5 March 2022 Episode 246 One health aspects

Dear colleagues,

I decided to review some aspects of the importance of the human/animal interface, because of some recent papers on the origin of SARS-CoV-2, on the role of minks and other (peri-) domestic animals and on a potential novel wild animal reservoir (the North-American White-Tailed Deer). Obviously, many questions  on these “One Health” aspects of SARS-CoV-2 are still open and should be investigated to prevent new zoonotic epi-pandemics …

Par 1 ANIMAL ORIGIN?  

1.1 Short reminder Ep 237-1: Holmes Cell Sept 2021: clearly defends the “natural” zoonotic origin

The phylogenetic tree and the early epidemiological investigations clearly show a link to the Huanan market and not to the Wuhan Institute of Virology (WIV)

This phylogenetic pattern is consistent with the emergence of SARS-CoV-2 involving one or more contacts with infected animals and/or traders, including multiple spill-over events, as potentially infected or susceptible animals were moved into or between Wuhan markets via shared supply chains and sold for human consumption.

There is a significant evolutionary gap  between SARS-CoV-2 and the closest related animal viruses:

for example, the bat virus RaTG13 collected by the WIV has a genetic distance of 4% (1,150 mutations) to the Wuhan-Hu-1 reference sequence of SARS-CoV-2, reflecting decades of evolutionary divergence

RmYN02, RpYN06, and PrC31—are closer in most of the virus genome than RaTG13  (particularly ORF1ab) and thus share a more recent common ancestor with SARS-CoV-2

The animal origins of many well-known human pathogens, including Ebola virus, hepatitis C virus, poliovirus, and the coronaviruses HCoV-HKU1 and HCoV-NL63, are yet to be identified, while it took over a decade

to discover bat viruses with >95% similarity to SARS-CoV and able to use hACE-2 as a receptor

Escape unlikely:

• No data to suggest that the WIV—or any other laboratory—was working on SARS-CoV-2, or any virus close enough to be the progenitor, prior to the COVID-19 pandemic.
• There have been no reported cases related to any laboratory staff at the WIV, and all staff in the laboratory of Dr. Shi Zhengli were said to be seronegative SARS-CoV-2 when tested in March 2020.
• Bat virus RaTG13 from the WIV has reportedly never been isolated or cultured and only exists as a nucleotide sequence assembled from short sequencing reads.
• Amplification in Vero E6 cells, a process that consistently results in the loss of the SARS-CoV-2 furin cleavage site

1.2. New data

Ep 246-1: Goa in Res Square  26 Feb 2022 presents data from 1380 environmental and animal samples collected after the closure of the Huanan Seafood market on Jan 1st 2020.

• Table 1 summarizes the 73 PCR positive samples on ground, sewage system, water drain, surfaces of door (even inner door of a freezer) and even “blood on the ground”. These viral sequences showed 99.980% to 99.993% identity with the human isolate HCoV/Wuhan/IVDC-HB-01 and two were completely identical with human Wuhan-Hu-1, from the early stage of COVID-19 outbreak
• On the contrary, no positive result was obtained from 457 animal samples including animal bodies, stray animals and their feces, with some stray animals  sampled until March 30th.  Table 2 gives an overview.

From this analysis, it is very clear that the Huanan market was involved in the transmission in Dec 2019, but no animal origin could be proven.

Ep 246-2:  Worobey in Zenodo 26 Feb provides a “spatio-temporal analysis” on the market in Dec 2019 to gain more insight

• The first human cases  confirm again the earlier Cell analysis that the Huanan market was indeed the “epicenter”

Next Worobey zooms in on the animals sold at Huanan market arguing that at least 10 species may be susceptible to SARS-CoV-2, based on live susceptibility, ACE-2 binding and/or serology

Worobey then points to raccoon dogs, which are noteworthy because they were associated with the emergence of SARS-CoV-1 (26) and have been shown to be both susceptible to infection with SARS-CoV-2 and capable of transmitting the virus (12–14).

However, as one can see by comparing the list of sampled animals (Table 2 of Ep 246-1) and those that are susceptible to SARS-CoV-2 (Table 2 of 246-2), there is little overlap and raccoon dogs were not amongst the investigated animals.  Worobey writes: it is apparent that by the time the Huanan market was closed on 1 January, 2020, and animal sampling at the market began, the SARS-CoV-2-susceptible live mammals that we know had been on sale there in the preceding months (Tables 2 and S3) were no longer present. 

Worobey concludes:  There were likely (!?) multiple infected animals at the Huanan market, leading to SARS-CoV-2 positive environmental samples (Fig. 4C) and at least two introductions of SARS-CoV-2

(i.e., lineages A and B) into humans (companion paper by Pekar et al.). Human cases clustered in (Fig. 5B) and near to (Fig. 1B) the Huanan market, with SARS-CoV-2 quickly spreading to wider Wuhan (Fig. 1E) and then nationally and internationally,  as summarized in Fig 6:

Ep 246-3: Pekar Zenobo 26 Feb 2022: SARS-CoV-2 genomic diversity early in the COVID-19 pandemic points to emergence via repeated zoonotic events.  

- The first zoonotic transmission likely involved lineage B viruses and occurred in late-November/early-December 2019

- The introduction of lineage A likely occurred within weeks of the first event.

Hence, as with SARS-CoV-1 in 2002 and 2003, SARS-CoV-2 emergence likely resulted from multiple zoonotic events.

(Sorry, I admit that I cannot really follow the detailed reasoning in this paper, therefore I just copy the summary figure)

(recCA= recombinant Common Ancestor and MRCA = most recent common ancestor)

Ep 246-4: Amy Maxmen Nature Briefing 27 Feb summarizes this evidence.   There is no doubt that the Huanan Market was the scene of a massive amplifying event, but the final proof of animal origin is not provided:

• Worobey (first author of Ep 246-2) himself in the past did not exclude a “lab leak”, but now states: it’s extremely improbable that two distinct lineages of SARS-CoV-2 could have been derived from a laboratory and then coincidentally ended up at the market.
• Munster (Rocky Mountains Laboratories):  is not completely convinced of two spillover events because the virus might have evolved from one lineage into the other in a person who was immunocompromised.  He says that searching for SARS-CoV-2 and antibodies against it in blood samples collected from animals sold at the market, and from people who sold animals at the market, could provide more- definitive evidence of COVID-19’s origins.
• CLEARLY, THIS SHOULD HAVE BEEN DONE, but it wasn’t and presumably will not be done any more…..   

Par 2 REVERSE ZOONOSIS (and secondary animal-to-human transmission):

1. 1. Reviews

Ep 246-5A: Kumar Pramod Veterinary World Oct 2021

Ep 246-5B: Goraichuck Virus Res Zoonotic and Reverse Zoonotic Transmissibility of SARS-CoV-2

Due to high conservation of ACE-2, a large number of mammals are potentially susceptible to SARS-CoV-2. but only a few species of fish, birds, or reptiles.  Species carrying ACE-2 sequence with K31, Y41, N90, and K353 are likely to be susceptible to infection by SARS-CoV-2 (including Homo sapiens, Macaca mulatta, Felis catus, Rhinolophus sinicus, Meloidogyne javanica, and Pelodiscus sinensis) while others should be less susceptible or resistant to infection (Fischhoff et al., 2021).

Note that some “adaptive” mutations are “divergent” i.e. unique to a particular species and others are “convergent” i.e. shared by several species (sometimes including humans).

Ep 246-5C: Peacock bioRxiv 3 Jan 2022 Effect of omicron on potential range of animal susceptibility

Receptor usage was screened using pseudoviruses expressing the indicated Spike proteins into BHK-21 cells expressing the indicated ACE2 protein. Viral entry was measured by assaying luciferase activity (RLU) using the BrightGlo reagent

As can be seen, there are clear shifts as compared to the D614G strain (which caused the first wave of the pandemic):

• Rats, mice and horseshoe bats have become relatively more susceptible
• Hamsters, cats, dogs and cattle have become relatively less susceptible

(Minks are lacking in this comparison)

1. 2.  The case of farmed minks

Ep 246-6 A: Fenollar Front Microbiol April 2021

Ep 246-6 B: Burkholz Infect Genetics Evolution 2021 Paired SARS-CoV-2 spike protein mutations in humans and minks

1. 3. The special case of wild white-tailed deer in North America

Ep 246-7 A: Chandler PNAS Nov 2021: prevalence in Northern USA

Ep 246-7 B: Marques medRxiv 27 Feb 2022 : Evolutionary Trajectories of SARS-CoV-2 Alpha and Delta

Variants in White-Tailed Deer in Pennsylvania

Of 93 nasal swab samples from “hunter harvested” deers, analyzed by RT-qPCR, 18 (19.3%) were positive for SARS-CoV-2.

Whole genomic sequencing of 7 deer samples indicated that they were derived from either alpha or delta human SARS-CoV-2.   Remarkably, the alpha lineage persisted in deer after its displacement by delta in humans, and deer-derived alpha variants diverged significantly from those in humans, consistent with a distinctive evolutionary trajectory in deer.

Ep 246-7 C: Pickering medRxiv 25 Feb 2022:  Highly divergent white-tailed deer SARS-CoV-2 with potential deer-to-human transmission in Canada (South-western Ontario).

This paper has two topics:

• Identification of a highly divergent SARS-CoV-2 lineage in deer with 76 consensus mutations including 37 previously associated with non-human animal hosts, 23 of which were not previously reported in deer.
• An epidemiologically linked human case from the same geographic region and sampling period, pointing to a potential case of deer-to-human transmission.

Maximum-likelihood (ML) phylogeny of White-Tailed Deer (or Odocoileus) derived viral genomes (and associated human sample) and a representative sample of the global diversity of human and animal-derived SARS CoV-2 (n=3,645). 

(Unfortunately the color for “human” SARS-CoV-2 is close to the “WTD,” therefore I pointed with an arrow to the single human case that is phylogenetically intermingled with WTD sequences)

1. 4.  Role of cats?

Searching for evidence of cat-to-human transmission, I found only 1 editorial and 1 small evidence-based paper.

Ep 246-8 A: Bessiere in Viruses April 2021 finds little arguments in a small scale study of any b potential for cat-to-human transmission:

•  The SARS-CoV-2 genome was detected by RT-qPCR in two cats out of five, which suggests that human-to-cat transmission is not an infrequent event. Given the variability of clinical manifestations, COVID-19 in cats is likely under-diagnosed.
• However, viral shedding was weak and transient, which hints that, even when infected, cats probably play a limited role in COVID-19 epidemiology.

Ep 246-8 B: Totton Zoonoses Public Health 2021 discusses how difficult it will be to prove cat-to-human transmission, because in each scenario, it supposes that the cat owner has very limited human contact. 

Nevertheless, if typical adaptive mutations occur in cats, it should be possible to differentiate between a “human” derived and a ‘”cat” derived SARS-CoV-2 ???

SOME GENERAL CONCLUSIONS

1. There is now a lot “circumstantial” evidence that the Huanan market was crucial in the early days of this pandemic: the virus was everywhere in that environment, but the true link with live animals sold or their traders has not been established. Although the precise animal species has not been identified nor human “patient zero”, all evidence points to a very likely zoonotic origin.
2. Multiple mammal species have been found susceptible, usually in domestic or peri-domestic environments (including farms and zoos), but more recently also in the wild.  
3. The case of massive “reverse zoonotic” infections in farmed minks and the sizable numbers of “reverse-reverse” infections back to humans has been nicely documented, including a series of adaptive mutations.  This type of adaptation also occurs in other animals: some are divergent, other convergent.   
4. The case of white-tailed deer shows that important reservoirs could be constituted in the wild, which evolve independently from the evolution in humans, and which also could be back-transmitted to humans.
5. Although cats (and dogs) have been shown to be susceptible as well, there is no clear evidence of back-transmission
6. The evolution of the virus in humans has not prevented transmission to animals (see the fact that several  human variants of concern are found in animals), but it may influence the relative susceptibility of different animal species (see the comparison between D614G and omicron.

Hopefully, a better knowledge of animal reservoirs and the transmission cycle will mitigate the gaps between animal and human spread in the future.

Best wishes,

Guido