Sunday, September 25, 2016

Does Resistance to One Antiviral Indicate Resistance to Another

            Infection by the Hepatitis B virus causes the liver disease Hepatitis B, which can lead to liver failure and cancer.  A simple vaccine can reduce chances of infection, but in places with widespread poverty many people can’t afford the cost of a vaccine.  Subsequently, in such places infection rates remain consistently high leading to an estimated 240 million people worldwide who are chronically infected and 686,000 people who die as a result of Hepatitis B every year.[1]  Due to the epidemic of chronically infected Hepatitis B patients, antiviral drugs have been developed to help prevent the onset of Hepatitis B.  One particular type of antiviral is called a nucleoside analogue.  Such antivirals work by inhibiting viral replication through the use of an alternative DNA subunit, which can be incorporated into viral DNA and subsequently blocks a virus’ own replication machinery.[2]  Nucleoside analogues are exceedingly useful therapies until mutations in the virus’ genetic material causes resistance to the antivirals. 
            A paper[3] published in the journal Nature in July of 2016 by Liu et al. details the usefulness of a specific nucleoside analogue, Entecavir, for treatment of a chronic Hepatitis B infection.  The researchers examined the effectiveness of Entecavir treatment in participants who had never been treated with a nucleoside analogue compared to those who had been treated with one, either lamivudine (LAM) or adefovir dipivoxil (ADV).  The study was conducted over 5 years on 89 individuals from China who were infected with Hepatitis B for at least 6 months and were only treated with Entecavir.  Statistical analysis was used to determine if Entecavir lead to viral remission as associated with prior treatment with LAM, prior history of LAM-resistance, prior history of partial virus resistance to ADV, and prior history of primary treatment failure to ADV. 
            The results of the study indicated that anyone who had never been treated with a nucleoside analogue before exhibited complete viral remission with only a minimal chance of developing resistance.  This is opposed to patients previously treated with a nucleoside analogue, who still responded well to Entecavir but at a lower rate of remission.  The sample group was further characterized by experience with LAM and ADV.  Interestingly, individuals previously treated with LAM, with or without developing resistance, were successfully treated for Hepatitis B, as were ADV patients with partial resistance.  However, ADV-experienced patients who had failed previous treatment had a significantly reduced probability of achieving viral remission when treated with Entecavir. 
            The study by Liu et al. provides important support for reducing antiviral resistance as a result of overuse.  Chronic Hepatitis B can be effectively treated with antivirals, like Entecavir, but for those who failed the first line treatment, doctors should carefully choose the next course of therapy.  By not just prescribing any antiviral when someone has an infection, hopefully the current issue with widespread antibiotic resistance can be avoided, or at least minimized, in regards to antivirals.  The results of the paper strongly support conscientious and judicious use of antiviral drugs.  
            The treatment distinction suggested by Liu et al. for patients with different histories is also indicative of the future of medicine.  Previous means of treating a disease involved a one-size fits all approach.  For example, the paper discusses how ADV was the primary means of treatment for chronic Hepatitis B in China for the past decade.  If a patient was diagnosed with Hepatitis B, he or she was most likely given ADV without deliberation.  However, as the results of the study indicated, such an approach is not effective due to acquired resistance.  Thus, it is important for those in the healthcare profession to consider a patient’s personal history prior to treatment in order to successfully treat an infection, instead of relying on a generalized approach. 
In order to facilitate the growth of personalized medicine, basic research into the conditions affecting the success of a particular treatment is necessary.  Liu et al. provided important information on a specific drug, but the study was limited by size, as acknowledged by the researchers.  Future research should be more wide spread, meaning more antivirals examined in a larger number of participants with different conditions.  Such work would allow for the development of a comprehensive set of guidelines regarding the use of antivirals.  Analysis of a large set of treatment results could be the beginning of a global initiative for stemming antiviral resistance by providing personalized treatment.

[1] http://www.who.int/mediacentre/factsheets/fs204/en/
[2] https://livertox.nlm.nih.gov/NucleosideAnalogues.htm
[3] Liu, K. et al. A five years study of antiviral effect of entecavir in Chinese chronic hepatitis B patients. Sci. Rep. 6, 28779; doi: 10.1038/srep28779 (2016).

Zika Virus Causes Problems for Monkeys, Too

Zika virus is currently a worldwide concern. Spread by mosquitoes and through sexual contact, the virus has been highlighted in the news since May 2015, when it was first detected in Brazil.i Since then, it has taken the world by storm: as of September 22, 2016, it has affected 47 countries/territories in North and South America, with over 275,000 suspected or confirmed cases in Brazil alone.ii (To view the interactive map, please click here.) With all of the fear and news coverage surrounding the outbreak, researchers are racing to learn more about this critical illness. A new paper, published on September 12, 2016, claims to have some of the answers.
Zika virus infection is often asymptomatic, but for pregnant individuals, the consequences can be devastating. Zika virus is associated with microcephaly (small head) and other fetal malformations.iii In addition, Guillain-Barré Syndrome, a type of autoimmune-induced muscle weakness, has been associated with Zika infection.iii In this research article, Adams Waldorf et al. (2016) primarily investigated the virus’s effects on fetal brain development. For this experiment, the team injected a Cambodian strain of the virus (strain FSS13025, Cambodia 2010) into five locations on the forearms of a pregnant pigtailed macaque monkey (see image of pigtailed macaque below). The monkey was had been pregnant for 119 days, approximately equivalent to 28 weeks of human pregnancy. The fetus was viewed by ultrasound weekly, and cesarean section was performed 43 days later (equivalent to 38 weeks in human pregnancy). The results were simply astonishing.

Within 10 days of the virus injection, the developing fetus began to encounter problems. The fetus showed lesions (damaged areas) on its brain, which evolved differently on the left and right hemispheres. In the left hemisphere, the fetus showed loss of brain volume, and even ventricular collapse (complete destruction of a ventricle, a hollow area within the brain). In the right hemisphere, the lesions simply increased in severity over time. White matter, one of the types of brain tissue consisting mainly of nerve fibers, stopped growing over time, while gray matter, another type of brain tissue consisting mainly of nerve cell bodies, continued to grow. The picture below shows some of this damage, indicated by red arrows (the numbers at the top signify the number of days after injection/the number of days since conception).

After cesarean section, the fetus was autopsied for further investigation. Evidence of the virus was detected in the brain, which showed significant underdevelopment of white matter. Brain lesions were observed throughout. These findings are very similar to reports of Zika virus in human fetuses, as seen by magnetic resonance imaging (MRI) after birth.
As groundbreaking as this study is, there are several flaws in its design. Only a single subject was investigated, and this subject was a pigtail macaque monkey. Meanwhile, a previous study conducted on rhesus macaques failed to produce similar results.iv  Therefore, the result of this pigtail macaque study could be coincidental or unrepresentative of typical Zika effects. Also, this study used five injection sites, giving a very high dose of the virus, which may not be reflective of an ordinary bite from a virus-carrying mosquito.
Future studies should include larger sample sizes, and should include several different types of macaque monkeys or similar species. In addition, future research could explore the mechanism causing the observed symptoms. How does the virus cause damage to the developing fetus? Lastly, more research needs to be conducted to determine if the pigtailed macaque could be a good model on which to test new medications for Zika virus. If pigtailed macaque monkeys respond very similarly to humans, then perhaps potential new vaccines could be tested on them, before the vaccines move to human trials.

Paper: Waldorf, K. M. A., Stencel-Baerenwald, J. E., Kapur, R. P., Studholme, C., Boldenow, E., Vornhagen, J., ... & Armistead, B. (2016). Fetal brain lesions after subcutaneous inoculation of Zika virus in a pregnant nonhuman primate. Nature Medicine.

Other sources:
iBBC News (August 31, 2016). Zika outbreak: What you need to know. Retrieved September 25, 2016, from http://www.bbc.com/news/health-35370848
iiEpidemic Diseases - Zika in the Americas. (September 22, 2016). Retrieved September 25, 2016, from http://ais.paho.org/phip/viz/ed_zika_countrymap.asp
iiiWorld Health Organization. 2016. Zika virus, microcephaly and Guillain-Barré syndrome. World Health Organization, Geneva, Switzerland. Retrieved September 25, 2016 from http://apps.who.int/iris/bitstream/10665/204961/1/zikasitrep_7Apr2016_eng.pdf?ua=1
ivDudley, D. M., Aliota, M. T., Mohr, E. L., Weiler, A. M., Lehrer-Brey, G., Weisgrau, K. L., ... & Gellerup, D. D. (2016). A rhesus macaque model of Asian-lineage Zika virus infection. Nature communications, 7.

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.

Saturday, September 24, 2016

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.

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.

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

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

More on Interferon: