First Discovery of "Multicomponent" Animal Virus

In a recent article in the journal Cell Host & Microbe, scientists have discovered the first clear evidence of a multicomponent virus that can infect animal cells. Ranging back into the 1970’s, examples of multicomponent viruses, those that have their genome separated into multiple distinct segments, have been found commonly within both plants and fungi.¹ Typically, most animal viruses package their entire genome into a single virus particle, which alone has the potential to infect cells and replicate itself within them. Multicomponent viruses, however, have numerous unique particles that collectively make up the viral genome, and at least two or more of these different types of particles are required for successful infection and replication within cells. While many scientists believe that this segmentation may allow for the virus to have more control over genome expression and variation, a higher rate of infection is logically required so that all of the various particles may be successfully transmitted into the cell. Presumably, this requirement may be one reason why multicomponent viruses are previously unknown to animals, as compared to more stationary plants and fungi. In this study from the U.S Army Medical Research Institute of Infectious Diseases, two new examples of multicomponent pathogens were discovered, one named Guaico Culex virus (GCXV), isolated from mosquitoes in Central and South America, and one isolated from red colobus monkeys in Uganda, a variant of the Jingmen Tick virus (JMTV). The discovery of these multicomponent viruses and their ability to infect animals has interesting significance both in human health and in the complex and relatively unknown evolutionary history of viruses.

In order to determine the multicomponent nature of these viruses, the researchers sequenced isolates from the mosquitoes and found five distinct particles for GCXV, although the fifth particle was only identified in some of the samples.

Figure 1: Identification of distinct viral particles for GCXV in infected cells²

Interestingly, when these particles were transfected into cells in various combinations, it was found that only segments one through four were necessary for infection and replication, while segment five was not necessary and did not seem to provide any significant improvement in infectivity.² This was shown through the use of viral plaque formation, which observes the regions of cell death within a cell culture that are caused by viral infection. Whereas in a non-segmented virus, plaques can be formed in the presence of just a single particle, in multicomponent viruses, all of the necessary particles are required in order for these plaques to form. For the JMTV isolates, four distinct segments were found, and were present in all of the samples isolated from both the red colobus monkey and from ticks.² Ultimately, the sequencing and transfecting of these segments supports the multicomponent nature of these viruses, with the JMTV variant remarkably representing the first known example of a multicomponent virus infecting primates. While the extent of multicomponent animal viruses is yet unknown, the existence of this primate-infecting virus alone suggests that there may be other segmented animal viruses, which may have relevant applications in human and animal health as research expands and develops.

The discovery of these viruses also has significance evolutionarily, as both of these multicomponent viruses, GCXV and the JMTV variant, fit well with a recently classified group of segmented viruses called “jingmenviruses”. Viruses in this category are all segmented into at least four different virus particles, have been found in ticks and other insects across China and South America, and are highly similar (80-90%) to GCXV and the JMTV variant on a genetic level.³ From this information the researchers reasonably suspect that all of the jingmenviruses share a segmented common ancestor. Numerous structural differences among these viruses, however, along with certain genetic differences, suggest incredible diversity within the clade, much of which is yet to be discovered and classified. Another major finding was that GCXV and other jingmenviruses are genetically similar to the unsegmented category of viruses called flaviviruses, although whether the evolutionary pathway diverged from an unsegmented or a segmented common ancestor is yet unknown. While viral evolution is highly complex and unknown to a large extent, the information provided by these newly discovered multicomponent viruses is another important piece in the evolutionary puzzle.

Figure 2: Potential evolutionary relationships and pathways between Jingmenvirus and Flavivirus²

In summary, this study reported the discovery of two new multicomponent viruses, including the first identified example of a virus of this type that can infect animals. Both evolutionarily and health wise, the discovery of these segmented viruses has important implications for future research, as scientists continue to connect together the evolutionary pathways of viruses and work to counter infectious diseases in humans. Ultimately, this study was just the first insight into this new class of viruses, and further examples of animal-infecting multicomponent viruses are needed to determine their prevalence among animals. Based on the isolates extracted from the red colobus monkey, identification of other animal-infecting multicomponent viruses may be found in other monkeys in Africa and South America, especially in places with high numbers of common jingmenvirus vectors like ticks and mosquitoes. Additionally, further examples of these viruses may aid in determining the evolutionary relationship of the Jingmenvirus and Flavivirus clades, particularly through the use of bioinformatics and comparisons of their genomes. Overall, the incredible variation of viruses ranges well beyond our knowledge, and identification of novel classes of viruses such as these are exciting developments in a field with much left to discover.

1 Fulton, W.R. (1980). Biological significance of multicomponent viruses. Annual Review Phytopathology 18, 131-146.

2 Ladner, J.T., Wiley, M.R., Beitzel, B., Kramer, L.D., Tesh, Robert B., Palacios, G., et al. (2016). A multicomponent animal virus isolated from mosquitoes. Cell Host & Microbe 20, 357-367.

3 Shi, M., Lin, X.D., Vasilakis, N., Tian, J.H., Li, C.X., Chen, L.J., Eastwood, G., Diao, X.N., Chen, M.H., Chen, X., et al. (2015). Divergent viruses discovered in arthropods and vertebrates revise the evolutionary history of the Flaviviridae and related viruses. J. Virology 90, 659–669.

Cancer Fighting Recombinant Mumps Viruses

Mumps Virus Symptoms:
Swollen Cheeks and Jaws

            As of August 23rd, only 1786 cases of the mumps virus have been reported this year in the United States (1). Although relatively scarce in the United States, the mumps virus frequently infects human populations throughout Europe, Asia, and Africa (1).The mumps virus is a member of the Paramyxoviridae virus family, characterized by its negative-sense single-stranded RNA genetic component, helical protein coating, and lipid envelope. Measles virus is the more commonly known member of the Paramyxoviridae family. Mumps results in noticeably swollen cheeks and jaws, fevers, headaches, muscle aches, lethargy, and loss of appetite. However, the majority of individuals infected with mumps will not show any symptoms. A person infected with the worst of mumps’ symptoms will typically recover in a matter of days. In the worst of cases, the mumps virus may cause inflammation in the brain, testicles, ovaries, and tissue surrounding the spinal cord. Like the common cold, the mumps virus is highly contagious, spreading by both saliva and mucus. Overall, the mumps virus is quite wimpy and easily forgettable; however, recent findings by Arun Ammayappan, Stephen Russell, and Mark Federspiel (2016), surmise that the mumps virus may prove itself as a powerful tool in cancer therapy.

                  Right behind heart disease, cancer is the second leading cause of death in the United States. The use of viruses to cure cancer has been of recent interest in the 21st century. By rational logic, it appears counterintuitive to fight a disease, like cancer, with a pathogen that causes disease itself, like a virus. However, certain viruses, called “oncolytic viruses,” have been found to specifically infect and destroy cancer cells, while leaving normal cells intact. Viruses take advantage of the differences in the interferon responses (which strongly inhibit viral replication), between normal cells and cancer cells in order to solely kill the latter (2). Last October, the FDA approved the first ever oncolytic virus therapy for patients with advanced melanoma (3). This drug, Talimogene laherparepvec, is a genetically modified herpes virus that replicates in and destroys cancer cells producing a protein called granulocyte-macrophage, colony-stimulating factor. Although the first cancer-fighting virus to be approved by the FDA, Talimogene laherparepvec was not the first virus used to treat cancer patients.

In fact, during the 1970s and 1980’s, Dr. Asada, a Japanese physician injected Urabe mumps virus, isolated from an infected child, into cancer patients as a form of treatment. Out of the 90 patients that Dr. Asada treated with the mumps virus, the tumor sizes lessened by more than half in 37 patients, and tumor growth halted in another 42 patients (4). Since Dr. Asada’s initial trial, two more successful trials have been performed using the same strain (Urabe) of mumps virus (5). The second trial involved treating terminally-ill cancer patients of various forms with the Urabe mumps virus, and the third trial involved pre-immunizing advanced gynecologic cancer patients with the mumps virus before the complete cancer treatment. Dr. Asada’s success with the mumps virus led Ammayappan et al. to resurge the idea of the mumps virus as a weapon against cancer.

 Ammayappan et al. began their studies by cloning an aliquot of the original Urabe strain of mumps virus used by Dr. Asada in the 1970’s and 1980’s. The researchers developed a reverse genetics system for the mumps virus, which allowed the authors to manipulate the mumps virus by inserting specific genes into the virus that encode for particular proteins, yielding a “recombinant virus.” The authors inserted a GFP protein into the mumps virus (rMuV-UC-GFP), thus, illuminating the cell-infecting mumps virus under fluorescent microscopy. By quantifying the amount of fluorescence from the GFP protein, one could determine relative levels of viral infection in particular cell lines. The authors were also able to engineer other unique mumps viruses, including mumps viruses containing an intact luciferase gene (rMUV-UC-LUC), a human sodium iodide symporter (NIS) gene (rMUV-UC-NIS), and an interferon-𝛽 gene (rMUV-UC-mIFN𝛽), to study various aspects of the mumps virus’ ability to replicate and spread in-vivo (in a live organism). The researchers cultivated their recombinant mumps viruses in Vero cells, which lack an intact interferon response, allowing the viruses to replicate freely for use in cell culture and live mice experiments.

The propagated rMuV-UC-GFP recombinant virus was used to infect various mouse and human tumor cell lines to identify the infectivity of the virus in-vitro (outside living organisms). Fluorescent microscopy demonstrated that rMuV-UC-GFP robustly infected most human tumor cell lines, including human myeloma (Kas6/1), plasma cell leukemia (ARH-77), human multiple myeloma (MM1), ovarian carcinoma cells (SKOV3), adenocarcinoma alveolar basal epithelial cells (A549), and cervical cells (HeLa). In mice, the rMuV-UC-GFP infectivity was much lower among mouse tumor cell lines. The neuroblastoma (N2A) and colon cancer (CT-26-LacZ) cell lines showed the highest amount of infected cells, but significantly less infection than human tumor cell lines. Furthermore, the researchers succeeded in infecting rat tumor cell lines, C6 and RG2 glioma cells, finding significant levels of infection in the RG2 tumor cell line, more than any mouse tumor cell line. Ammayappan et al’s in-vitro studies of rMuV-UV-GFP in various tumor lines demonstrate the virus’ high levels of infectivity in human and rat tumor cell lines, but low level of infectivity in mouse tumor cells lines.

Figure 2a from Ammayappan et al. (2016): Green fluorescence indicates viral infection from rMUV-UC-GFP.
 Grey images illustrate tumor cells without the use of fluorescence to indicate infectivity.

 In order to identify factors other than the interferon response responsible for the lack of recombinant mumps virus replication in certain tumor cell lines, the researchers treated less permissive cell lines with a Janus kinase inhibitor called Ruxolitinib, which restricts the interferon response. As expected, the interferon inhibitor, Ruxolitinib, allowed the mumps virus to more robustly infect rat glioma, mouse lung carcinoma, and colon carcinoma cells; however, unexpectedly, plasmacytoma and human multiple myeloma cells still showed diminutive levels of viral infection. This lack of infection in the presence of Ruxolitinib suggests that certain tumor cells restrict mumps virus infection by other cellular mechanisms besides the interferon response.

 Finally, in order to test the oncolytic properties of the recombinant mumps viruses in-vivo (in a live organism), the recombinant viruses were used on mice implanted with human myeloma cells (KAS6/1). Human myeloma cancer cells implanted just under the skin of mice were allowed to develop into tumors for about five days before the mice were injected with one of the recombinant mumps viruses. The researchers used the original clone of the Urabe mumps virus (MuV-UC), an altered mumps virus that replicates more quickly than the original Urabe strain (rMuV-UC-L₁₃₃₂₈-GFP), and an altered mumps virus with a luciferase gene (rMuV- UC-LUC), which allows the virus to be observed under bioluminescent imaging due to the luciferase gene’s luminescent  products created during the virus’s replication. After sixty days, the MuV-UC treated mice demonstrated significant tumor suppression in all five mice subjects, and one observed a complete response, which is a complete absence of detectable tumor cells. Two mice had a complete response to the rMUV-UC-L₁₃₃₂₈-GFP isolate, and one complete response and a great extent of tumor suppression were noted in the mice injected with rMuV-UC-LUC. Using virus antigens, the researchers were able to detect mumps virus proteins in the harvested tumors after days 7 and 12, ensuring that each virus replicated well in the tumors. Altogether, the recombinant mumps viruses demonstrated the ability to suppress and degrade human myeloma tumor cells when implanted into mice, reinforcing the potential for the virus to be used in oncolytic therapy.

Overall, this initial recombinant mumps virus study by Ammayappan et al. (2016) suggests that recombinant mumps viruses kill human most tumor cells quite well, at least when implanted in mice, and the retain potential for future use in cancer therapy. Obviously, the ability of the mumps virus to serve as an oncolytic agent without untargeted side-effects in humans remains uncertain. Much more reproducible experimentation of the recombinant mumps viruses in-vivo must be performed. One major adversity of yielding reliable results from the in-vivo experiments on mice was the inability of the recombinant mumps virus to significantly infect mouse cells. Because the rat tumor cell lines demonstrated a much higher degree of susceptibility to mumps virus, rats may serve as a more realistic and reliable model for testing the results of mumps virus infection in human cancer cells versus non-cancer cells. Furthermore, the FDA’s neurovirulence safety test (2005), which measured nervous system damage in rats caused by mumps virus, predicted that rat-based mumps virus infections adequately parallel the potential neurovirulence of the mumps virus in humans (6 & 7). Other areas of further study include performing recombinant mumps virus experiments in-vitro and in-vivo with even more cancer forms, identifying other specific modes of viral defense in tumor cells besides the interferon response, and analyzing the compatibility of the recombinant mumps virus with other cancer therapies, such as radiation therapy or chemotherapy. Such further findings will either strengthen or weaken the possibility of recombinant mumps virus as an oncolytic agent, produce more efficient models for testing potential oncolytic viruses, and provide a greater understanding of the ability of similar viruses to alleviate cancer by targeting the degradation of tumor cells while leaving normal, healthy cells unscathed.

Paper:

http://www.nature.com/articles/mto201619

Ammayappan, A, Russell, S, Federspiel, M (2016). Recombinant mumps virus as a cancer therapeutic agent. Molecular Therapy- Oncolytics 3: 16018

1.     “Mumps Cases and Outbreaks.” CDC, August 23, 2016. https://www.cdc.gov/mumps/.

2.     Singh, PK, Doley, J, Kumar, GR, Sahoo, AP, Tiwari, AK (2012). Oncolytic viruses & their specific targeting to tumour cells. Indian J Med Res 136: 571-584

3.     Lawrence, L. “FDA Approves First Oncolytic Virus With New Melanoma Therapy.” Cancer Network. http://www.cancernetwork.com/melanoma/fda-approves-first-oncolytic-virus-new-melanoma-therapy.

4.     Okuno, Y, Asada, T, Yamanishi, K, Otsuka, T, Takahasi, M, Tanioka, T et al. (1977). [Mumps virus therapy of neoplasms (2)]. Nihan Rinsho 35: 3820-3825.

5.     Okuno, Y, Asada, T, Yamanishi, K, Takahashi, M, Tanioka, T et al. (1978). Studies on the use of mumps virus for treatment of human cancer. Biken J 21: 37-49.

6.     Rubin, SA, Pletnikov, M, Taffs, R, Snoy, PJ, Kobasa, D, Brown, EG et al. (2000). Evaluation of a neonatal rat model for prediction of mumps virus neurovirulence in humans. J Virol 74: 5382-5384.

7.     Rubin, SA, Afzal, MA, Powell, CL, Bentley, ML, Auda, GR, Taffs, RE et al. (2005). The rat-based neurovirulence safety test for the assessment of mumps virus neurovirulence in humans: an international collaborative study. J Infect Dis 191: 1123- 1128.

Photographs:

https://phil.cdc.gov/phil/details.asp?pid=130

Figure 2 from Paper (Ammayappan et al., 2016)

Additional Resources:
http://www.mayoclinic.org/diseases-conditions/mumps/basics/definition/con-20019914

http://www.ncbi.nlm.nih.gov/books/NBK8461/

Cancer Research Institute Video: “Oncolytic Virus Therapy: Dynamite Cure for Cancer Cells” https://www.youtube.com/watch?v=zwlCkVnUgWQ

More on Interferon:

https://www.britannica.com/science/interferon

Building the "Trojan Horse" to Combat Ebolaviruses

    From it's discovery a short four decades ago, Ebola viruses have directly affected the lives of more than 30 000 people (1).  Despite the many historical outbreaks, the recent outbreak in Western Africa in 2014 marks the largest and most complex yet officially bringing the infection across seas to the United States.  As a result, a "Public Health Emergency of International Concern" was declared and an international response to combat Ebola virus has commenced.

     Ebola virus is a severe and often fatal disease in humans with an average case fatality rate of about 50% (2).  The disease can be contracted through various forms of direct contact with anything that has been contaminated or infected with the virus.  Evidence suggests that bats are the "reservoir hosts", transmitting the disease to other animals or humans (3).  Affected individuals experience a range of symptoms including severe headache and weakness as well as fever and muscle pain.  The illness is caused by infection with a member of the Filoviridae family of viruses, genus Ebolavirus.  To date, there are five identified species of Ebola virus with four of which causing disease in humans (2).  Nonetheless, the recent outbreak in 2014 caused specifically by the Zaire ebolavirus has driven scientific research to find protection against any or all of the Ebola viruses to promote the well-being and health of the world's population.  

     In order to understand the scientific foundation for the development of therapeutics, one must become familiar with the viral entry process of the Ebola viruses.  In terms of "viral entry", this is the point in the infection cycle in which the Ebola virus genetic material is released from a vesicle (lysosome) located inside the target cell.  The released viral genetic material can then undergo replication and transcription/translation to form more viral components and ultimately assemble more virions that can be released to commence the infection process in other target cells.  In other words, "viral entry" in the case of Ebola viruses does not refer directly to it's physical entry into the target cell, but rather the release of the viral genetic material from within an internal compartment of the target cell.  

(5)

     Generally, the sequence of events for viruses similar to Ebola the  are as follows: a viral adhesin (in Ebola's case, it's glycoprotein) attaches the virion to the target cell membrane through receptor interactions, the virion is engulfed via endocytosis now encapsulating the virion in an endosome, the virion is delivered to another vesicle known as a lysosome, then the environment within the lysosome triggers a conformational change of the protein (Ebola's glycoprotein) that will ultimately undergo a series of conformational changes to fuse the virion to the lysosomal membrane by interacting with a membrane receptor, allowing the virion to release its genetic material outside of the lysosome.  Very simply put, an Ebola virus finds a way into the cell via endocytosis and then finds a way out of the lysosome for it's genetic material via fusion with the lysosomal membrane to reach the cytoplasm. 

     Filoviruses, the family that Ebola viruses are members of, display a unique characteristic in terms of this entry process.  Once the endosomal materials, including the virion, are within the lysosome, a protease cleaves the glycoprotein (GP) of the virion resulting in a GP that now reveals affinities for molecules that it could not interact with prior.  Specifically, the cleaved GP on the virus in the lysosome now has a receptor-binding site that can interact with the Niemann-Pick C1 (NPC1) intracellular receptor (the uncleaved version of GP cannot interact with NPC1).  This fusion event located in the lysosomal membrane is what ultimately leads to the release of the viral genetic material from the lysosome, and thus this cleavage event of the GP is essential for filovirus infection.  An excellent visual representation for this process can be found from the Albert Einstein College of Medicine at "How Ebola Virus Infects a Cell".  

      

     Now that there is an understanding of viral entry, a discussion can be had regarding a developed therapy for Ebola viruses.  Researchers have developed what's known as "ZMapp", which consists of three antibodies that target the Zaire Ebola virus glycoprotein.  These antibodies block the ability for the glycoprotein to interact with the receptors it needs to for successful infection (ie. reaching the cytoplasm).  The research team found that the ZMapp concoction of antibodies can rescue nonhuman primates already infected with the Ebola virus (4).  As this brings light to the potential of antiviral therapies, there still exist a great concern for another viral epidemic to occur from another species of Ebola virus.  In other words, ZMapp targets the Zaire Ebola virus responsible for the 2014 epidemic but has yet to prove successful with others.  This is because ZMapp is designed to recognize a "binding site" (epitope) specific to the glycoprotein of the Zaire Ebola virus, and thus cannot target the other Ebola viruses' glycoproteins.  As a result, there is a need to be prepared for any epidemic by another filovirus by constructing broad protection immunotherapies (A.Z. Wec et al., 2016). 

     A team of researchers have recently brought a "Trojan horse" antibody strategy to life that preliminary results have shown potential for combating a whole scope of filoviruses.  The idea in this therapeutic method is to ultimately attack the cleaved glycoprotein in the lysosome and/or the NPC1 receptor in the lysosomal membrane to block the entry of viral genetic material into the cell, by bypassing the obvious obstacle that the lysosome is a closed off compartment within the cell.  The "Trojan horse" title to this therapy describes how the researchers are utilizing the virus itself to deliver the aforementioned antibodies to the lysosome.  This is done by coupling the cleaved GP or NPC1 targeting antibody to an antibody targeting a "binding site" (epitope) broadly conserved across filoviruses in the uncleaved version of the glycoprotein.  Using a well-known dual variable domain strategy, DVD-Ig, this coupling of antibodies can be achieved (A.Z. Wec et al., 2016).  In a simple sense, this "Trojan horse" therapy is a very hopeful strategy for combating filovirus infections because it attacks the unique characteristic (cleavage of the GP in the lysosome) of filoviruses in their mechanism for successful viral entry.  Blocking the interaction of the cleaved GP with the NPC1 receptor by either blocking the cleaved GP or the NPC1 receptor disables the ability to release the genetic material into the cytoplasm.  This therefore disables the ability to replicate and create more viral components to survive and assemble more virions, combating infection. 

     Preliminary results have shown, in short, that the DVD-Igs are localizing successfully within the lysosome, and that the DVD-Ig targeting the cleaved glycoprotein is fully protective against the human Sudan Ebola virus in a model system.  Although more work needs to be carried out before such a therapy is "perfected" and could be used on the human population, the recent work has demonstrated that broad protection against ebolaviruses is a strong possibility.  If this is a possibility, then this also demonstrates that broad protection against other infectious families of viruses is a possibility... and that building a shield against epidemics is a likelihood. 

__________________________________________________________

Primary Article: 

A. Z. Wec et al. (2016).  A "Trojan horse" bispecific antibody strategy for broad protection against 

     ebolaviruses.  Science 10.1126/science.aag3267.  

Works Cited:

1.  http://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/index.html 

2.  http://www.who.int/mediacentre/factsheets/fs103/en/

3.  http://www.cdc.gov/vhf/ebola/about.html

4.  Qui et al., (2014).  Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp.  Nature 514: 47-53.  doi:10.1038/nature13777.

5.  http://www.newswise.com/articles/researchers-find-key-used-by-ebola-virus-to-unlock-cells-and-spread-deadly-infection

Links found within text:

http://www.bbc.com/news/world-africa-28755033

http://www.cdc.gov/vhf/ebola/resources/virus-ecology.html

http://www.virology.ws/2010/07/22/the-virus-and-the-virion/

http://www.ncbi.nlm.nih.gov/books/NBK9831/

https://micro.magnet.fsu.edu/cells/endosomes/endosomes.html

http://hyperphysics.phy-astr.gsu.edu/hbase/biology/lysosome.html
http://www.nature.com/nature/journal/v477/n7364/full/nature10348.html

https://www.youtube.com/watch?v=BHQUp-R0q9U

http://www.nature.com/nature/journal/v514/n7520/full/nature13777.html

http://www.ncbi.nlm.nih.gov/pubmed/22735951

New method of vaccine delivery?

http://www.youtube.com/watch?v=BGRy5VU-LfI

Researchers take advantage of immune cells in the skin and solve some major roadblocks to vaccine delivery. Could this be applied to the mucosa? Could this replace the needle and syringe?

Tony Song