a big difference in the amount of virus material being collected.

Moreover, insufficient viral material in the specimen collected can also lead to false-negative results. A patient in the early state of infection will shed much less virus; tests taken at this stage have a higher chance of showing negative results. In addition, if the virus is drawn toward the lower respiratory tract for example, then a test from throat or nose swab may miss the virus.^6,7 Samples collected from tracheal aspirate or sputum are alternatives for a highly suspicious patient with negative test result.

However, there is the possibility that the tests are accurate and the patients do not have coronavirus at the time of testing, according to Dr MacDermott of King’s College London. The early signs of coronavirus are very similar to other respiratory viruses. The patients may not be actually infected with the new coronavirus, therefore the test result is negative. But they can became infected and later test positive for the coronavirus.⁷

What we can learn so far from the countries worldwide in their handling of the outbreak of COVID-19?

Since the outbreak in South Korea in late February, the country has now managed to test more than 10,000 people a day for the newly emerged coronavirus, using kits provided by 4 local biotech companies, with sensitivity rates of over 95%. This powerful, fast testing ability is mostly attributed to its painful experience in handling the outbreak of Middle East Respiratory Syndrome (MERS) in 2015. Since then, the country has set up a system to allow rapid approval of testing kits for viruses that may cause pandemics.⁸ While the shortage of test kits in Japan and the US has jeopardized the containing of the virus in those countries, the effective collaboration system between the regulator and the local biotech companies in South Korea has provide a good example for countries worldwide in handling the outbreak of new disease.

The number of patients with COVID-19 is now surging in European countries over the last two weeks. Let us hope that these countries do not have bureaucratic processes that prevent them from providing a high capacity of tests that can quickly identify and treat COVID-19 patients.

References

1. https://web.archive.org/web/20200306034835/https://www.sciencemag.org/news/2020/02/united-states-badly-bungled-coronavirus-testing-things-may-soon-improve

2. https://web.archive.org/web/20200307043124/https://www.ncbi.nlm.nih.gov/genbank/sars-cov-2-seqs/

3. Roujian Lu, Xiang Zhao, Juan Li et al. Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet 2020; 395: 565–74.

4. https://web.archive.org/web/20200303000654/https://www.who.int/emergencies/diseases/novel-coronavirus-2019/technical-guidance/laboratory-guidance

5. XingzhiXie, Zheng Zhong, and Wei Zhao et al. Chest CT for Typical 2019-nCoV Pneumonia: Relationship to Negative RT-PCR Testing. Radiology, Published Online:Feb 12 2020. https://doi.org/10.1148/radiol.2020200343

6. “What actually happens during a coronavirus test?” Arman Azad, CNN, 5 March 2020. https://web.archive.org/web/20200306034011/https://edition.cnn.com/2020/03/04/health/coronavirus-test-what-happens-explainer/index.html

7. “Are Coronavirus tests flawed?” James Gallagher. BBC news, 13 February 2020.

8. “Virus Testing Blitz Appears to Keep Korea Death Rate Low”. Heejin Kim, Sohee Kim, and Claire Che. Bloomberg, 4 March 2020.

Coronavirus (1) Behind the questions and answers from WHO

The outbreak of the new coronavirus COVID-19 has caused much concern. The World Health Organization has set up specific web pages, https://web.archive.org/web/20200217030319/https://www.who.int/health-topics/coronavirus, and https://web.archive.org/web/20200215214737/https://www.who.int/emergencies/diseases/novel-coronavirus-2019/advice-for-public/myth-busters to answer common questions about coronaviruses in general and the newly emerged one in particular. I would like to share with you the scientific factors on some of their sayings and my thoughts.

"Coronaviruses (CoVs) are a large family of viruses that cause illness ranging from the common cold to more severe diseases such as Middle East Respiratory Syndrome (MERS-CoV) and Severe Acute Respiratory Syndrome (SARS-CoV). A novel coronavirus is a new strain that has not been previously identified in humans." (WHO)

Coronaviruses are enveloped viruses with a positive sense, single-stranded RNA genome. With genome sizes ranging from 26 to 32 kilobases in length, CoVs have the largest genomes for RNA viruses. Not all coronaviruses cause severe diseases. Human coronaviruses 229E (HCoV-229E), OC43 (HCoV-OC43), NL63 (HCoV-NL63), and HKU1 (HCoV-HKU1) have been circulating in humans for a long time. These viruses cause syndromes related to the common cold.

SARS-CoV, MERS-CoV and COVID-19 have recently been introduced to the human population and therefore the humans are immunologically naive to the viruses. These newly-emerged viruses can cause serious respiratory tract infections and high mortality rate (SARS, ~10%¹; MERS, > 35%²). For the COVID-19, we don't yet know the average mortality rate. According to a report on Lancet,³ the mortality rate of the 99 patients who were among the earliest discovered cases was ~11%. According to the official reports from the government of China, the mortality rate of the confirmed cases is about 2.1%.

"Coronaviruses are zoonotic, meaning the viruses are transmitted between animals and people. Detailed investigations found that SARS-CoV was transmitted from civet cats to humans and MERS-CoV from dromedary camels to humans. Several known CoVs are circulating in animals that have not yet infected humans." (WHO)

There are 7 strains of CoVs that can infect humans while more than 200 strains of coronaviruses infect animals. The coronavirus family is composed of 4 genogroups:1. alpha; 2. beta; 3. gamma; and 4. delta. Groups 1 to 4 infect birds and a variety of mammals, while coronavirus groups 1 and 2 are known to infect humans. SARS-CoV, MERS-CoV and COVID-19 belong to group 2. Recent studies have suggested that bats are the natural reservoir of a range of coronaviruses.^4,5,6

Both SARS-CoV, and MERS-CoV are believed to have originated from bats, with civet cats⁷ and dromedary camels⁸ are respective intermediate transmitters of the viruses to humans. According to a virologist at the University of Hong Kong, Kwok-Yung Yuen, who co-discovered the SARS virus, the best way to prevent the outbreak of another new emerging disease is "to avoid disturbing wildlife habitats and never put wildlife into markets. Respecting nature is the way to stay away from the harm of emerging infections."⁹

"Can I catch COVID-19 from my pet?" "No, at present there is no evidence that companion animals or pets such as cats and dogs have been infected or have spread COVID-19."(WHO)

For enveloped CoV to enter into host cells, spike (S) glycoprotein on CoV, is needed to mediate the virus entry. The spike protein is a principle cell entry protein responsible for attachment and membrane fusion.¹⁰ The protein receptors on humans for spike protein identified so far are all ectopeptidases. These include angiotensin-converting enzyme 2 (ACE2) for SARS-CoV¹¹ and COVID-19¹² and dipeptidyl peptidase 4 (DPP4) for MERS-CoV.¹³ The protein receptors are highly expressed in the epithelial cells of the respiratory and enteric tissue, making them attractive targets for viruses to enter the host.

The ACE2 is also expressed in cats and dogs, indicating the potential of pets contracting COVID-19. In fact, several types of coronaviruses can cause illness in animals and spread between animals and people. Therefore, we should avoid letting our pets wander around infectious areas, and wear facemask if we care for a sick pet.

However, it is not necessary to abandon our pets for fear they may transmit COVID-19. There have not been any reports of pets or other animals becoming sick with COVID-19. In fact, for a type of virus to jump from one species to another species and successfully replicate and spread , it must undergo a series of mechanisms* which most commonly results in the complete absence of the disease in the targeted host species. The jump from bats to humans does not mean it is likely to occur again and infect another species.^#

"Can COVID-19 be caught from a person who presents no symptoms?" "Understanding the time when infected patients may spread the virus to others is critical for control efforts. Detailed medical information from people infected is needed to determine the infectious period of COVID-19. According to recent reports, it may be possible that people infected with COVID-19 may be infectious before showing significant symptoms. However, based on currently available data, the people who have symptoms are causing the majority of virus spread."(WHO)

According to a preprint posted on medRxiv, the incubation period of COVID-19 can be as long as 24 days. This generates more difficulty to exclude the possibility of the infected person having a second unrelated contact. This also increases the possibility of coronavirus becoming a pandemic disease, like the influenza but with higher mortality rate, affecting everyone globally. The development of vaccine for the coronavirus may reduce the harm it causes to humans.

"Is it safe to receive a package from China or any other place where the virus has been identified?" "Yes, it is safe. People receiving packages are not at risk of contracting the new coronavirus. From experience with other coronaviruses, we know that these types of viruses don't survive long on objects, such as letters or packages."(WHO)

Information on the survival data of several coronaviruses, on different environments, that infected humans can give us an idea of what and where we should be mindful to avoid contracting the new coronavirus. Below is some useful information from some of the reports:

- HCoV-229E was found not fully inactivated for at least 7 days after deposition on different environmental surfaces at ambient temperature and relative humidity condition of approximately 50%.¹⁴ This is alarming as the minimum infective dose of respiratory viruses can be very low.¹⁵

-Among the frequently-touched surfaces in a classroom, the doorknob was found to have high content of HCoV-229E.¹⁴ This reminds us to be mindful of doorknobs in public places.

-In outbreaks units, SARS-CoV nucleic acids were detected on air samples, surfaces and inanimate objects. This suggests droplets and aerosol generation by a patient with SARS-CoV, and that surfaces could be sources of virus transmission.¹⁶

-SARS-CoV can survive for 36 hours on stainless steel.¹⁷

-SARS-CoV on a polystyrene surface showed slower inactivation.¹⁸

-In suspension, SARS-CoV retained its infectivity for up to 9 days; in the dried state, survival time was 6 days.¹⁸

-Most SARS cases were the result of direct transmission via respiratory droplets during close personal contact, and adequate respiratory protective measures were shown to be effective.¹⁹

-At the Amoy Gardens high-rise housing estate in Hong Kong, transmission probably occurred through SARS-CoV shed in the faeces of a patient.²⁰ The spreading of virus can be minimized if we flush with the toilet lid closed.

*Viruses can be carried from one type of animal to another in a variety of ways. By far the most common result is the complete absence of disease. This is because when replication in the new host does occur, the innate defenses from the new host can suppress the infection. Moreover, the adaptive responses of the new host can eliminate the illness even if the infection from the virus overcomes innate defenses. Very rarely, virus replication evades innate and adaptive immune responses and causes overwhelming disease.

^#For a virus to replicate and spread, it must be able to 1) bind to a cell-surface molecule; 2) carry out membrane fusion; 3) deliver critical components into the cytoplasm of the host; 4) avoid triggering apoptosis and highly suppressive type I interferon response; 5) interact successfully with cellular cofactors to replicate its genome and structural proteins; and 6) carry out virion assembly and exit the cells.

Therefore successful transfer of a virus to a new host species is not a simple random process.

References

1. Drosten C, Gunther S, and Preiser W, et al. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med. 2003; 348:1967-1976.

2. Baharoon S and Memish ZA. MERS-CoV as an emerging respiratory illness: A review of prevention methods. Travel Med Infect Dis 2019-Review. PMID 31730910

3. Nanshan Chen, Min Zhou, Xuan Dong, et al. Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study. The Lancet Published online: January 30, 2020. DOI: https://doi.org/10.1016/S0140-6736(20)30211-7.

4. Li W, Shi Z, Yu M, et al. Bats are natural reservoirs of SARS-like coronaviruses. Science 2005; 310:676-9.

5. Lau SK, Woo PC, Li KS, et al. Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats. Proc Natl Acad Sci U S A 2005;102:14040-5.

6. Simmons NB Order Chiroptera. In: Wilson DE, Reeder DM, editors. Mammal species of the world. Baltimore: Johns Hopkins University Press; 2005. p. 312-529.

7. Guan Y, Zheng BJ, He YQ, et al. Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China. Science 10 Oct 2003: Vol. 302, Issue 5643, pp. 276-278.

8. Raj VS, Farag EA, Reusken CB, et al. Isolation of MERS coronavirus from a dromedary camel, Qatar, 2014. Emerg Infect Dis. 2014; 20(8): 1339-42.

9. Cyranoski D. Bat cave solves mystery of deadly SARS virus - and suggests new outbreak could occur. Nature 2017: 552, 15-16.

10. Li F. Structure, function, and evolution of coronavirus spike proteins. Annu Rev Virol. 2016 Sep 29; 3(1): 237-261.

11. Li W, Moore MJ, Vasilieva N, et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 2003, 426:450-454.

12. Zhou P, Yang XL, Wang XG, et al. A Pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 2020, DOI: 10.1038/s41586-020-2012-7.

13. Raj VS, Mou H, Smits SL, et al. Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC. Nature 2013, 495:251-254.

14. Bonny TS, Yezli S, and Lednicky JA. Isolation and identification of human coronavirus 229E from frequently touched environmental surfaces of a university classroom that is cleaned daily. American Journal of Infection Control 46 (2018) 105-7.

15. Yezli S, Otter JA. Minimum infective dose of the major human respiratory and enteric viruses transmitted through food and the environment. Food Environ Virol 2011; 3: 1-30.

16. Booth TB, Kournikakis N, Bastien J, et al. Detection of airborne Severe Acute Respiratory Syndrome (SARS) coronavirus and environmental contamination in SARS outbreak units. J. Infect. Dis. 191:1472-1477.

17. World Health Organization. 2003. First data on stability and resistance of SARS coronavirus compiled by members of WHO laboratory network. World Health Organization, Geneva, Switzerland. http://www.who.int/csr/sars/survival_2003_05_04/en/index.html.

18. Rabenau HF, Cinatl J, Morgenstern B, et al. Stability and inactivation of SARS coronavirus. Med. Microbiol. Immunol. 2005, 194:1-6.

19. Seto WH, Tsang D, Yung RW et al. Effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (SARS). Lancet 2003 361:1519-1520.

20. Hong Kong Department of Health (2003) Outbreak of severe acute respiratory syndrome (SARS) at Amoy Gardens, Kowloon Bay, Hong Kong.http://www.info.gov.hk/info/ap/pdf/amoy_e.pdf