Stuff from the literature: very SARS-like coronavirus in Chinese horsehoe bats...

The smoking bat for SARS-CoV?

Xing-Yi Ge and colleagues from China, USA, Australia and Singapore described some new severe acute respiratory syndrome (SARS)-like coronaviruses (SL-CoVs) in bats, publishing in Nature last month.

These discoveries were especially notable (not that any new virus discovery isn't) because they displayed more "SARS-like" properties than many earlier so-called SARS-like CoVs. One could grow in the same line of lab cells and also in human cells, it could be visualized by electron microscopy and it could use the same receptor as the SARS-CoV (angiotensin converting enzyme II; ACE2) . Plus, they were genetically very similar.

The bat species was confirmed by gene sequencing to be Rhinolophus sinicus, family Rhinolophidae; the Chinese rufous horseshoe bat.

Throat and faecal samples (anal swabs and faeces) were screened using RT-PCR with primers towards the conserved RNA-dependent RNA polymerase region (RdRp) and new primers were designed to detect other regions of any discoveries. 27 of 117 samples were CoV POS and had sequences determined.

Two novel (and 5 previously identified) SL-CoVs, each with a 29,787+ base pair RNA genome and sharing 95% nucleotide identity with the Tor2 strain of the SARS-CoV which is higher than previous SL-CoVs from China. The receptor binding domain (RBD) of the new CoVs shared 85-96% amino acid identify with the SARS-CoV. These were called:

1. RsSHC014
2. Rs3367

Vero cells were used to attempt growth of SL-CoV virions that were first concentrated from samples. This succeeded for one sample, a variant of Rs2267 (99.9% nucleotide identity with Rs3367) and they named this isolate SL-CoV-WIV1. This success is something that hasn't been achieved with the majority of recently identified bat CoVs.

WIV1 also grew, although less efficiently, in:

• human alveolar basal epithelial (A549) cells
• pig kidney (PK-15) cells
• R.sinicus kidney (RSKT) cells

...but not in...

• Human cervix (HeLa) cells
• Syrian golden hamster kidney (BHK21) cells
• Myotis davidii kidney (BK) cells
• Myotis chinensis kidney (MCKT) cells
• Rousettus leschenaulti kidney (RLK) cells
• Pteropus alecto kidney (PaKi) cells

So we have much more convincing evidence that the SARS-CoV is likely to have originated from a bat.

h/t to @MERS_inSAUDI

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No symptoms but still shedding virus?

Click on image to enlarge.
A stylized trace of the temperatures during a PCR cycle.
D-denaturation, when primers and double-stranded
DNA (dsDNA) are reverted to single strands of DNA;
A-annealing, when primers 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 MERS-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 (MERS-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 MERS. 

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 MERS-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 MERS-CoV? We have some camel seroepidemiology studies which I've previously described here and here. Human studies?

1. In the study that found MERS-CoV-like neutralizing antibodies in Egyptian camels, no human sera from Egypt (815 from 2012-13 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 MERS-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 MERS-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 MERS-CoV.

Work like that mentioned for SARS largely remains to be done for MERS. 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 MERS-CoV cases. 

What does a positive PCR result mean in an asymptomatic MERS-CoV case? Still can't answer that. Are contacts seroconverting as an indication of MERS-CoV infection? Still can't answer that. How many mild or asymptomatic MERS-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 MERS-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

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Does MERS-CoV delay the antiviral response against it?

Lau and colleagues from the University of Hong King recently wrote about MERS-CoV's ability to delay the induction of proinflammatory cytokines in human cells. These are potent defensive chemicals that cause and accelerate an inflammatory response to viral infection.

While inflammation is part of kicking out the virus, it can get out of control, especially with more "foreign" viruses, like those that are still zoonotic and not as co-evolved with humans. Excessive inflammation can lead to tissue remodelling and damage.

In their paper in the Journal of General Virology, the authors compared the results of growing viruses in 2 different cell systems, which was need to account for differences in tropism by the different viruses. Key findings include:

• The lower airway cell-line, CaLu-3 was used for MERS-CoV and SARS-CoV because they produce good titres
• IL-1�, IL-6, IL-8, TNF-a, IFN-� & IP-10 mRNA levels were increased by MERS-CoV and SARS-CoV compared to uninfected cells
• Proinflammatory cytokines shown in bold above were induced more by MERS-CoV infection than by SARS-CoV
• Innate antiviral cytokines TNF-a, IFN-� & IP-10  were induced more by SARS-CoV infection
• MCP-1 (a chemokine) and TGF-� (anti-inflammatory cytokine) remained unimpressed by infection
• At 48-hours, MERS-CoV infection induced less IL-8 or IFN-� protein than did SARS-CoV

• Embryonal lung fibroblasts (HFL) to grow MERS-CoV and compare it to HCoV-229E (which does not grow well in CaLu-3 cells), an infrequently identified but well characterised "common cold" CoV.

• The CaLu-3 results suggested MERS-CoV generates an attenuated innate immune response  the response which induces inflammation. SO HCoV-229E was added as it produces a strong innate response through IFN-�
• IL-1�, IL-6, IL-8, TNF-a, IFN-� & IP-10 mRNA levels were increased by MERS-CoV and HCoV-229E
• HCoV-229E was a stronger inducer than MERS-CoV of all but TNF-a, which triggered more by MERS-CoV
• MCP-1 and TGF-� again remained unchanged

The authors conclude a delayed innate immune response by MERS-CoV infection compared to SARS-CoV. 

This study contrasted with that by Kindler and colleagues which I reviewed earlier. The reason may be because Kindler only sampled for immune analyses up to 12-hours, rather than the 30-hours used by Lau. This would certainly lend weight to claims of a "delayed" induction by MERS-CoV infection since in this study no IFN-� was produced either virus at 12-hour, the first rise in mRNA was apparent at 24-hour post-infection (Lau's protein data suggest there may have been a tiny amount of IFN-� translation at 12-hours, but Kindler did not measure protein). 

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MERS-CoV, SARS-CoV, HCoV-229E: comparative culture and immunology

The following study in mBio by Kindler and colleagues from Switzerland, Germany, Denmark and the Netherlands came out in February but, as I'm trying to brush up for a talk next week, it's an important one to add to my recent list of quick reviews.

The authors used human bronchial airway epithelium cultures (HAE), also known as air-liquid interface (ALI)* cultures, to grow and examine the immune responses resulting from growth of, the Middle East respiratory syndrome coronavirus (MERS-CoV), the severe acute respiratory syndrome (SARS) CoV or HCoV-229E. 

These cultures start life as scraped/brushed/biopsied primary cells that are then "grown out" in special culture flasks in the presence of the right solution of hormones and chemicals, so that they can mimic a true mature epithelium - multilayered, mucous-producing and with beating cilia and tight cell:cell junctions.

So, at 6-hours post-infection with either virus, RNA was purified and run through a next generation sequencing protocol to attempt assembly of the genome of culture virus. Even after culture and using a genome to assemble against, only 0.006% (1,616/24,053,494) of reads could be ascribed to the virus.

Some key points of the culture findings:

• MERS-CoV reached peak levels of replication after 48-hours
• SARS-CoV peaked 72 to 96-hours after infection.
• MERS-CoV infected mostly non-ciliated cells (supporting other findings)
• No induction of interferon (IFN)-� resulted from infection by any CoV
• Only marginal expression of proinflammatory cytokines (TNF-a most active) resulted, mainly by HCoV-229E infection, at 6-hours, suggesting equivalent adaptation of the virus to growth in HAE cultures and that 
• Human bronchial epithelium, in the absence of dendritic and other cells, does not mount a strong innate immune response to the CoVs used.
• Uninfected HAE cultures respond quickly to treatment with IFN-a (a type I IFN) or IFN-?3 (type III IFN), with upregulated expression of RNA from IFN-stimulated genes (ISGs; Mx1, 2'5'OAS, Stat1, Mda5 and Rig-I)  
• Addition of IFN-a or IFN-?3 to try and "protect" sick cells (by pre-incubating with IFN then infecting them) reduced viral genome replication compared to no treatment for MERS-CoV, SARS-CoV and HCoV-229E.

*Thanks for Ron Fouchier and Ronald Dijkman for clarifying HAE are grown under ALI conditions.

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How MERS may be SARS, but we don't really know

On July 26th, Prof Christian Drosten wrote in Lancet Infectious Diseases about some similarities and differences between the diseases Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS), each linked to a zoonotic coronavirus (CoV) infection.

Drosten is senior author of the 2 publications papers describing gold-standard MERS-CoV laboratory diagnostic methods, all PCR-based, which afford excellent diagnostic and genotyping capabilities upon the user. Unfortunately we have not yet seen much use of the genotyping assays. He has also co-authored papers on the MERS-CoV receptor, viral replication, its naming, discovery of antibodies in camels and MERS case reports. He has an even bigger list of diverse publications on the SARS-CoV.

Drosten is well positioned to say that at first glance it is not the same beast but that we have many things to learn before we can be sure of that. 

Reviewing data from Assiri and colleagues from the same issue of Lancet, he noted that MERS and SARS have some similarities. Cases often presented with fever as a classifying symptom at presentation. Upper respiratory tract symptoms were not common (4-40% of cases had something that could identify an upper airway disease) and so most cases could be clinically differentiated from the common cold.

A major difference from SARS has been the high level of comorbidities associated with MERS cases. However, this needs to be interpreted with caution since for example, a third of people in a pre-MERS study of Saudi Arabian outpatient visits had diabetes, including more than half over the age of 50-years. In that context, the proportion of MERS-CoV positives among his group in the Kingdom of Saudi Arabia (KSA) population may not be so over-represented. It may simply flag the opportunistic nature of the virus. 

MERS also differentiates itself from SARS in its rapid progression to a fatal outcome; again this may be related to the population it is affecting the most; older males with comorbidities. Mechanistically, MERS-CoV differs in its cellular receptor (DPP4 vs ACE2 for SARS-CoV) and its greater replicative efficiency and ability to infect a wider range of cell types in the lower airways compared to SARS-CoV. And then there's the spelling, nucleotide and amino acid sequence differences too!

Drosten also poses some questions: 

1. Can we rely on samples from the upper respiratory tract in which viral loads can be low enough to muddy the waters of result interpretation?
2. We need serological assays and we need to define them using characterized cases. Then we need to roll them out at the population level to better define those icebergs that seem to be everywhere these days

Without further data, we're still left to ponder what would SARS have been like if the CoV got into the KSA? In fact, what do other respiratory viruses do to those with comorbidities in the KSA?

There are similarities and differences between MERS and SARS and between their viral causes. More work is needed. 

The best way to answer the question posed in Drosten's article would have been a direct comparison of the impact of the two viruses in the same population. Thankfully for the hosts, there has been no significant overlap between the 2 outbreaks so far.

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Time for the bat signal? The need for an animal model for Middle East respiratory syndrome coronavirus.

Elizabeth Devitt notes in Nature Medicine, that unlike its cousin, the severe acute respiratory syndrome coronavirus (SARS-CoV), some important features of MERS-CoV including its transmission, incubation period, and ability to spread systemically within the host, have not been able to be defined for the MERS-CoV using non-human models, because the virus does not like to infect the same animals. 

When the MERS-CoV infects a larger non-human animal, the rhesus macaque monkey, the disease it produces, while still defined as pneumonia and proving the casual link between MERS-CoV infection and disease, resolved faster and was not as severe as that in humans. These animals are also not easy to work with. I wonder if older monkeys with comorbidities have been looked at in particular? [UPDATE: The macaques above live to about 25-years]. It is this population in which MERS is most severe. Nonetheless, the monkey studies provide an excellent vehicle on which to test the usefulness of 2-drug an antiviral approach (Falzano et al, described earlier) that can clear MERS-CoV infections in vitro.

While cell/tissue culture methods using primary human airway cells have proven extremely useful for looking at cellular biology, antiviral effects, and immunobiology related to MERS-CoV infection, something with legs will be needed for future vaccines and to address the list above. We've seen many examples of how animal models massively improve our understanding of influenza virus pathogenesis, if an example is needed.

Also according to Devitt, Ian Lipkin is still wading through the data from samples collected from a range of animals that may be the natural hosts for the MERS-CoV in Saudi Arabia. Meanwhile, we recently learned of another CoV (PML/2011) found in the fecal pellets from a South African Neoromicia cf. zuluensis bat in 2011. PML/2011's nearest CoV relative was the MERS-CoV - its closest viral relative found to date (at least in the conserved RdRp region used by the authors).

This all begs the question, is there a bat animal model? CoVs, but also studies of other viruses like Hendra and Nipah, would benefit from a well-defined model based on these critters. That is, if they can be worked with and if they show any signs of these diseases - which they may not. My very quick skim of the literature found that bats used for neurological studies and for Hendra virus.

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