Under-the-Radar Human Evolution

Designer babies have been discussed as a possibility for decades.  With the recent advancement to genome editing technology referred to as “CRISPR”, the discussions about the ethics of have been amplified.  This is because CRISPR has reduced hurdles to human genome editing in terms of cost and technical ease.  Three days ago, the following video was posted to YouTube:

Who needs peer-reviewed publications when you have YouTube?

This unorthodox reveal of the existence of genetically modified humans was shocking to the world at large, yet predictions of rogue scientists “skipping over ethical concerns and going for it” have been floating around for years.  More than one TED Talk has addressed the ethical concerns of designer babies.  Here is one for our last Amazon Review assignment of the year.


The ethical dilemma of designer babies | Paul Knoepfler

For more thoughts on this subject, try out any of the following movies:


How CRISPR lets us edit our DNA | Jennifer Doudna

Genetic Engineering Will Change Everything Forever – CRISPR

Great Ape Father Figures

For Evolution

Show notes from this video on YouTube:

“At the Senkwekwe Center for mountain gorilla orphans in Congo, a handful of Virunga National Park rangers live around the clock with four juveniles whose parents were killed. The rangers see their families only every few weeks and are very close to their charges. Chief caretaker André Bauma along with his team have hand-raised the gorillas since they were first brought to the center. The first gorilla, Ndakasi, was found when she was just two months old, near the body of her murdered mother. Bauma cared for her like a human child—letting her sleep on his chest for warmth and bottle-feeding her to help build her strength. After that, three more orphaned gorillas joined “the family” at the center. Since no mountain gorilla orphan has ever been successfully returned to the wild, they will always depend on humans.”

Are these gorilla caretakers interacting appropriately with the gorillas?  Are they teaching the orphans how to be parents similar to wild gorillas?  Keep these questions in mind as you read about this new study that documented gorilla father behavior over a few decades.

Biology from an Engineering Perspective

For Evolution

Chapter 8 of The Tangled Bank discusses evolutionary adaptations that came about due to different types of mutation.  First, if genes are duplicated within a genome, the copies might come under the control of a new promoter.  This could change the rules about when and how much a gene is expressed.  Second, gene recruitment can occur if mutations lead to a different protein function.  The protein could acquire a second function while maintaining the first function or the old function could be lost and a new function takes its place as the purpose of that gene.

Together, these examples drive home the idea that gene expression control (example: promoter properties) and gene coding information (example: protein function) can be thought of as separate “parts”.  Synthetic biology is based on the idea that the parts could be mixed and matched to intentionally lead to a specified function.  Genetic engineers can build ever increasingly complex processes by connecting more parts together.  As an introduction to this, we will watch “Synthetic Biology: Programming Living Bacteria” by Christopher Voigt.

Rapid Evolution

For Introduction to Evolution

In chapter 6 of The Tangled Bank by Carl Zimmer, we learned that there is a simple equation to describe the rate of evolutionary change:

Rate of evolutionary change = “heritability of trait” x “strength of selection”

In the Rapid Evolution video, TREY the Explainer provides a couple of textbook examples.  The three stories covered in 20 minutes include finches in the Galapagos Islands, wall lizards, and stickleback fish.  These examples show rapid evolution (and even speciation) within a few decades time.

Washington Fish

Stickleback fish from Lake Washington in 1957 and 2006

Image credit: Unknown.  (Was used in Seattle Times article)

Monkeys Carry Astroviruses of “Other Animals”

Submitted by Sydney Jennings

This paper is a summary of the article, Non-Human Primates Harbor Diverse Mammalian and Avian Astroviruses Including Those Associated with Human Infections. The purpose of this paper was to explore the idea that Astrovirus (AstV) infections are species-specific, meaning that human Astroviruses (HAstVs) can only cause infection in humans and avian Astroviruses can only cause infection in birds, etc. Through genetic analysis of sequences derived from a highly conserved RNA-dependent RNA polymerase gene, this paper provided solid evidence that non-human primates (NHPs) can harbor a wide variety of mammalian and avian Astrovirus genotypes, including those that are only associated with human infections1.

Astroviruses, along with the rotaviruses are the leading cause of gastroenteritis in children, the elderly, and immunocompromised people1. Astroviruses are small, nonenveloped, positive sense, single-stranded RNA viruses with a star-like appearance, that are transmitted through a fecal-oral route to a wide range of hosts, including calves, piglets, dogs, cats, and mink1,2. These viruses enter cells via adsorption to a receptor that has not yet been identified, and the virus is then internalized by endocytosis2. Although these viruses are most commonly known to cause diarrhea, they have also been found to cause a variety of diseases including nephritis, hepatitis, and encephalitis, or the virus can be asymptomatic depending on the species1.

For a long time, it was thought that bat Astrovirses (BAstV) were most closely related to AstVs from other animals than any other AstVs. However, a recent phylogenic analysis suggests that AstVs in non-human primates are evolutionarily much closer to AstVs in other animals than are bat AstVs1. Additionally, recent studies have shown that diverse AstVs genotypes similar to animal-origin AstVs have been found in children with diarrhea, that more than 25% of nonhuman primates tested had human Astrovirus (HAstV) antibodies, and that there is a recombinant non-human primate Astrovirus with parental relationships to a common human Astrovirus1. These recent findings have provided evidence that non-human primates can be infected by many diverse Astrovirus genotypes, some that are not specific to non-human primates, disproving speculation that Astrovirus infections are species-specific1.

Since 2008, the number of animal hosts shown to be infected with AstVs has quadrupled to include at least 30 mammalian species and 14 avian species with a correlating genetic increase in genetic diversity, which has led to the division of the Astroviridae family into two genera, Mamastrovirus (MastVs) and Avastrovirus (AAstVs)1. Even though AstV infection was thought to be species-specific, phylogenic analysis showed that a single host may be susceptible to infection with divergent AstV genotypes1. One example of this would be that humans can be infected with serotypes HAstV1-8, or recently identified serotypes HastV-MLB1-3, HMO AstVs A-C, and HastV-VA1-4 viruses1. The recently identified HAstVs are much closer genetically to animal AstVs than they are normal HAstVs1. However, diverse MAstV and AAstV genotypes have not been detected in any animal hosts, but potential human-mammalian recombination events have been detected, indicating that the species barrier may have been crossed at some point1. It is known that non-human primates are susceptible to a variety of serotypes of enteric viruses, similar to those of humans, such as rotavirus and norovirus, but there has been no data yet on this type of non-human primate and AstV relationship, which influenced the experiment this paper is discussing1.

In Bangladesh and Cambodia, multiple species of non-human primates including three types of macaques and Hanuman langurs have thrived for centuries on heavy interactions with human, by ranging freely through villages and religious sites and being found in captive settings1. Through analysis of sequences derived from the highly conserved RNA-dependent RNA polymerase gene, it was found that non-human primates harbor a variety of MAstVs, including genotypes previously only associated with human infections1. Additionally, the presence of antibodies to HAstVs further supports that thought that non-human primates are susceptible to infection by HAstV genotypes1.

Fecal samples were obtained from non-human primates in Bangladesh and Cambodia between 2007-2008 and 2011-2012 and the RNA was screened using a pan-astrovirus RT-PCR targeting a 422 length nucleotide segment from the highly conserved RdRp gene mentioned earlier1. Of the 879 samples taken, shown in the figure below, only 68, or 7.7% of the fecal samples tested positive for Astrovirus genotypes1. The results of table below show that there were positive results for the presence of HAstVs in all the different sample contexts from Banglasdesh and over half of these sample contexts tested positive for mammalian Astroviruses, while only one tested positive for avian Astroviruses. The results from the Cambodia samples didn’t provide much information, as there were only 6 positive samples obtained from non-human primates living in Cambodia.

A deeper look showed that the non-human primate that was tested, percent similarity to the closest identified sequence, and a proposed nomenclature for the identified genotypes. A preview of the table can be seen in the figure below, and the complete table can be found in the original article. It can be seen in the preview of this table that there is up to 88% similarity to HAstV1 genotypes, proposing the idea that these genotypes found in the non-human primates were actually genotype of HAstVs1.

To answer the idea proposed above, researchers did a sequence analysis to create a phylogenic tree representing the different genotypes that were found in the positive samples. This sequence analysis revealed that HAstV, MLB, and VA genotypes were detected in the positive non-human primate samples1.

The phylogenetic tree above shows that 11.7% of the samples were 79%-84% similar to HAstV, VA and MLB reference viruses1. Interestingly, these genotypes were detected in the non-human primates, in 2007, before the official identification of the viruses between 2008-20091. The non-human primate sequences FCB5, MCB35, and MCB37 branched off the human subclades of focus, forming a unique clade1. In addition to the non-human primate and HAstV relationships depicted in the following phylogenetic tree, the results show that 23.5% of the samples were similar to MAstV isolated from diverse animal hosts such as dogs, pigs, and sheep1. The last interesting piece of information that was obtained from the phylogenic tree sequence analysis was that 4.4% of the positive samples were clustered with AAstVs, however, the subclade formed for these AAstVs was distinctly different from the previously identified AAstV genotypes1.

From the phylogenic tree produced above, the data showing the similarity of the positive sample genotypes to the HAstV-1 reference viruses is so large that the results could not be shown in the original tree. An extension of the original phylogenic tree, which can be seen below in part A of Figure 3, focused specifically on the HAstV genotype similarities. Additional approaches were done to obtain more information on the positive sample genotypes. These approaches included genome walking, 3RACE, and deep sequencing on all RdRp-positive samples1. As part of these extensive approaches, about 300 nucleotides were obtained from the 5’ end of MAStV/Hoolock gibbon/ Bangladesh/BG36/ 2007ORF21. An analysis of this nucleotide sequence showed a high relatedness between the non-human nucleotide sequence and a HAstV genotype1. Researchers were also able to obtain about 900 nucleotides of MAstV/Rhesus macaque/Bangladesh/BG31/2007 ORF2 and confirmed that it clustered in a clade that includes viruses obtained in cows, pigs, and deer1. The results for the 900-nucleotide analysis can be seen in part C of figure 3.

The results from these other approaches suggested that normal HAstVs and abnormal HAstVs such as MLB, VA, and HMO can be detected in non-human primates1.  The results from part A show that 60.3% of the positive samples were 98-100% similar to HAstV-1 reference viruses1. In addition to the high relatedness to HAstVs in part A, it can also be seen that non-human primate sequences BG31, BG41, and MBG260 cluster within known cow, pig, and deer RdRps if BG35 is excluded from the alignment1. The ClustalW of the BG36 nucleotide sequence confirmed that it was 84% similar to HAstV MLB1 capsid sequences1. It can be seen that BG31 capsid was shown to align within the cow, pig and deer clade mentioned above, creating the hypothesis that non-human primate BG35 is a possible recombinant AstV1. Below is a drawing of the possible way that a recombination could happen with this type of virus3.

Astrovirus drawing.jpg

Figure 1: Drawing of the possible scheme that could occur for a virus of this type.

It can be seen that the recombinant has different aspects of both viruses, this type of recombination has a high probability for becoming highly pathogenic if one of the original strains is pathogenic itself and the virus is able to infect the host 3.

Lastly, it can be seen, that turkey Astrovirus type-2 (TAstV-2) is genetically distant from the mammalian viruses being observed and no TAstV like sequences were found in any of the analyses, so the TAstV-2 was used as a control AstV for the testing of non-human primate sera for the presence of antibodies against HastV-1 and MLB capsid proteins by ELISA1.

There were 90 sera samples from Bangladesh that were able to be tested and 48 from Cambodia1. Of these 138 samples, 44 (31.9%) of the samples tested positive for AstV antibodies, and the majority of these positive samples1. Additionally, 72.7% of the AstV antibody positive samples were positive for specific antibodies against HAstV-1 and 27.3% of the samples were positive for the abnormal MLB capsids antibodies1.

The studies of this paper provide solid evidence for the argument that non-human primates can harbor a diverse range of Astroviruses. An even more important idea that came from this paper was the fact that human Astrovirus strains can be harbored in non-human primates. The evidence that supports these ideas is that diverse AstV genotypes were found in the originally tested fecal samples, the high similarities to HastV reference viruses, the possible recombinants with other animal viruses, and the detection of HastV antibodies in a majority of the sera samples tested. The paper addressed some possible further studies, which are, to determine that HastV in non-human primates is due to actual infection and if such infections are asymptomatic or associated with a clinical disease1.

References:

 

  • Oxford, J., Kelam, P., and Collier, L. (2016). Human Virology. New York: Oxford University Press

 

Protecting Picornavirus Genomes from RNAse Degradation within Endosomes

Submitted by Winter Kemppainen

The research article, Picornavirus RNA is protected from cleavage by ribonuclease during virion uncoating and transfer across cellular and model membranes,1 outlines the results from a study that investigated how the RNA of Picornaviruses is transferred from the virion into the host cell’s cytoplasm without degradation by ribonucleases. The research focuses specifically on the well-known poliovirus, belonging to the enterovirus genus. This summary includes background information regarding Picornaviruses and their entry into host cells, a review of the questions and models posed by the study, and a discussion of the results presented in the article.

Picornaviruses belong to the family, Picornaviridae, that contains six different genera. Picornaviruses are small, icosahedral viruses. Their virion diameters measure 18-30 nm in length. The viruses are non-enveloped, meaning the virion capsids are not enclosed in a protective membrane. The genomes of Picornaviruses are made up of single-stranded, positive-sense RNA (+ssRNA). The +ssRNA acts as mRNA in host cells to encode a single polyprotein that later gets cleaved into smaller, functional proteins after transcription.2

Non-enveloped Picornaviruses, such as poliovirus, enter their host cell via receptor-mediated endocytosis. This process occurs by virion attachment at specific receptor proteins located on the cell membranes of the host cells. Polioviruses bind to CD155 Poliovirus Receptors (PVR) of their host cells. It is important to mention that the PVRs alter the size of the virions from 160S to 135S after attachment, resulting in a 4% enlargement.1 Once bound to the outer cell membrane of the host, the virion is brought into the cell by an inversion of the cell membrane to create a vesicle for the virion, termed an “endosome” by the authors.

During the endocytotic process, the virion is not the only particle brought into the host cell. The serum within which the virion was travelling through before host cell attachment, and enzymes within the serum also get enveloped by the endosome and delivered into the cell. One of the enzymes that typically included is ribonuclease A (RNase A). Ribonucleases are enzymes that catalyze the breaking up and degradation of RNA molecules.3 The role of RNases are to regulate mRNA expression and protect cells from foreign nucleic acids.

It is important to note the significance of the RNase A as it gets brought into host cells along with viruses carrying RNA. This study aims to answer four questions involving the possible interactions between the endosome, virion, and RNases during RNA transfer to the cytoplasm:

“a) is the RNA released from random positions on the particle, only a random <10% of which are adjacent to the membrane?

  1. b) does the attachment process induce a polarization of the particle so that RNA is only released from a position adjacent to the membrane?
  2. c) can RNA released into the endosomal lumen traverse the membrane to reach the cytoplasm?
  3. d) is the RNA protected during transmission across the membrane from RNases that might be present in the endosomal lumen?”1

The answers of these questions have the potential to shed light on the unclear mechanism of how the Picornavirus viral RNA enters host cell cytoplasm without degradation by ribonucleases.

The article poses three possibilities for RNA translocation. The first possible model involves disruption of the endosomal membrane to release the contents of the vesicle into the cell cytoplasm. The second model involves the disruption of the endosomal membrane caused by the insertion of viral peptides. The third model suggests peptides interact with the endosome membrane and the virion to create a channel through which the RNA travels through.1 The results of this study suggest the first two models are improbable, while the third model provides a probable explanation for Picornavirus RNA transfer.

The study first tested the ability of viral RNA to be translocated into a vesicle by virus uncoating. Cryoelectron tomographic imagery was used to capture viral +ssRNA insertion into an in vitro liposome (mimicking an in vivo endosome).1

The liposomes contained poliovirus receptors on their outer membranes to facilitate virus attachment. The entry of viral RNA into the receptor-decorated liposomes supports the idea that RNA is able to travel through channels or pores, and that membrane disruption is not a necessary step in RNA translocation.

The research study also found that the translocated RNA in the liposomes was insensitive to RNase A.1 YoPro-1 fluorescence was used to monitor nucleic acid binding inside and outside of the liposomes, while RNase A was added only to the outside of the liposomes. Fluorescence microscopy was used to observe RNA survival in the presence of RNase A.

The preservation of the viral RNA across the liposome membrane in the presence of RNase A further supports that polioviruses are able to transfer their genomes across a lipid membrane without degradation by ribonucleases. The channels through which viral RNA travel are able to protect the nucleic acid from potentially fatal environments.

The study expanded the research by attempting in vivo experiments to determine whether or not viral RNA was translocated successfully in cultured cells and protected from RNase A in similar ways observed in the in vitro experiments. HeLa Ohio cells were infected with poliovirus in the presence of RNase A. The extracellular fluid was marked with dextrans, a red fluorescent marker. The polioviruses were marked with fluorescent dye Cy2 that fluoresces green. When overlapped, the two markers fluoresce yellow. The results of the experiment were viewed with fluorescent microscopy.

The results of the in vivo experiment indicate that extracellular fluid and material is taken into the cell along with the poliovirus during endocytosis. Assuming RNases could be present in the extracellular fluid and could be taken into the cell with the viruses, the possibility of a protected gateway through which viral RNA can travel through to avoid degradation is even more likely.

Finally, the infectivity of poliovirus was tested while the virus particles were covalently linked to RNase A.1 Linkage of the virus to the ribonuclease ensured that the RNase would be taken into the host cell with the virus. The poliovirus was fluorescently labeled with Cy2 marker. The RNase was fluorescently labeled with DyLight-594 marker. Fluorescence microscopy was used to show the interaction between the poliovirus and RNase A enzymes inside a host cell. The results indicate that the polioviruses were unaffected by the RNase A and were still able to infect the host cell. This result further supports the hypothesis that viral RNA insertion is protected from RNase A degradation.

The results of the study suggest plausible answers to the four questions posed regarding RNA translocation. The experiment suggests that RNA release may have certain directionality, of which could be a result of the altered virus particle interacting biochemically with the cellular membrane. Questions a) and b) cannot be fully answered according to these results, but they provide a strong foundation for further research. The answer to question c) is that RNA can travel through endosomal lumen, but in the presence of ribonucleases, needs to be protected. The answer to question d) is yes, RNA is protected during transmission to the cytoplasm. The experiments indicate that while RNase A is present and able to catalyze the degradation of RNA, the poliovirus remains viable within the host cell and continues with infection. At this point, infection can only be possible if the viral RNA is protected from the RNases.

While the results of the study were not capable of answering all the questions posed in full clarity, the model in which Picornaviruses insert their +ssRNA into the host cell’s cytoplasm was narrowed down quite clearly to the third model: that channels are formed to allow the viral RNA to translocate safely to the cytoplasm.1 Figure 1 outlines a simplified prediction of this channel-formation model.

Polio genome protection.jpg

Fig. 1. Simplified model of Poliovirus RNA translocation inside host cell. As poliovirus binds to the CD155 Poliovirus Receptor on the cell membrane, endocytosis takes in the virus along with RNase As within the extracellular serum. A channel forms between the endosome membrane and the virus particle to allow the +ssRNA to leave the endosome and enter the cytoplasm.

The other two models involving membrane disruption may be ruled out in this case, because the results show that RNase A is capable of degrading viral RNA. If membrane disruption were to occur, the RNases would be released along with the viral RNA into the cytoplasm, and there would degrade the RNA before transcription could proceed. Based on the results from this study, picornavirus RNA must be protected at all times from RNase A to successfully infect its cellular hosts.

The study admits that there is still debate on how protein channels could form between virus particles and the endosomal membrane. However, this research provides some clarity on how Picornaviruses translocate their +ssRNA to infect their hosts. This study has laid the foundation for further research to be done to try to determine the mechanisms of viral RNA protection as it gets transferred to the host cell cytoplasm.

 

References

  1. Groppelli E, Levy HC, Sun E, Strauss M, Nicol C, Gold S, et al. (2017). Picornavirus RNA is protected from cleavage by ribonuclease during virion uncoating and transfer across cellular and model membranes. PLoS Pathog 13(2): e1006197.
  2. Collier L, Kellam P, Oxford J.(2016). Picornaviruses: Polio, hepatitis A, enterovirus, and common cold. In Human Virology(5th ed., pp. 82-91). Oxford, UK: Oxford University Press.
  3. (n.d.). Ribonucleases (RNases) | Thermo Fisher Scientific – US. Retrieved September 22, 2018, from https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/thermo-scientific-restriction-modifying-enzymes/modifying-enzymes-thermo-scientific/ribonucleases-rnases.html

Vesicle-Cloaked Noroviruses

Submitted by Lyndsi Leprowse

The topic of this discussion are Caliciviruses, and this discussion goes into specific detail about that of a certain member of the Caliciviridae family, called Norovirus. The family of Caliciviridae consists of seven species that are divided among five genera, which are: lagoviruses, neboviruses, noroviruses, sapoviruses, and vesiviruses1. Of the five genera in this family, only two infect humans, one of which is the Norovirus. According to the book, in developing countries 200,000 deaths per year are caused by noroviruses, and nearly 1,000 in the US, all in children that are under five years old1. The Caliciviridae family is made up of non-enveloped viruses that are icosahedral, and are 27-40 nanometers in diameter1.

The genome of the Norovirus is polyadenylate, and it is a positive-sense single-stranded RNA that is roughly 7.3-7.5 kb in size1. The 3’ end is polyadenylated, and the 5’ end of the genome has the virus protein VPg attached1. VPg then acts as a cap substitute and recruits factors that initiate translation of mRNAs1. As for how this virus can be transmitted, the most common causes of transmission are the faecal-oral route, inhalation of aerosols from vomit, and point source outbreaks from contaminated food and water1. Noroviruses are also more likely to occur during the winter, however, different Caliciviruses can be seen at all times of the year1.

The paper, Vesicle-Cloaked Virus Clusters Are Optimal Units for Inter-organismal Viral Transmission, discusses how both rotaviruses and noroviruses might enter a cell. While this paper discusses both Rotavirus and Norovirus, I will mainly only be going into detail about the Norovirus. The purpose of this paper was to determine how exactly Rotavirus and Norovirus are shed from the body. It is well known that Caliciviruses are shed in the body through the faecal matter, however, this paper is trying to determine exactly how the Rotavirus and Norovirus are shed and then passed onto the next host after being shed.

While this study shows how these two viruses are released, it is important to note that the two viruses are also very different in size. For this study, it was determined that both rotaviruses and noroviruses are released by extracellular vesicles. However, since the size of the norovirus is anywhere from 27-40 nanometers in diameter, and rotaviruses are around 75 nanometers in diameter, it has been determined that the rotaviruses are released in extracellular vesicles, while noroviruses are released by exosomes. However, exosomes are actually a type of extracellular vesicle.

To begin their experiment, stool samples from infected patients were taken and incubated using TIM-4-coupled beads2. After incubating, the sample was run on an SDS-PAGE/western analysis using anti-human norovirus VP1 antibody. After analyzing picture A in figure 3 of the article, it can be seen that the stool sample had the presence of the human norovirus capsid protein VP1 that was associated with the phosphatidylserine (PS) vesicles2. Once the infection was found within the stool sample, the sample was then placed under a negative-stain electron micrograph and these images show that the norovirus was contained within a small exosome2.

Norovirus vesicles.gif

Figure 1: This figure is an example of how a TIM4 antibody attaches to the VP13.

From the data found from the first two images in figure 3 of the paper, it was concluded that the human norovirus was shed non-lytically into the human stool by escaping inside of exosomes that came from the infected host2. To determine whether or not this information was accurate, more studies needed to be done. To continue to research this, the cells of a mouse phage called RAW264.7 were infected in culture with non-lytic murine norovirus (MNV-1). The purpose of this experiment was to test the permeability of the membrane of the mouse phage. The results of this test showed that approximately one hundred percent of the MNV-1 cells left the RAW264.7 cells without breaking the membrane2.

After the results of the third experiment confirmed the first two experiments, the next experiment was done using extracellular MNV-1 that was enriched in small exosome size PS vesicles, and were then centrifuged at 104 x g, and 105 x g. Both the supernatant and the pellet from both of these centrifugation techniques were then incubated with either Annexin V-coupled magnetic beads (ANX) or control (CTL) magnetic beads, and then run on a SDS-PAGE or western blot analysis using anti-murine norovirus antibody VP1, which was also the antibody that was used during the first SDS-PAGE/western analysis2. From the results that are shown in figure 3, it can be determined that the norovirus is not actually present in the supernatant, but it is present in the pellet, and it also seems to show that the presence is more noticeable at 105 x g. This indicates that it is possible that whatever was found at 104 x g was actually debris. Just as with the first two figures, they took the results from the SDS-PAGE/ western of the 105 Annexin V bead and observed them through a negative-stain electron micrograph, and it revealed small exosome-size vesicles containing MNV-1 particles2.

MNV-1 were then treated with acute levels of GW4869, and the MNV-1 levels were then reduced, which was similar to multivesicular body (MVB) derived exosomes2. The replication from this experiment was not affected, and the lipid analysis of the vesicles from MALDI-TOF/MS showed that there was a presence of bis(monoacylglycerol)phosphate (BMP), which is a lipid enriched in MVBs and MVB-derived exosomes2. They then took the sample to test whether or not the exosomes containing the norovirus were infectious. To do this, they inoculated RAW264.7 cells and human enteroid cell cultures with either human norovirus or MNV-1-containing exosomes2. An increase in human norovirus genome copies was measured after the vesicle inoculum was washed off of the enteroids, and this showed that the exosomes were infectious, as well as the MNV-1-containing exosomes when inoculated into new RAW264.7 cells2.

From this information, another test was done to determine the infectivity of MNV-1 without the receptor. It was found, however, that without the MNV-1 receptor, CD300lf, the cells could not be infected. CD300lf is a member of a family of PS receptors2. This result was tested to rule out a simple vesicle membrane-host plasma membrane fusion as a way of delivering the virus to the host2.  The cells were then inoculated with CellBrite Fix 488-labeled fluorescent MNV-1 exosomes, and then z-sectioning showed that there was approximately a 50% decrease in the cells that were treated with the anti-CD300lf antibodies compared to those that were treated with the immunoglobulin G (IgG) antibodies2. The study then suggests that CD300lf could serve a dual purpose: the first being to allow exosome internalization into an endocytic compartment through the interaction with the vesicle PS lipids, and also binding to the MNV capsids and mediating genome transfer into the cytosol2.

The results of this study showed that rotaviruses and noroviruses that were not surrounded by a vesicle were less likely to cause a problem, whereas if the viruses were in larger populations inside vesicles, have a higher chance of infection rate and overcoming replication barriers. This result shows that if viruses are clustering together in higher populations inside of vesicles and then moving from host to host, then infection is more likely to occur. Also, if the viruses are clustering inside of vesicles then they are more likely to use more than just vesicles, and more than these two viruses are likely to spread this way as well. The results of this showed that in order to disrupt the clustering among viruses, antiviral therapeutics need to be used2.

References

  1. Oxford, J., Kellam, P., and Collier, L. (2016). Human Virology. New York: Oxford University Press
  2. Santiana, M., Ghosh, S., Ho, B.A., et al. (2018). Vesicle-Cloaked Virus Clusters Are Optimal Units for Inter-organismal Viral Transmission. Cell Host & Microbe 24, 208-220.
  3. Graff, J. Virology. September 25, 2018.

Dating Older Humans

For Introduction to Evolution – Chapter 3 Amazon Review

In chapter 2, we learned about a few important historical figures that contributed to the pre-Darwin field of evolution.  William Smith’s “map that changed the world” provided the first example of “biostratigraphy” where the relative ages of fossils are inferred from the layer of rock/deposits the fossil is found.  Now, in chapter 3, we spent some time discussing radiometric dating, a method based on monitoring radioactive decay.  (See the illustration of alpha and beta particle release during the decay of radioactive lead…that started out as radioactive thorium.)800px-Thorium_decay_chain_from_lead-212_to_lead-208.svg

YouTube is cluttered with anti-evolutionists attempts at undermining the validity of radiometric dating.  There is more than one (or two) way to determine the age of fossils, so first read “Everything Worth Knowing about…Scientific Dating Methods” by Gemma Tarlach and take brief notes on the various methods.

Next, dig into the “Fossil from Arabian Desert…” also by Gemma Tarlach to see an example of fossil dating using the uranium time series method.  Draw a timeline of events described in this article.  Then write a standard Amazon Review.

Image credit: Eugene Alvin Villar for the Philip Greenspun illustration project.  CC BY-SA4.0.