Influenza A (H7N9) virus update...

Xinhuanet has the latest H7N9 figures from China's National Health and Family Planning Commission. With no new cases reported this month, and just one from July reported in last month's update, the 12 provinces or municipalities that have hosted infections resulted in:

• 134 cases (I have 136; presumably the Taiwan case and asymptomatic Beijing boy)
• 45 deaths, up form from last month
• 87 discharged cases leaving 2 still in hospital
• PFC at 33.1%

I've updated by VDU H7N9 page with these numbers also.

Thanks to Crawford Kilian's Tweet for making me aware of this report.

Concise overview of MERS from mid-2013...

Publishing in the Oman Medical Journal, Blakhair and colleagues summarized the state of play for MERS back in July 2013.

While the numbers may have changed since then, the article is as relevant now as it was. Not much new data has been accrued to address the knowledge gaps listed in July despite 3-months and about 50 papers having passed.

The final summary is particularity useful I thought...

The fact that our current knowledge on this virus is sparse should not induce unnecessary panic or fear, instead it should promote vigilance and a state of preparedness. Over reaction to the current situation may lead to significant clinical, economic and epidemiological impacts among others.

The article also provides VDU's first ever citation in the scientific literature. Cool.

Reference...

1. The Struggle Against MERS-CoV (The Novel Coronavirus). Oman Medical Journal. 2013. 28(4):226-7
   http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3725253/pdf/OMJ-D-13-00242.pdf

The tip of the VirusBerg...

The VirusBerg.
Click to enlarge. If there are more cases with mild/less
 symptomatic disease then the denominator grows
and the proportion of deaths to survivors, the PFC,
shrinks. At the start of an outbreak or emergence of
a new pathogen, it is usually the most severely ill or
those ill with a specifically identifiable disease
that attract the attention of specimen collectors &
testers. Later in the event as resources are freed up
and panic reduces, testing of other population
groups may find more cases of less severe disease.
 This may not eventuate though and the PFC may
 remain high confirming a serious threat to human
health. Feel free to use graphic. Cite this blog
and Ian M. Mackay.

I've talked about the Denominator problem over on my "main" site, Virology Down Under's H7N9 page, but I thought it worth revisiting for 2 reasons: (1) our understanding of Middle East respiratory syndrome (MERS-CoV)  fatalities may still affected by a possible bias in our understanding of MERS-CoV transmission because of a continued focus on testing just the "tip" of the VirusBerg and (2), I made a new graphic that I like more than the old one!

We see a high PFC for MERS for 2 reasons that spring to my mind:

1. There are no, or very few, mild cases of MERS. Cases are usually severe and most are being caught. We know there are some mild cases and we know that contacts of confirmed cases are infrequently MERS-CoV positive. I'm assuming contacts are tested with the same rigour as those in hospital due severe MERS (e.g. lower airway sampling with follow-up testing). 
2. Although we don't see much transmission from cases to contacts we do see some and this may be enough to maintain a small number of community transmission events. The second reason then is that we are only testing cases with severe disease, many of whom die as a result of complications due to the initial infection. Prospective screening of a sample of the well general community and those with "common colds" might identify more cases that would reduce the PFC.

+1 to HA and NA numbering-new fruity bat flu

Now I need to remake this tree!

Thanks to a new study in PLoS Pathogens by Tong and colleagues (they who brought us the last new influenza numbers from bats), we have a new haemagglutinin (HA) and a new neuraminidase (NA) to add to the list. These are also from a fruit bat (flat faced (Artibeus planirostris) in fact.

That makes 18 HAs and 11 NAs to play mix-and-match with. I wonder if we'll one day see the avian and bat genes mix up?

Mycophenolic acid added to the study list of anti-MERS-CoV therapeutics?

In a recent online release from the Journal of Infection, Chan, colleagues from Kwok-Yung Yuen's group and collaborators describe their search for compounds with broad-spectrum activity against both influenza viruses and coronaviruses (CoVs); the latter being especially rare.

Testing a pre-existing 1,280 compound panel of potential drugs yielded 10 hits with activity against MERS-CoV  (EMC strain). Only mycophenolic acid (an inhibitor of T and B lymphocyte and antibody production) fit the bill for a likely compound from this panel. It is also active against other viruses and is used clinically to prevent solid organ transplant rejection. 

Mycophenolic acid is available, can be given orally or parenterally, dosage is convenient, serum concentrations exceed those needed to be active against MERS-CoV and most tertiary care facilities can monitor drug levels. The risk of immunosuppression is of concern however.

Other agents showed some anti-MERS-CoV/EMC activity in the Vero cell culture assays. Ribavirin, interferon (IFN)-α, and IFN-β were active

Combining mycophenolic acid with  (IFN)-β produced an additive effect.

Further studies sound likely.

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-1β, IL-6, IL-8, TNF-α, IFN-β & 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-α, IFN-β & IP-10  were induced more by SARS-CoV infection
• MCP-1 (a chemokine) and TGF-β (anti-inflammatory cytokine) remained unimpressed by infection
• At 48-hours, MERS-CoV infection induced less IL-8 or IFN-β 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-β
• IL-1β, IL-6, IL-8, TNF-α, IFN-β & 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-α, which triggered more by MERS-CoV
• MCP-1 and TGF-β 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-β 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-β translation at 12-hours, but Kindler did not measure protein). 

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)-β resulted from infection by any CoV
• Only marginal expression of proinflammatory cytokines (TNF-α 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-α (a type I IFN) or IFN-λ3 (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-α or IFN-λ3 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.

The MERS-CoV receptor story to date..

The Middle East respiratory syndrome (MERS) coronavirus (CoV) uses the dipeptidyl peptidase 4 (DPP4; also called CD26) molecule as its cellular contact for entry into the host.

The receptor was described in Nature by Raj and colleagues (led by Bart Haagman) in March 2013, part of a Dutch, German, Swiss and Saudi Arabian team. Some of this group, led by Christian Drosten, had previously found that MERS-CoV strain EMC did not use the angiotensin converting enzyme 2 (ACE2) as a receptor, which the SARS-CoV does. 

Haagman's group went on to use a modified form of the S1 domain of the Spike (S) protein to probe cell types in search of those that bound the protein, and allowed MERS-CoV replication. This included African green monkey (Vero) and human liver (Huh-7) cell lines and to a lesser extent, kidney cells from the Pipistrellus pipistrellus bat. Binding or infection failed in COS-7 African green monkey kidney cells. 

Drosten's earlier study identified that the baby hamster kidney (BHK) or African green monkey kidney (MA104) cell line could not support MERS-CoV/EMC while a rhesus monkey kidney (LLC-MK2 cell line) and bat cells or cell lines from Rousettus aegyptiacus kidney (RoNi/7),  Rhinolophus landeri lung (RhiLu), Pipistrellus pipistrellus kidney (PipNi/1 and PipNi/3), Carollia perspicillata kidney (CarNi/1), Myotis daubentonii kidney (MyDauNi/2), porcine (PS) and human kidney cancer (769-P) cell line could. The diversity of this cellular tropism is unique among the known CoVs.

Haagman's group next purified proteins from permissive cells by capturing them using the modified S1 domain, resulting in the discovery of a 100 kilodalton (kDa) protein which was characterised using mass spectrometry. DPP4 shows high amino acid and nucleotide sequence similarity across multiple species. 

DPP4 was sought and found on Vero, Huh-7 and human non-ciliated bronchial epithelial cells (?rather than ciliated cells found more widely throughout the upper and lower respiratory tract) or COS-7 cells. DPP4 is also found on human epithelial cells in the kidney, small intestine, liver and prostate as well as stimulated lymphocytes. That creates a number of spots of linkage between MERS-CoV replication and signs and symptoms of MERS.

When COS-7 cells were made to express DPP4, MERS-CoV/EMC could now bind. Blocking access to DPP4 using specific antisera resulted in blockage of cellular infection by MERS-CoV/EMC. Blockage did not result when the enzymatic function of DPP4 was inhibited using specific drugs.

But what is DPP? It's a few things. It's a protein that spans the cellular membrane, existing as a dimer (2 of itself somehow associated); it cuts dipeptides from hormones and chemokines to make them bioactive (do stuff); it has a role in glucose metabolism; it is abundant; it is involved in T-cell activation, movement of cells and organism around the body, cell adhesion, cell death (apoptosis), regulation of tumour growth. Reducing the levels of DPP4 expression or inhibiting virus binding may be useful therapeutic research areas for the future.

If the virus is limited in its tropism to cells of the deeper airways, it's transmission may also be limited because it is a little more difficult to cough and sneeze material from deep in the lungs compared to a virus happy to replicate itself in the nasal cavity and surrounds.

So to the latest advance.  Cui and colleagues, in the Virology Journal this month, cleverly looked at the receptor molecule in bats (from which CoVs often spring) as a way of saying something about the relationship between host and virus; namely MERS-CoV. Because the viruses and their receptors are predicted co-evolve, signatures can be identified in the genes for the receptor and for the bit of virus it interacts with.

In particular they found that 6 amino acids were very conserved among a batch of bat DPP4 sequences. 3 of these are predicted to occur where the MERS-CoV receptor binding domain (RBD) interacts with DPP4 protein, suggesting "co-evolutionary history" between bats and MERS-CoV. 

The authors concurred with others in concluding that MERS-CoV has an origin in bats because it has evolved to maintain its interaction with the bat DPP4.

Also check out Mike Coston's comments and Crawford Kilian's thoughts on this recent paper.

Editor's Note #11: Blog biology...

Just like a living thing, if untended, a blog can fade away.

At least mine did while I was away on holiday. There's always a cycle - the weekends are quite - but while I was away the (mostly) unique visitors stopped visiting. 

As you can see, VDU's blog doesn't get thousands of hits per day anyway - a niche site at best. 238 visits was a peak day during the past month.

VDU, warts and all!

How mice lie....

In a nice 9:40 video presentation to TEDMED 2013, H. Shaw Warren brings us up to speed on some of the shortcomings of using mice to understand our complex human immune responses to bacterial infection.

Some key points he makes are:

1. We use inbred mice. They "all look the same" because they are. That does not capture the diversity of responses to infection that we must deal with in trying to understand human infections
2. Mice are resistant to infection and inflammation requiring lots of material o be injected. This is not what happens in the wild (100-100,000x more resistant than humans)
3. Trauma and burns can be studied by examining gene responses in humans. These studies found poor correlation with mouse models.
4. Mice look very different from us, why wouldn't their immune responses be equally different?
5. Mice have evolved in environments rich in microbial exposures
6. Mice have large litter sizes and short gestational periods which would increase adaptive evolutionary cycles - perhaps they have adapted to tolerate larger inocula than humans
7. Mouse model success often determines whether a candidate drug proceeds to human trials. Some of these may work in humans
8. All of the drugs studied in mice that have worked, have failed in humans (I did not know that)
9. Mice still useful for gene, gene pathways, techniques and toxicity studies but extrapolating to complex human inflammatory disease networks may be a stretch
10. The scientific community should raise the bar in justifying a link between human and mice responses before proceeding

Very nice talk.

Thanks to @MsWZ for tweeting link