Middle East respiratory syndrome coronavirus (MERS-CoV): camels, camels, camels!

Two studies in Eurosurveillance, an editorial note, A Lancet Infectious diseases report and a comment point 2 hairy toes toward camels as a harbour and source in some capacity, for MERS-CoV, or MERS-CoV-very-like, infections ticking over around the Arabian peninsula. All in the space of a week!

First up, Hemida and colleagues from Saudi Universities, China and the United States describe the search for neutralizing antibodies in animals in a 12-Dec Eurosurveillance article. Great to see Saudi Uni researchers involved. I've mentioned this virus neutralization assay and its intent before. This new study builds on that from Perera and colleagues who looked at camels and some other animals.

Some major findings from this study include:

• Dromedary camels (n=310), sheep (n=100), goats (n=45), cattle (n=50) and chickens (n=240) from MERS-CoV hotspots in Saudi Arabia (Riyadh and Al Ahsa) were tested with the pseudoparticle neutralization (ppNT) test
• 280 camel sera (90% of camel sera) were positive using the MERS-CoV ppNT test. No other animal sera reacted in this test
• 96% of camels had MERS-CoV (or a close relative)-reactive antibodies by 1-year of age; two-thirds of camels that were younger than 1-year of age reacted, which suggests acquisition of these infections accrues rapidly during that 1st year, or maternal antibodies remain in the offspring
• 54 randomly selected camel sera (18% of all camel sera) were diluted out and tested using ppNT and a standard MERS-CoV microneutralization test (MNT). High levels of calf antibody specific to BCoV did not block MERS-CoV infection nonetheless there were some similar titres to both viruses in some of the camel sera. A ≥4-fold higher amount of antibody reactivity towards 1 virus compared to the other defined which was the most likely virus reacting. It's possible (likely?) that camels have "seen" (been infected by) both viruses or similar viruses at some time. Some of the subset of camel sera had high levels of antibody only to MERS-CoV (or a close relative)
• Cows did not have any sign of MERS-CoV-reacting antibodies in their sera; they did have BCoV reactivity though
• It was not stated whether the camels were ill or healthy at sampling

So other animals were not neutralising-antibody positive but young Saudi camels, like Omani, Spanish [retired to the Canary islands] and Egyptian camels before them, had acquired and reacted to infection by MERS-CoV (or a close relative) according to these validated antibody-detection tests. Regular sampling of an animal cohort is one suggested future direction.

Secondly we have Reusken and colleagues from the Netherlands, Jordan and Germany look at animals from the first known site to harbour MERS-CoV infections in April 2012 at a hospital in Zarqa city in Jordan. This was published online 12-Dec in the same issue of Eurosurveillance. Just fyi, Prof Marion Koopmans is senior author on this study and on the study below.

Some of the key points include...

• Sera from 3-14-month old dromedary camels (n=11), goats (n=150), sheep (n=126) and cows (n=91) were tested by an antibody microarray method, used previously by this group, and the results confirmed by identifying antibodies with the ability to neutralize MERS-CoV infection.
• The lower levels of antibody than seen in an earlier study may reflect leftover maternal antibody protection, although the authors note than maternal camel antibodies wane within 2-months of birth and that adult camels had higher levels of antibodies
• This study cited a reference noting that apart from cows, camels, goats and sheep are major sources of meat and milk in the region, ~1 sheep/pilgrim or ~1 camel/7 pilgrims is slaughtered in Saudi Arabia for the Hajj which equates to ~3,000,000 animals!
• 11/11 camel and 6/126 sheep sera had antibodies that reacted with MERS-CoV but, in additional testing, the sheep sera were not able to neutralize infection by MERS-CoV
• 23/91 cows and 128/150 goat sera reacted with the human CoV, OC43 (antigenically related to BCoV); no sera reacted with SARS-CoV
• A broadly reactive CoV, or "pancoronavirus", PCR method was used to screen camel faeces; 3 BCoV sequences were obtained, but no sign of MERS-CoV RNA in the faeces hinting that there was not an active infection at the time of sampling. This last point assumes that MERS-CoV is excreted from the camel gut during/after an acute infection. The next study may not support that assumption.
• It was not stated whether the camels were ill or healthy at sampling

Add young camels from Jordan to those from Saudi Arabia, Oman and retired Spaniard animals as possibly having been infected by MERS-CoV (or a...you know, similar thing) or at least having antibody acquired from their mothers. 11/11 POS may yield some more data to narrow down the age of acquisition; 3-months and seropositive could suggest MERS-CoV acquisition at or very close to birth, or simply remaining protective maternal antibody. Perhaps camel farms and farmers should be a next stop for detailed testing. 

In an Editorial note, the Eurosurveillance Editors note that these data do not define the primary source for human acquisition is still unclear.

Thirdly we have Haagmans and colleagues from the Netherlands, Qatar and the United Kingdom describing the study of the Qatari farm camels and temporally related human infection, from which MERS-CoV was detected back in late November. This article was published online by the Lancet Infectious Diseases (17-Dec).

Some key findings here include...

• The article's introduction suggests that the genetic diversity of human MERS-CoV viruses determined to date is the result of multiple zoonotic acquisitions 
• This study started with a 61-year-old Qatari male (61M; FT#144) farm owner who had not travelled outside Qatar and his 23-year-old male (23M; FT#150) employee
• 61M was RT-PCR POS (upE assay) on a sputum sample (collected Oct-13) and 23M on a throat swab (collected Oct-17) and subgenomic (ORF1b and nucleocapsid [N]) sequencing at the Public Health England confirmed the detection to be MERS-CoV
• MERS-CoV genomic sequences from the 2 human cases were placed on GenBank and called Qatar_3_2013 [61M] and Qatar_4_2013 [23M] as were camel sequences from the subsequent experiments
• Sera, rectal swabs and flocked nasal swabs were collected from all of the farm's 14 camels as well as 5 stool samples from 3 cages, by a team wearing personal protective equipment. Samples were shipped to the Netherlands for upE, N and ORF1a RT-PCR testing
• Vero and Huh-7 cells were inoculated with swabs that had been added to viral transport medium onsite. A single culture from Camel#7 was upE RT-PCR positive at day-4 after inoculation but no culture yielded infectious virus
• 5/14 camel nose swabs were MERS-CoV PCR positive using upE, N and ORF1a assays
• Sequencing of a fragment of Spike gene yielded 100% identity with other Saudi MERS-CoV sequences; sequence only differing by 1 base from the original isolate, MERS-CoV/EMC.
• All camel sera were antibody positive using an immunofluorescence test on MERS-CoV/EMC-infected cells
• There was no "direction" to the acquisition of MERS-CoV. Whether the camels infected the humans or the humans infected the camels could not be determined from this outbreak
• The authors conclude that detailed cases histories are important to identify animal exposures. These might not otherwise be though important in a cursory question and answer of a patient, their family or contacts

This study adds very important data that indicate a recent or resolving MERS-CoV infection in camels. No positivity was found from gastrointestinal samples. Despite no isolation of infectious MERS-CoV, the detection of RNA is an acceptable surrogate for the presence of "live" virus in an animal or person (even if it could not propagate in vitro). So the camel story has some very important new chapters added in this series of studies.

In a Comment in LID, Ferguson and Verkhove note how the One health concept is exemplified by not only this publication; as it has been by the entire MERS-CoV story. The comment also notes the need for much more study, passive and active surveillance of human and animal disease/movements and better and faster reporting to link these, or any other, animals back to the cases that are spread across a very broad geographic region. They hold Haagmans and colleagues' article up as an example of how to get more answers and prevent sustained MERS-CoV transmission among humans from developing in the future.

Tracing and testing camels imported into the region from Africa for use as food may also open a new front to identify the transmission potential of MERS-CoV (or a similar beastie) in camel infections. Testing of pneumonia causes at these other sites both for virus and antibodies against virus is probably also warranted. 

As usual, new data bring new questions and so many papers in only a week makes for lots of questions.