New MERS-CoV Case: UAE

A new case in an 82-year old male (82M), the 3rd in the United Arab Emirates (3.7% of cases). The previous 2 having been diagnosed externally, but seeming to have originated from the UAE.

This brings the MERS-CoV case total worldwide to 82 including 45 deaths; a (PFC of 54.9%. Of those with data, 66% of case are male as are 76% of fatalities. Of those cases with age data available, 49% =60-years (ratio of Male:Female 2.3:1).

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Another H7N9-in-ferrets paper.

This was on my "to read" pile and got moved to the "forgot to read" pile.

Kreijtz and colleagues from the Osterhaus group described inoculating ferrets intratracheally with 10⁵-10⁸ 50% tissue culture infective doses influenza A(H7N9) virus A/Anhui/1/2013. Some interesting results include...

• Ferrets showed much more pathology than in the studies below; from 2-days onwards ferrets had breathing difficulties as well as fever and weight-loss and some animals died on day-3 post inoculation. Perhaps due to the instillation of virus into the lower airways rather than intranasally?
• Infectious virus and viral RNA could be identified from the upper (nasal turbinates, pharynx, tonsils) and lower respiratory tract (trachea, bronchus, bronchial lymph nodes, lungs). It was most interesting to note that virus came back up the airways to replicate in the upper respiratory tract.
• The authors note that Anhui/1 can bind both a2,3 & a2,6 sialoside molecules (supporting Tumpey's findings below). It's a2,6 (human-like) sialoside preference explains the upper respiratory tract findings, while the human deep lung's expression of a2,3 explains the virus causing pneumonia in the ferrets.

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Where is sialic acid in different animals?

Some reviews (this and this) from a year or more ago went into this question. Tabulated for some animals below. There often seems to be at least some of the non-dominant receptor in each tissue so thing are not all or nothing.

Host	Sialoside	Tissues expressing most of it..
Human	a2,3	Eye, conjunctivae, tear duct, ciliated respiratory cells (minority) including cuboidal bronchiolar cells, type II pneumocytes
Human	a2,6	Non-ciliated respiratory cells (majority) including nasal mucosa, paranasal sinuses, pharynx, trachea, bronchi
Pig	a2,3 & a2,6	Respiratory epithelium
Ferrets	a2,3 & a2,6	Respiratory epithelium
Chickens	a2,3 & a2,6	Respiratory and intestinal epithelium
Quail	a2,3 & a2,6	Respiratory and intestinal epithelium
Duck	a2,3	Intestine, trachea

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Do "asymptomatic" animals tell us anything about a virus?

Are animals that get inoculated with a known quantity of infectious virus, but that don't seroconvert (produce antibodies) or host any viral replication, considered to be asymptomatic? Can we define that state in animals and if not, why not?

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H7N9 less efficient by droplet - but occurred in over 30% of ferrets.

Belser and colleagues from Tumpey's group describe their findings after putting two H7N9 viruses (A/Anhui/1/2013 and A/Shanghai/1/2013) into mice and ferrets. Some highlights from the Nature paper released yesterday... 

• Mice inoculated intranasally showed greater disease with human than with avian H7N9 virus 
• Ferrets inoculated intranasally (106 plaque-forming units) did not generally show signs of severe disease, although 2/8 were euthanased because of severe lethargy. 
• H7N9 was detected mainly in nasal turbinate, tracheal and lung tissues of ferrets but also in brain, olfactory bulb, intestinal tract and rectum. Spleen, kidney and liver were virus-negative. H3N2 was found in nasal tissue but not elsewhere. 
• Direct contact between ferrets was an efficient method of transmission (100% of naive ferrets placed in a cage with inoculated ferrets were infected and developed antibodies) but ferrets housed in neighbouring cages with perforated walls were less frequently infected (33% were virus positive and seroconverted). 100% of H3N2 were positive by both modes of transmission. 
• Bird and "human" H7N9 viruses grew better at 37�C (human lower respiratory tract temperature) than 33�C (human upper respiratory tract temperature) in polarized Calu-3 (derived from human bronchial epithelium) cells. They still grew at both temperatures though. 
• H7N9 produced a lot more virus in Calu-3 cells by 24-hours than seasonal H3N2 (human) influenza virus 
• The MDCK cell line required exogenous trypsin to be added for formation of H3N2 and H7N9 plaques 
• H3N2 grew well at either temperature 
• While ocular inoculation of mice with H7N9 or H3N2 did not produce consistent virus replication at the site, virus did grow in the nose at days 3 and 6. Suggest the tear ducts could be a path for self-inoculation as is seen with rhinoviruses. 
• Limited droplet transmission supports the lack of sustained human-to-human transmission seen in China. 
• Shanghai/1 preferred the more avian a2,3-sialosides (sialic acids that end a carbohydrate group which sticks out of a cell's glycoprotein or glcyolipid) whereas Anhui/1 did not favour either, having a mixed a2,3/a2,6-sialoside preference 

In another paper received a little later but released yesterday by Nature, Watanabe and colleagues from Kawaoka's group find the same increase pathogenicity in mice, same proportion of droplet transmissions, using the same human H7N9 strains. Some differences though... 

• The authors also included macaques as a non-human primate and pigs, quails and chickens
• This paper found A/Anhui/1/2013 replicated in the lower respiratory tract (1 or more of tracheal, bronchial or lung tissue) of intranasally infected chickens and quail and also in the upper respiratory tracts of miniature pigs, ferrets and macaques 
• Macaques had a lot of severe lung inflammation starting as early as 3-days after inoculation. 
• In MDCK cell culture (with trypsin?), the human H7N9s grew as efficiently at 33�C as H1N1pdm09 (A/California/4/2009). What caused that difference or did Watanabe not compare both temperatures? IN bronchial epithelial cells however, they took 24-hour longer at 33�C to get to the same viral titres as at 37�C, The H1N1pdm09 reached higher titres than H7N9 at the cooler temperature but was as efficient at the warmer temps. 
• Antibody studies revealed no H7N9 immunity among 500 people in Japan 
• Evaluation of anti-influenza antivirals in mice; Favipiravir out-performed Oseltamivir, Zanamivir and Laninamivir by limiting weight-loss (a sign of illness in mice - they go off their food) and limiting viral loads. 
• Anhui/1, Shanghai/1 and Hangzhou/1 H7N9 HA proteins all preferred a2,6-linked sialosides (human-like); Anhui/1 and Hangzhou/1 most strongly with a special liking for the sialosides covering bronchial epithelial cells. The Shanghai/1 strain bound equally well to a2,3- or a2,3-sialosides. This seems quite different to Belser et al. Technical issue - perhaps to do with the preparation of samples? 

These results are not that different from those previously described by Zhu and colleagues in May, covered earlier on VDU.

If 33% of naive humans challenged with H7N9 might became infected, we might have an explanation for the sporadic nature and low number (considering the potential for exposure) of H7N9 cases in China.

Its also worth noting that even if 33% of humans were infected, and the current PFC remained true (32%) that predicts around 10% of cases dying. Of course its not that simple, and if models and gut feelings can be trusted, there are likely to be many more cases of mild or asymptomatic disease out there that affect the PFC.

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New H7N9 numbers and Case Fatality Rate/Ratio/Risk [UPDATEDx2]

The latest Influenza A(H7N9) virus numbers are out and we can see the deaths have ticked up. We have no public information on these or many of the past releases either. With 132 cases (presumably this still does not include the Taiwan case or the asymptomatic Beijing boy, so I maintain 134) with 43 deaths.

My data (which suffer from the public reporting process; I do not mean to imply that WHO or other experts do not have these details) suggest we are missing:   

• Case details (which cases) on 13 deaths (30% of fatalities)
• 5 dates of onset 
• Date of fatality in 1 known case 
• 56 dates of hospitalisation 
• 38 dates of discharge 

I am going to use a new term on VDU, to avoid "Case Fatality Ratio". Mine is being called the Proportion of Fatal Cases (PFC)[1]...just by me mind you!

The PFC is a percentage calculated as the currently known number of fatalities divided by the number of total lab-confirmed cases including fatalities, regardless of whether surviving cases are inpatients (hospitalized) or outpatients.

At writing, the current H7N9 PFC was 32.1%

The PFC is just a number - it's not meant to imply that it includes every case that ever happened (it never could) and does not account for those cases who will die directly or indirectly as a result of their infection later on, but who may be alive at the time of calculation. 

The PFC is a snapshot to be used before an outbreak is done and dusted. It is meant as a guide to what is happening right now using the data we can get our hands on. Sometimes that's lots of data (as it was with H7N9 - but has not been for a while now) or very limited data (as it is with MERS-CoV cases).

The Case Fatality Ratio/Rate/Risk (CFR) makes use of the number of recovered cases in its denominator.[2] So it's important to know survivor numbers. As suggested above, this requires that all the people who will recover from their infection, have recovered (and been discharged) from their infection. 

Using the CFR early in an emerging virus/disease outbreak, when what usually brings in outbreak to our attention is death, is great for selling papers, but not helpful realistic in a bigger picture sense. 

The CFR is most useful at the end of an epidemic/pandemic, but not so much when data-in-hand is poor during the early days of many outbreak. 

Of course, some will take a PFC and multiply it by the world's population as an estimate of how many are going to die if the virus reaches pandemic levels. That's not helpful or accurate. Just accept it as that snapshot of what's happening now.

References...

1. J. P. Dudley and I. M. Mackay. Age-Specific and Sex-Specific Morbidity and Mortality from Avian Influenza A(H7N9). J. Clin. Virol. 2013. Sept.
2. http://en.wikipedia.org/wiki/Case_fatality_rate

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