MERS-CoV: alpacapalooza...

In the search for the animals that may be another reservoir for, or just support infection by, Middle East respiratory syndrome coronavirus (MERS-CoV), a few studies have looked at furry little alpacas (Vicugna pacos). 

We already know from Eckerle and colleagues' work that cells derived from alpacas have the required receptor molecule used by MERS-COV (DPP4) and that they can support replication of MERS-CoV in the lab,[1] But are cells in flasks different from furry animals in the wild?

Three articles came out in the June 2016 issue of Emerging Infectious Diseases looking at alpacas and MERS-CoV. There's no way to tell who submitted when in this journal (ongoing grr).

A Colorado State University team sought an easier - but still relevant - animal model than camels to work with.[2] They infected 3 (called A1, A2 and A3) alpacas with 10⁷ plaque forming units (PFU; a cell-specific measure of the amount of infectious virus in a diluted sample) of the HCoV-EMC/2012 variant of MERS-CoV via 3ml of diluted virus per nostril. 3 days later, they housed 3 more uninfected alpacas (A4-A6) with the infected ones.  

After 70 days, A1-A6 were infected again ("challenged"), in the same way. Three other alpacas were infected the same way but euthanized 5 days after infection and their tissues collected for analysis. Nasal swabs were collected before infection and then daily from all living animals for 5 days post-infection and on day 10. A4-A6 were also swabbed 3 times per week to day 19. This could all have been a bit more clearly demonstrated using a timeline by the way.

None of the infected animals had a fever or observable nasal discharge and their appetites and activity remained constant; they didn't seem to be affected by infection with MERS-CoV. Infectious MERS-CoV was shed by A1-A3 to day 5 and transmission occurred to the A4-A6 arrivals:

• A4 was found to shed virus for 1 day, 
• A5 shed no virus and 
• A6 shed across 8 days. 

Lots of variation even in a controlled environment like this.

When furries A1-A6 were challenged with a fresh infection, A1-A3 developed antibodies which protected them from infection (because no virus was shed). A4-A6 shed a little infectious virus for at most 2 days. A4-A6 also developed neutralizing antibodies but took longer than A1-A3 to do so. 

A wild transmission occurred from inoculated A1-A3 to naive A4-A6. This was a lower dose than the original inoculum, and seemed to elicit a milder antibody response in animals A4-A6 as well. 

Virus was found in the nose, larynx and trachea of A7-A9 but not the lungs.

The Australian/Singapore team sought an animal model of MERS with a better temperament and more manageable size than the camel.[3] 

Under biosafety level 3 conditions, they used a camel MERS-CoV variant (Al-Hasa_KFU-HKU13/2013) and exposed each alpaca to 10⁶ 50% tissue culture infectious doses (TCID50; another cell-specific measure of infectious virus quantity), monitored for 21 days then challenged as described above.

Blood as well as nasal, oral, rectal and urogenital swabs were collected over time and tested by sensitive RT-PCR, culture and for the presence of neutralizing antibodies.

The furries once again did not develop a fever (animal No.2 had a raised temperature though) or a respiratory illness.
Not a great model of disease, perhaps good for transmission?

Infectious virus was isolated from oral upper and deep nasal swabs but not from urogenital or rectal swabs. RNA detection by RT-PCR followed this same pattern. After challenge and in the presence of antibodies which had appeared from day 10-12, viral RNA could not be detected anywhere in any animal.

Neutralizing antibody did not appear until 21 days in animal No. 1, 10 days in No. 2 and still hadn't appeared at day 35 in No. 3. But, it was apparently unnecessary for protection from reinfection in this study of alpacas.

These 2 studies used 10⁶-10⁷ cell-specific quantities of MERS-CoV to infect the alpacas, but, when sought, less was produced by the newly infected animals - except in one of the Australian/Singapore animals where the peak of 10⁶ TCID50 detected equalled the input dose. This finding suggests the laboratory inoculum may not be relatable to real-world amounts of virus produced by an infected animal source. It may however, just be how much is needed to get a model system infected.

The authors all agreed that alpacas could be a good model for MERS-CoV in camels and that animal infections supported the finding of the initial alpaca cell culture work. But that culture link is not quite so straightforward.

Deliberately infected goats, sheep and horses (using the HCoV-EMC/2012 variant of MERS-CoV) - also animals whose cells had supported MERS-CoV in the laboratory - showed little or no sign of viral replication and the animals mostly remained healthy (some nasal discharge was seen from 2 of 4 horses).[5]

Despite some signs of neutralizing antibody developing in goats and a sheep, the same Colorado team were not convinced that any of these animals would be likely hosts for MERS-CoV in the wild.

Cells in a flask are not always the most realistic model for animal transmission I guess.

The final alpaca article was from a Qatar/Netherlands team who tested alpacas in a region of Qatar where MERS-CoV is found to be enzootic among camels (spreading naturally among the animals).[4] 

Hobby alpaca and camel herds were the subjects of this study. They had been kept about 200m apart in the same farm and cared for by the same animal workers.

Blood samples were tested from 15 alpacas and 10 dromedary camels; nasal swabs were also collected from the 10 camels. Nasal, rectal and oral samples were only collected from a subset of the alpacas for antibody testing and sensitive RT-PCR.

MERS-CoV neutralizing antibody was present in 15 of 15 alpacas and 9 of the 10 camels according to a 90% plaque-reduction neutralization test. This indicated past natural infections had occurred.

Antibodies were also detected that suggested past infection by dromedary betacoronaviruses and camelid alphacoronaviruses but this was not unusual nor unexpected.

No swabs were positive by RT-PCR indicating that no animals were infected at the time of sampling. The authors did not know when, how or how often MERS-CoV may have naturally infected the alpacas.

So we can add alpacas to camels on the short list of animals that can host MERS-CoV infection.

References...

1. Replicative Capacity of MERS Coronavirus in Livestock Cell Lines
   https://wwwnc.cdc.gov/eid/article/20/2/pdfs/13-1182.pdf
2. Infection, Replication, and Transmission of Middle East Respiratory Syndrome Coronavirus in Alpacas
   http://wwwnc.cdc.gov/eid/article/22/6/pdfs/16-0192.pdf
3. Experimental Infection and Response to Rechallenge of Alpacas with Middle East Respiratory Syndrome Coronavirus
   http://wwwnc.cdc.gov/eid/article/22/6/pdfs/16-0007.pdf
4. MERS-CoV Infection of Alpaca in a Region Where MERS-CoV is Endemic
   http://wwwnc.cdc.gov/eid/article/22/6/pdfs/15-2113.pdf
5. Inoculation of Goats, Sheep, and Horses with MERS-CoV Does Not Result in Productive Viral Shedding
   http://www.mdpi.com/1999-4915/8/8/230

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.