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

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.

Chapter 1 Student Questions

For Virology

  1. Did having bacteria and air-borne contamination with cultures of viruses skew results? Were they trying to correlate a grown virus with a certain illness, and did contamination ever throw off their results?
  2. The EM microscope has been a valuable tool for the identification and measuring of viruses. During the time it took for the EM to become more commonplace in the usage of measuring viruses, what other methods were used and how were the sizes of the viruses determined? What are the advantages of the EM compared to the other methods that were used? What were the patterns that were found?
  3. Why did virologists refer to a virus as a poisonous liquid in the nineteenth century?
  4. Why can’t hepatitis c be grown in the lab via conventional methods? 
  5. What is a nanopore sequencer? 
  6. The Greek Goddess Hygeia was worshiped at the Parthenon 3500 year ago for her exemplary what?
  7. What causes infantile paralysis, also known as polio?
  8. Why did virologists refer to a virus as a poisonous liquid in the nineteenth century?
  9. What is Reverse Genetics and how does it contribute to virology?
  10. Did having bacteria and air-borne contamination with cultures of viruses skew results? Were they trying to correlate a grown virus with a certain illness, and did contamination ever throw off their results?
  11. The book mentioned that antibiotics caused a great advance in the propagation of viruses because the antibiotics inhibited airborne contaminants, but it also mentioned that cells are now cultured without antibiotics. Why would pharmaceutical groups stop using antibiotics in their cultures if they have worked so well to inhibit contaminants in the past?
  12. What are the studies of phylodynamics and phylogeography?
  13. What is the term for a commercial treatment of goods, still used today, soon discovered after Louis Pasteur documented the fermentation process of yeasts? (Hint: Fine Wine)
  14. How was the first virus, tobacco mosaic, discovered and what does it infect?
  15. How do scientists identify viruses?
  16. Does the recent addition of antibiotics (1940s/50s) to culture mediums which inhibit contamination in large-scale vaccine production, affect the cell cultures negatively?
  17. How did the understanding of a virus’ replication cycle lead to the creation of viral controls?
  18. Have there been any statistical studies on the rate at which antigenic drift occurs in influenza viruses? If so what was concluded?
  19. The EM microscope has been a valuable tool for the identification and measuring of viruses. During the time it took for the EM to become more commonplace in the usage of measuring viruses, what other methods were used and how were the sizes of the viruses determined? What are the advantages of the EM compared to the other methods that were used? What were the patterns that were found?

Fit to Fight the Flu

Influenza virus, the causative agent of flu, has a genome composed of segmented, single-stranded, negative sense RNA.  This combination leads to challenges for your immune system because this virus can evolve through occasional genetic shift and continuous genetic drift.  Let me explain.

To cause a genetic shift, two different strains of influenza infect the same animal (and, importantly, the same cell within the animal) simultaneously.  The outcome can be that the segments from the two strains of influenza mix and match to form a novel virus (aka a reassortant virus).  For illustration purposes, I have drawn a cell that is infected with one virus (all red genome) and a second virus (all yellow genome).  The result is virus progeny that have red and yellow genome segment combinations called reassortments.  The new virus may be a human influenza virus and an animal virus, so the progeny would be a new form of influenza that would be a novel threat to everyone in the world.  If you hear about pandemic flu viruses, these viral reassortants would be the culprits.

Reassortant virus

Genetic drift, the accumulation of variant genomes, is also a problem for our immune systems due to new virus mutants that result from errors made by RNA polymerases.  Vaccines developed for the northern hemisphere’s winter are designed to “teach” the immune system about new variants dominating in the southern hemisphere’s during their “winter” (aka summer in the northern hemisphere).  You see, unlike polymerases that read DNA to make RNA, polymerases that read RNA to make RNA lack proofreading mechanisms where, upon making a mistake, the polymerase backs up in a 3’ to 5’ direction and fixes the error (1).  In summary, the genetic drift that occurs annually in the southern hemisphere are the mutations northern hemisphere populations need to be ready to detect.

When vaccines miss the mark regarding the most likely variants of flu coming to the northern hemisphere, the innate immune system has increased importance in protection against severe disease.  This branch of the immune system responds to general microbial molecules, such as a bacterium’s cell wall, rather than to specific, genome-encoded molecules such as proteins.  Regardless of which strain of flu virus causes an infection, the innate immune system is ready to resist the uncontrolled replication of the virus.

This brings us to the article we are going to discuss, “IFITM3 restricts the morbidity and mortality associated with influenza”, that was published in the journal, Nature, in 2012 (2).  Being in a high-tiered journal, one of my first questions usually is, “Why was this study published in _______?”  In this particular case, I believe it is because the story presented in this manuscript works its way from animal models, to clinical genetic and epidemiological data, and finishes with human cell cultures that support the mouse and human findings.

IFITM3 was known to be involved in innate immunity responses and, specifically, was highlighted in a previous study that used a genomic screen to identify proteins that function to restrict virus replication (3).  Not only did this original study show that IFITM3 restricts the replication of influenza (family Orthomyxoviridae), but also protects against a couple of flaviviruses (Dengue and West Nile).

Mice aren’t always a great model for influenza infections because they are resistant to many influenza strains.  Certain strains such as “PR/8” are virulent in mice because these strains were selected for virulence as they went through many rounds of infection in mice.  The authors of the study showed that even virus strains that are typically nonlethal in mice cause such extreme sickness that the mice unable to make IFITM3 lost over 20% of their weight (an indication that the mice needed to be euthanized to prevent unnecessary suffering).  The disease severity was not associated with a difference in viral replication in the first two days, but instead, the IFITM3 mutant mice were not able to restrict virus replication as well in the third and fourth day of the infection.  This is consistent with the idea that IFITM3 acts within the innate branch of the immune system since adaptive immune responses would take effect later in the infection.

When the lungs of the infected mice were examined, traces of the virus was found throughout the lungs.  Moreover, the pathology of the lungs of IFITM3-lacking mice included edema and bleeding with an infiltration of an innate immune cell knowns as neutrophils (aka polymorphonuclear cells).  None of these observations was too surprising as severe influenza infections are often associated with this type of pathology.

While many studies would be expected to end with the mechanism of the protein in question being discovered.  For IFITM3, the mechanism(s) of protection against influenza virus does not appear to be settled and has been the topic of more recent research.  Instead, this study ends with a look at IFITM3 mutants in the human population.

Through sequencing many human genomes, scientists have been able to identify specific base pair mutations within the human populations.  Technically, these mutations are known as single nucleotide polymorphisms (SNPs).  At the time of the publication, 28 IFITM3 SNPs were known.  Interestingly, the authors went on to sequence the IFITM3 gene for 53 individuals that had been hospitalized by either the 2009 H1N1 pandemic flu or the seasonal flu from that year.  Incredibly, the SNP that caused the loss of a portion of the IFITM3 protein was found far more frequently in these hospitalized patients than in the population as a whole.

With a likely important SNP in hand, the authors conjured up some human cell lines that had the “normal” IFITM3 protein or the shortened IFITM3 protein caused by the SNP in question.  Consistent with their mouse and human data, the mutant IFITM3 cells supported higher levels of influenza infection that their normal counterparts.

A question to ponder after reading this paper is, “Why does the shortened IFITM3 mutant exist in the population?”  Some answers include:

  • Evolutionary pressure hasn’t caused this mutant to be lost in the population yet.
  • The shortened form of IFITM3 has a beneficial quality…just not in the case of viral infections.
  • The selective pressure from the viral infections has not been strong enough to counteract genetic drift of this human genotype.

References

  1. Steinhauer, D. A., Domingo, E., and Holland, J. J. (1992) Lack of evidence for proofreading mechanisms associated with an RNA virus polymerase. Gene. 122, 281–288
  2. Everitt, A. R., Clare, S., Pertel, T., John, S. P., Wash, R. S., Smith, S. E., Chin, C. R., Feeley, E. M., Sims, J. S., Adams, D. J., Wise, H. M., Kane, L., Goulding, D., Digard, P., Anttila, V., Baillie, J. K., Walsh, T. S., Hume, D. A., Palotie, A., Xue, Y., Colonna, V., Tyler-Smith, C., Dunning, J., Gordon, S. B., Smyth, R. L., Openshaw, P. J., Dougan, G., Brass, A. L., and Kellam, P. (2012) IFITM3 restricts the morbidity and mortality associated with influenza. Nature. 484, 519–523
  3. Brass, A. L., Huang, I. C., Benita, Y., John, S. P., Krishnan, M. N., Feeley, E. M., Ryan, B. J., Weyer, J. L., van der Weyden, L., Fikrig, E., Adams, D. J., Xavier, R. J., Farzan, M., and Elledge, S. J. (2009) The IFITM Proteins Mediate Cellular Resistance to Influenza A H1N1 Virus, West Nile Virus, and Dengue Virus. Cell. 139, 1243–1254