More information on the neurotropic coronavirus GX_P2V

new form of SARS-CoV-2 coronavirus has been reported. The researchers ^[1] at Beijing University of Chemical Technology say this virus, named GX_P2V­(short_3UTR), was isolated from pangolins and spontaneously mutated in passage culture. They made eight clones, named C1 to C8. They randomly selected C7 for testing and found that, unlike the parent virus, the C7 clone moved from lung to brain within six days. It caused 100% death in transgenic animals with human ACE2 receptor, known as humanized mice.

Many virologists have commented that taking random viruses and cultivating them in human cells or humanized mice, which gives them a chance to adapt to human biology, is a very bad idea.

Sequence differences

C7 differs from the parent GX_P2V mainly by the deletion of a 104 nucleotide segment in the 3′-untranslated region and by a number of point mutations. The mutations reported in the preprint are shown in the table below.

Partial view of peptide sequence comparison of spike proteins from SARS-CoV-2 and GX_P2V. The line shows a high degree of homology but also a vertical shift at the point on SARS-CoV-2 where the furin cleavage site is located. This shows that the furin site was not a combination of site mutations, but a four-amino-acid insert in SARS-CoV-2

Most of the point mutations have the effect of making the protein product more non-polar, possibly increasing its ability to enter the cell passively.

Recall that spike cleavage of SARS-CoV-2 between residues 685 and 686 is necessary for infection. Cleavage of Spike produces two fragments, S1 and S2. S1 is a receptor-binding domain and S2 contains a fusion peptide, heptad repeat domains, and transmembrane domain. Spike protein attachment to cell surface receptors is followed by internalization into the target cell by the clathrin-mediated endosome pathway.

Two of the mutations in C7 are in the S2 region of the spike protein. There are two reasons this is of particular interest. The first is that cleavage of SARS-CoV-2 at the S1-S2 site was strongly enhanced by the insertion of a furin cleavage site that was likely engineered into SARS-CoV-2. Because furin is a ubiquitous enzyme, the furin cleavage site greatly increases the tropism of the virus, which means it can infect many more types of cells, including cells on the blood-brain barrier.

The mutations in S2 are not within the heptad repeats, transmembrane domains, or the fusion peptide domains. The mutation at 807 occurs immediately after the fusion peptide (amino acids 784–802 in GX_P2V), which mediates fusion with the target cell.^[4]

Secondly, in 2021 other researchers^[2,3] engineered a furin cleavage site into the S2 domain of IBV-YN, an avian coronavirus that does not infect humans. In the engineered form of the virus, called rYN-S2/RRKR, the PRGR↓S sequence in IBV-YN was replaced with an RRKR↓S to create a second furin-S2′ site. This produced severe neuropath­ology in animals, suggesting that mutations in the S2 spike domain somehow give the virus access to the brain.

The amino acid sequence comparisons below, calculated using the Smith-Waterman alignment algorithm, show that the furin cleavage site found in SARS-CoV-2 is not present in GX_P2V, but is present in IBV/YN despite its lower similarity to SARS-CoV and GX_P2V.

Furin cleavage sites are shown in lower case.
GX_P2V     DIPVGAGICASYHSMSSF----RSVNQRSIIAYTMSLGA
           ||| ||||||||          |||   |||||||||||
SARS-Cov-2 DIPIGAGICASYQTQTNSprrarsVASQSIIAYTMSLGA

GX_P2V     ICASYHSMSSFRSVNQRSIIAYTMSLG
           |      ....||.::.....:.:|.|
IBV/YN     IKLTKETrrfrrsTSENVTSCHYVSYG

The engineered furin cleavage site in IBV/YN, which produced severe neuropathology, is only two amino acids away from the K807N mutation in GX_P2V, suggesting that mutating S2 in this area may be important for brain infectivity. However, the 104 bp deletion could also be important.

SARS-COV QVKQI-YKTPPIKDF--GGFNFSQILPDPSKPSKRSFIEDLLFNKVTL
mutant GX                              N
GX_P2V   QVKQI-YKTPPIKDF--GGFNFLQILPDPSKPSKRSFIEDLLFNKVTL
         ..|.. |..|...:.  |.||...:|...|.|..|||||||||..|.. 
IBV/YN   STKPAGYNAPVFSNISTGDFNISLrrkrsSSPRGRSFIEDLLFTSVET

Tropism

From Fig 1D in the paper, it is clear that brain was most strongly affected and that infection spread from lung to brain by six days. The brain had by far the highest levels of virus, about 151 times higher than the lung (see table below). Other tissues, such as liver, had much lower levels of virus.

It is not clear what the researchers mean by their claim that the eyes of the mice “turned white.” C57BL/6J mice are not albino mice and normally have black eyes. They didn't perfuse the animals to remove adventitious virus, so there's probably a lot of it stuck to the endothelial cells on the blood-brain barrier.

If I were to speculate, I would guess that the mutation changed the affinity for ACE2 in such a way that it attaches to the BBB.

However, the only evidence that there was neuropathology at all is their unsupported qualitative claim that “shrunken neurons” were found. They found only minor changes in a panel of 31 cytokines and chemokines and no severe inflammatory reaction. For many cytokines, the mock-infected animals had much higher levels at 6 days than the infected ones. So how did the animals die? It's not clear. The authors suggest that their animal model was abnormal, with low triglyceride and cholesterol. Based on the quality of the data, they'll probably have trouble getting this published in a good journal.

The impression this paper gives is not one of careful, competent science, but of a kid playing with dynamite.

If these results are accurate, we could be one step away from a virus as infectious as Covid, but which attacks the brain. Such a virus would cause permanent cognitive dysfunction in anyone infected. It's meaningless to say this virus is so lethal it won't cause a pandemic: there are many uncertainties in their methods, and no way of knowing how humans might be affected.

It also demonstrates what could happen if a virus like this, passaged in human cells, was improperly handled. If it made its way into the wild and was subsequently “discovered” in another species, it would cause an immediate panic.

jan 17 2024, 4:55 pm. updated jan 18 2024, 6:37 am