More Monkey(pox) Business

A strange paper from the Wuhan Institute of Virology and the possibility of a silent epidemic spreading for years.

Note: Figures have been added back.

Curiouser and curiouser, the current monkeypox situation raises more questions than it answers.

Of course, COVID has made many- rightfully- skeptical about the origins of this sudden monkeypox arrival. Monkeypox has rarely been an issue outside of areas of endemic infection, and yet as of now there are cases springing up across the globe.

Previously I wrote that what we are seeing is likely to be some sort of zoonotic spillover effect: an animal reservoir somehow came into contact with a large group of people and monkeypox spread into this group.

Monkey(pox) Business

But recent events have raised some speculations as to the actual origins of this outbreak. It doesn’t help that there was an apparent paper coming from the Wuhan Institute of Virology from February 2022 indicating that the institute may have been conducting monkeypox research.

As with all things, it requires a bit of skepticism and a dive into the literature in order to figure out what is really happening.

The Wuhan Institute of Virology Paper

The following paper has been making its rounds online, pointing fingers towards Wuhan once again:

Efficient assembly of a large fragment of monkeypox virus genome as a qPCR template using dual-selection based transformation-associated recombination (Yang, et. al.)

Of course, this paper would appear rather scary: here we go with further gain-of function-research - if that was the intent of this study.

From what I have seen online, it appears many people have not read this study or looked at it close, but instead made assumptions based on the title alone or snippets taken and posted into online news sources.

This paper looked specifically at optimizing a technique called Transformation-associated recombination (TAR). TAR utilizes a unique feature of yeast with respect DNA repair, in which yeast cells are able to take up exogenous bits of DNA and incorporate it into its own genome. This incorporation requires splitting of the yeast genome, alignment of the exogenous DNA fragments with parts of the yeast genome called hooks, and then incorporation of this new DNA fragment.

Transformation-associated recombination (TAR) cloning is based on the well-known property of free DNA ends as efficient substrates for homologous recombination in yeast (Orr-Weawer et al. 1981). Assembly of two DNA molecules by recombination in yeast was first demonstrated by Botstein and colleagues (Kunes et al. 1985; Ma et al. 1987). This team reported that a yeast vector DNA, which is broken in a sequence absent in the yeast genome, is efficiently rescued by recombination with a homologous restriction fragment included during yeast transformation (Kunes et al. 1985).

The following diagram¹ outlines a bit of this technology, although the first 4 steps are the important steps to look out for.

From Kaprina, N. & Larinov V. “Step1: The diagram shows TAR cloning of a gene of interest from total genomic DNA with a TAR vector containing YAC and BAC cassettes and two unique targeting sequences (hook1 and hook2) (in green) homologous to the 5′ and 3′ end of a gene of interest. The size of hooks may be as small as 60 bp (Noskov et al. 2001). The hook sequences may either be both unique, or one of the hook sequences may be a common repeat (for example, an Alu repeat for cloning from human genomic DNA). The TAR vector DNA is linearized by a unique endonuclease located between the hooks to expose targeting sequences. If necessary, genomic DNA may be treated by CRISPR/Cas9 endonuclease before yeast transformation, which will greatly increase the yield of gene/region-positive TAR clones. Step 2: Genomic DNA and a linearized TAR vector are co-transformed into yeast Saccharomyces cerevisiae cells. Steps 3 and 4: Recombination between targeting sequences in the vector and the targeted sequences in the genomic DNA fragment leads to rescue of the fragment (or gene) as a circular TAR/YAC/BAC molecule.”

If all of this sounds confusing, the technology being used here is slightly similar to the restriction enzyme recombination technology you may have heard about in high school biology, where enzymes extracted from bacteria are used to clip genes from an exogenous source at specific regions of DNA. The cleaved DNA can then be incorporated into another organism’s genome creating a chimeric, recombinant genome-essentially a gain-of-function.

Taken from byjus.com. This diagram outlines the insertion of an exogenous gene into a plant through restriction enzymes and recombination. This gene can provide gain-of-function capabilities, such as insecticide capabilities or additional nutrient production for more enriched fruits and vegetables.

So it could be that this TAR technology is another step in recombination technology. The only difference here is that instead of utilizing restriction enzymes and ligation enzymes (ligases), TAR utilizes yeast cells own recombination capabilities in order to incorporate exogenous DNA fragments.

However, as the researchers noted in their article, TAR technology may run into issues in which the genome may recircularize without the new DNA fragment, or it may not incorporate the fragment correctly. In both instances, the recombination would fail, and so if there was a method to properly select for the desired recombinant product then recombinant yield would be improved.

In order to eliminate unwanted products, the researchers utilized one positive selection marker and one negative selection marker. The negative selection marker is not important for this discussion, but they essentially used a yeast genome encoding a gene that would produce a cytotoxic product when placed into the right environment. This gene would only work if the genome recircularizes without any exogenous DNA fragment inserted, and therefore the yeast cell that doesn’t properly recombine would kill itself off:

Thus, the PADH1-controlled expression of URA3 converts 5-fluoroorotic acid (5-FOA) in the culture medium into the toxic product 5-fluorouracil and kills yeast bearing the recircularized TAR vector. In contrast, if the TAR vector captures DNA using its hook through HR, then the insertion between PADH1 and URA3 is larger than 130 bp in length, and URA3 expression is abolished.

In order to check if the proper recombination occurs (positive selection marker), the researchers needed a way of detecting the recombination through the presence of specific genes. It is here where the researchers utilized monkeypox DNA fragments. Monkeypox detection is the same as SARS-COV2 which utilizes PCR to amplify specific genes, and therefore if the researchers inserted Monkeypox genes they can use PCR to detect if the recombination was a success. In fact, the researchers argue that this utilization of TAR technology could be used to detect for monkeypox. Here, the researchers chose two genes to look out for:

Quantitative polymerase chain reaction (qPCR) is the gold standard for the detection of orthopoxvirus (including MPXV). For pan-orthopoxviruses detection, the E9L (DNA polymerase) gene has been shown to be an excellent target for qPCR assays (Kulesh et al., 2004). For MPXV detection, Li et al. reported that the C3L (complement-binding protein) gene could be used as the qPCR target for the MPXV Congo Basin strain (Li et al., 2010).

Now, the researchers don’t help themselves out when they explain that they did not have any monkeypox genomes available- strange for an institute dedicated specifically to virology research to not have the capabilities of receiving monkeypox samples, and so where they came up with their monkeypox DNA fragments is concerning. The researchers themselves note that they constructed the fragment themselves, which may raise questions as to what extent their synthetic construction of a monkeypox genome can be.

Either way, it seems like such a strange comment to have made, as if to excuse why they are using fragments. Why make such a comment when you can just explain that you constructed the fragment artificially sans the lack of actual genome?

But for the sake of conversation in regards to gain-of-function research and monkeypox business, this is really the end of it when it comes to this study. The researchers were able to detect the E9L and C3L genes through qPCR, which suggests that their TAR methodology may have been useful, and could thus be used for monkeypox detection.

From Yang, et. al. “Standard curves from qPCR assays. Tenfold dilutions of the assembled MPXV fragment ranging from 1 ​× ​101–109 copies per reaction were measured using qPCR. The calculated Ct represents the average of 3 different assays. A best-fit line and the coefficients of determination (R2) of E9L (A) and C3L (B) are presented.”

But aside from these results there isn’t anything alarming. The researchers did not conduct any studies that would point to a more virulent, contagious monkeypox. In fact, the research here didn’t even utilize an entire genome, so the production of virus would not be possible from this study alone.

So why the cause for alarm from this study? It appears that many media outlets took to hearing “Wuhan Institute of Virology” and may have ascribed some type of nefarious action to this paper. But what’s concerning about the sudden pile-on of this study is that many people don’t appear to have actually read the dang study for themselves.

Take this article from The Daily Wire², which misquotes many parts of the paper. The “latter” that is quoted in the article refers to smallpox, not monkeypox. Viruses also don’t have chromosomes; higher-order organisms such as humans, mammals, and plants have chromosomes, not viruses.

The paper has also been misquoted in the concerns over this type of research. Sure, they attempt to assuage concerns by suggesting they only used 1/3 of a monkeypox genome, but the main concerns the researchers raised were about research conducted in another lab in which the entire genome of a virus was reconstructed and found to be infectious (emphasis mine):

TAR assembly has become essential for preparing infectious clones of large DNA/RNA viruses. Using pGF as a TAR vector, Shang et al. assembled a synthetic baculovirus with a genome length of more than 145 ​kb (Shang et al., 2017). To establish a reverse genetics system for SARS-CoV-2, a joint team successfully assembled the full-length cDNA clone of SARS-CoV-2 in less than a week using TAR assembly (Thi Nhu Thao et al., 2020). However, this DNA assembly tool applied in virological research could also raise potential security concerns, especially when the assembled product contains a full set of genetic material that can be recovered into a contagious pathogen. Recently, a group of scientists was funded by a biotech company to synthesize a full-length horsepox virus genome and recover it into an infectious virus (Noyce et al., 2018). Not surprisingly, such a controversial achievement has received enormous attention and raised global debate on its biosecurity implications (DiEuliis et al., 2017; Koblentz, 2017, 2018; DiEuliis and Gronvall, 2018). In this study, although a full-length viral genome would be the ideal reference template for detecting MPXV by qPCR, we only sought to assemble a 55-kb viral fragment, less than one-third of the MPXV genome. This assembly product is fail-safe by virtually eliminating any risk of recovering into an infectious virus while providing multiple qPCR targets for detecting MPXV or other Orthopoxviruses (Li et al., 2010).

So from what I can tell, this research isn’t quite the smoking gun that it has been made to be, but instead may be a consequence of lack of proper analysis. As soon as someone saw this study it may have begun to make its rounds without anyone conducting a proper assessment of the paper. By the time most outlets have reported on this study, it appears they are mostly quoting each other rather than reading the paper for themselves. It’s just another consequence of the media apparatus not putting in the legwork to actually read what they pretend to report on.

But let’s also be clear here: this does not mean that the Wuhan lab is without scrutiny. We’re still not getting any transparent information in regards to SARS-COV2, and any lab conducting gain-of-function research should be investigated for possibly creating more virulent pathogens.

As it relates to this study, the main concern is not the study itself, but why such a study would be necessary. Monkeypox is not endemic to China, and from what I can tell there is no evidence of cases reported there. So why create a diagnostic tool to test for monkeypox when no monkeypox is around? Maybe there is some foresight coming from the Wuhan lab that we are unaware of- the language in the above excerpt seems like some PR pivot, but as it stands the article itself is not one to draw concerns, but the implications may be something worth paying attention to.

From Silent to Outbreak?

So aside from that Wuhan lab study, what do we make of the current monkeypox situation?

Although this outbreak can be tied to a clade of outbreaks between 2017-2019 from Israel, Nigeria, and the UK there are several dozen single nucleotide polymorphisms (SNPs) found within the genome of the recent outbreak. SNPs are nothing out of the ordinary since they tend to just indicate mutations have occurred within a virus. For example the N501Y mutation found in many SARS-COV2 variants is an example of an SNP (the change in amino acids being derived from changes in the nucleotide sequence that lead to different codons).

What’s strange with these recent events is that the number of SNPs being seen is far above what is typically normal for a dsDNA virus. Such viruses are expected to have only a handful of SNPs every year. Instead, there appears to be several dozen over the short course of a few years.

The user arambaut has made these remarks on virological.com (emphasis mine):

The long term evolutionary rate of the related variola virus (VARV; the smallpox virus) has previously been estimated to be about 9x10-6 (with 95% credible intervals of 7.8x10-6 – 10.2x10-6) substitutions per site per year (Firth et al. 2010) translating into about 1-2 nucleotide changes per year for a nearly 200,000 nucleotide genome. This makes 47 substitutions in the space of 3-4 years an unexpectedly large number. As MPXV is considered a zoonotic virus with limited human to human transmission, this long branch may be evidence of adaptation to humans allowing for the sustained transmission that is now observed.

These mutations aren’t random, as most appear to be of a specific type of mutation. Most notably, the TC→TT mutation, as well as the GA→AA mutation in the reverse complement strand of the virus.

Nucleotide changes from prior Monkeypox samples and the current outbreak. Nucleotide changes are color-coded, and the numbers of mutations are seen above.

This has lead some researchers to suspect that these mutations may be related to an antiviral enzyme called Apolipoprotein B Editing Complex (APOBEC3). This family of enzymes act as deaminases (remove the amine functional group from bases) with the ability to induce mutations in a viral genome. APOBEC3, in particular, is a deoxycytidine deaminase which turns deoxycytidine (C) into a Uracil³.

From Refsland, Eric & Harris, Reuben. The deamination process of APOBEC3. A swap from a C to a U leads to an incorrect reading of the base during replication which replaces the original G in the complementary strand with an A.

Several questions have been raised about whether the activity of this enzyme may be responsible for the high level of mutations. As of now, there isn’t substantial evidence, although the correlation appears to be relatively high.

But what’s even more interesting is that this 2022 outbreak isn’t the first time that this high level of mutation was seen. In fact, a sample taken from a Maryland patient who traveled to Nigeria in 2021 and was infected also showed a higher than normal degree of mutations.

arambaut notes this in another comment on virological.com via a phylogenetic tree. The 2021 sample is boxed while the 2022 branch is shown below.

Phylogenetic tree showing the branching off points from a 2021 monkeypox sample and the current 2022 outbreak. The colorful dots indicate individual SNPs, which suggest that even in the 2021 MD sample (boxed) a high degree of mutations already arose compared to samples from prior years.

There’s also this comment made by arambaut that was very interesting:

If these APOBEC3 edits are specifically indicative of replication in humans as opposed to another host species then this would confirm this entire clade to be representative of the emergence of a human epidemic by 2017.

This could mean that monkeypox may have mutated undetected in an endemic population for quite some time, and it is only now through outbreaks in the West that we are beginning to take notice of this rapid mutation.

But why the sudden outbreak in the West? It could be a collection of many factors leading to the outbreak.

Years of reduced smallpox vaccination may have led to generations of orthopoxvirus-naïve people. Paired with possible immune dysfunction via COVID vaccination⁴, a large-scale event such as the raves and sex parties that occurred in Spain and Belgium, and a possibly infected patient partaking in these events, then we may see why this outbreak has occurred.

There still is no definite proof of this, but for now there also doesn’t appear to be evidence of foul-play. Unlike SARS-COV2, which has a strange 12-nucleotide insertion of the furin-cleavage site, it doesn’t appear that the genomic sequencing of these monkeypox cases have turned up any concerning insertions. The high mutation rates may have occurred through serial passage of monkeypox through APOBEC3-heavy cell cultures, but that would not necessarily explain the 2021 MD sample that already showed a high degree of mutations.

What is concerning is that many of the current sequences appear to differ in a few SNPs, which suggests that microevolution and rapid mutations are occurring- again, something that shouldn’t be happening with a dsDNA virus.

So for now we can only speculate as to why a virus that was considered to have been stable appears to be obtaining more mutations. More importantly, the change in protein expression through these mutations needs to be examined.

But again, until we obtain further evidence of what is happening we are only left speculating and wanting.

1. Kouprina, N., & Larionov, V. (2016). Transformation-associated recombination (TAR) cloning for genomics studies and synthetic biology. Chromosoma, 125(4), 621–632. https://doi.org/10.1007/s00412-016-0588-3

2. These criticisms of this paper have, of course, caused other outlets to “fact-check” these claims. Take this Newsweek article:

https://www.newsweek.com/scientists-dismiss-monkeypox-lab-leak-theories-1710007

This “fact-check” itself doesn’t provide anything substantive. It’s just another example of using the term “fact-check” and appeals to authority as a way of diverting differing viewpoints.

3. Refsland, Eric & Harris, Reuben. (2013). The APOBEC3 family of retroelement restriction factors. Current topics in microbiology and immunology. 371. 1-27. 10.1007/978-3-642-37765-5_1.

4. I’m still slightly hesitant to make this argument until I do further research.