Wednesday, 16 August 2017

This little MERS-CoV infected piggy had RNA, but that little piggy with indirect contact had none...


Back in April, a Spanish/Dutch collaborative study came out looking at whether pigs deliberately infected with Middle East respiratory syndrome coronavirus (MERS-CoV) might be able to transmit that virus to other pigs.[1] Turns out they can, but so weakly that the authors concluded pigs are unlikely to be a source of virus in the wild.

Alpacas have been use for transmission studies in the past - these furry four legs do seem to be able to host and transmit MERS-CoV once infected, and in the wild, and may have a role in keeping the virus alive among animals. They may also be a source for spillover into humans on occasion; check out MERS-CoV: alpacapalooza for a reminder.[2]

But what did this new study do?

First up they infected a  group of 5 pigs (P1, P2, P3, P4, P5) with a fair whack (107 TCID50 (50% tissue culture infectious doses) of the HCoV-EMC/2012 MERS-CoV virus variant by shooting 1.5ml of virus in saline up each nostril of the pig's noses. None of the pigs showed any measurable signs of illness - no temperature rise or respiratory signs. That result alone shows that otherwise healthy walking bacon is not a model of  MERS (the disease).

After 2 days, 5 uninfected pigs were added to the same cage to permit direct contact (touching snouts and other stuff that pigs do to each other when in the same space); P6-P10. This group were called 'direct contacts'. They represents your Mum & Dad - the ones who keep kissing you while you're in hospital with MERS (not that kids often get sick with MERS-CoV!).

A third group of piggies were housed in a separate pen, 30cm away, and called 'indirect contacts'. They represent that cousin who came to visit you in the hospital, but just sat in the corner playing PokemonGo on their tablet because they didn't really like you anyway but got dragged along by your Uncle.

A lot of different samples were taken from the pigs from before inoculation and at various intervals up until 26 days afterwards. The results from testing these samples break down as follows:

  1. Cell culture studies found that only the MERS-CoV inoculated pigs shed infectious virus and developed an antibody response to MERS-CoV spike protein (measured by S1 ELISA) or that were capable of neutralizing MERS-CoV in cell culture experiments.
    Neither of the 2 contact groups shed detectable infectious virus or mounted an immune response. 
  2. Sensitive molecular methods (RT-PCR [3]) found that the inoculated pigs all shed MERS-CoV RNA from day 1 after inoculation; 3 stopped shedding after day 7. 3 of 5 direct contact pigs shed MERS-CoV RNA between day 3 and day 5. These pigs entered the infected pig's pen at day 2-were they being infected by residual inoculum?
    A sample of virus left on the bench for a week and a portion tested each day in cell culture and by RT-PCR would have been useful to assure us that infectious virus from the original inoculum wasn't the only thing being detected in this study
  3. Some viral RNA was detected in air samples collected between the two pens and from some wall/surface swabs - but infrequent and in very small amounts
So, the study concluded that infected pigs may transmit MERS-CoV but that if they do, it's a pretty inefficient process. 

It's not clear to me whether the initial P1-P5 were successfully infected by MERS-CoV at all. The piggies had virus given to them, sure. But did that virus enter cells and replicate within those cells to assemble new viruses? If it did, were those new viruses infectious? Did new viruses release from the infected cells and infect new cells and the other pigs, or was this all detection of eh original inoculating virus? 

The immune responses detected only in the inoculated pigs may have been the response to inoculum, without MERS-CoV replication. The very small amounts of viral RNA detected in indirect contacts and in the air and on some surfaces could also be leftover inoculum spread from the noses of P1-P5. Without knowing how long MERS-CoV in saline remain infectious or detectable under their conditions, we cannot answer that. We do know that MERS-CoV can remain infectious for 2 days and be detected by RT-PCR methods for at least 5 days.[4,5] 

So all in all, the main contribution of this study to the furthering of MERS knowledge is its ability to rule out pigs as a useful animal model for disease or transmission.

References...
  1. Middle East respiratory syndrome coronavirus experimental transmission using a pig model
    https://www.ncbi.nlm.nih.gov/pubmed/28653496
  2. MERS-CoV: alpacapalooza
    http://virologydownunder.blogspot.com.au/2016/09/mers-cov-alpacapalooza.html
  3. Reverse transcription polymerase chain reaction (RT-PCR)...a primer
    http://virologydownunder.blogspot.com.au/2015/05/reverse-transcription-polymerase-chain.html
  4. Middle East respiratory syndrome coronavirus: how tough is it?
    http://virologydownunder.blogspot.com.au/2013/10/middle-east-respiratory-syndrome.html
  5. Korea contamination: Middle East respiratory syndrome coronavirus in the room..
    http://virologydownunder.blogspot.com.au/2016/09/korea-contamination-middle-east.html