Is Coinfection with Herpesvirus Responsible for Acyclovir Phosphorylation in Enterovirus Treatment?Angela Sowers

Department of Biological Sciences, York College of Pennsylvania

Introduction•In recent years, numerous outbreaks of enterovirus 71 (EV 71) have occurred in the Asia-Pacific region (Qui 2008).•EV 71 can result in HFMD (hand-foot-and-mouth disease), encephalitis, aseptic meningitis, upper respiratory illnesses, and paralysis (Shors 2009).•There is no FDA-approved antiviral for the treatment of enteroviruses (Chen et al. 2008).•Acyclovir, an antiviral used to treat herpesvirus, has successfully treated patients with enterovirus in studies conducted by Faulkner et al. (2005) and Shelley et al. (1988).•Acyclovir is activated by thymidine kinase, an enzyme found only in herpesvirus; enterovirus does not code for a thymidine kinase.•Recent studies have been conducted concerning acyclovir treatment for HIV, in which researchers showed that acyclovir only inhibited HIV replication in patients who were coinfected with herpesvirus.•It is possible that patients with enterovirus may be successfully treated with acyclovir if they are already infected with herpesvirus.

*Asia –Pacific region is in red

http://tpe-u.com/tpu/emea/en/aboutus/contact_persons/docId-2474054/Ansprechpartner_APAC.html?docPart=0

Objectives•To determine whether or not EV 71 phosphorylates acyclovir•To determine if acyclovir phosphorylated by HHV-6B (human herpesvirus 6B) inhibits EV 71 replication

Literature CitedChen, T., Weng, K., Chang, S., Lin, J., Huang, P. and Shih, S. 2008. Development of

antiviral agents for enteroviruses. Journal of Antimicrobial Chemotherapy 62: 1169-1173.

Elion, G.B. 1993. Acyclovir: discovery, mechanism of action, and selectivity. Journal of Medical Virology Supplement 1: 2-6.

Faulkner, C.F., Godbolt, A.M., DeAmbrosis, B. and Triscott, J. 2005. Hand, foot, and mouth disease in an immunocompromised adult treated with aciclovir. Australasian Journal of Dermatology 44: 203-206.

Lisco, A., Vanpouille, C., Tchesnokov, E.P., Grivel, J., Biancotto, A., Brichacek, B., Elliott, J., Fromentin, E., Shattock, R., Anton, P., Gorelick, R., Balzarini, J., McGuigan, C., Derudas, M., Götte, M., Schinazi, R.F. and Margolis, L. 2008. Acyclovir is activated into a HIV-1 reverse transcriptase inhibitor in herpesvirus-infected human tissues. Cell Host and Microbe 4: 260-270.

Monpoeho, S., Mignotte, B., Schwartzbrod, L., Marechal, V., Nicolas, J.C., Billaudel, S. and Ferre, V. 2000. Quantification of enterovirus RNA in sludge samples using single tube real-time RT-PCR. Biotechniques 29: 88-93.

Qui, J. 2008. Enterovirus 71 infection: a new threat to global public health? The Lancet Infectious Diseases 7: 868-869.

Shelley, W.B., Hashim, M. and Shelley, E.D. 1996. Acyclovir in the treatment of hand-foot-and-mouth disease. Cutis 57: 232-234.

Shors, T. 2009. Understanding Viruses. Jones and Bartlett Publishers, Sudbury, MA.

AcknowledgementsDr. Mathur, for her guidance and patience throughout this project.

Research Design

Set up tissue cultures: EV-71 only,HHV-6B only, coinfected culture

(EV-71 & HHV-6B)

Treat all tissue cultures with ACV(4.8 µg/day for 3 days)

Monitor ACV phosphorylation bydetecting the presence of ACV-MP

and ACV-TP (LC-MS/MS)

Monitor EV-71 replication in coinfected culture (Real-Time PCR)

Expected ConclusionHHV-6B must be present in order to phosphorylate acyclovir and cause it to inhibit EV-71 replication. Clinically, this would mean a patient would have to be infected with herpesvirus for successful treatment of enterovirus with acyclovir.

Expected Results

Figure 3: Example of a real-time PCR plot measuring EV-71 replication in the coinfected culture treated with ACV. EV-71 replication will decrease with each treatment over the course of three days.

Review of Literature•Acyclovir (ACV) is a guanine-like compound used to inhibit DNA polymerase, an enzyme necessary for DNA biosynthesis.

http://en.wikipedia.org/wiki/Aciclovir

http://www.scd.co.kr/upload/medicine/

•Initially, acyclovir is phosphorylated by a viral thymidine kinase (found in herpesvirus) and becomes acyclovir monophosphate (ACV-MP).•ACV is further phosphorylated by host cell enzymes, forming acyclovir triphosphate (ACV-TP); this is the active form that will incorporate itself into the DNA and prevent replication (Elion 1993).

•Recently, Lisco et al. (2008) tested the effectiveness of acyclovir treatment on HIV by treating infected tonsil tissue received from donors. They discovered that acyclovir inhibited HIV replication in these tissues. The researchers knew that HIV does not code for a thymidine kinase. Noting this, they tested their tonsil tissues for herpesvirus (HHV’s 1-8) and found that the donated tonsil tissues were all infected with at least one form of herpesvirus (mostly HHV-6). The researchers concluded that HHV must be present for successful acyclovir treatment of HIV.

Infect tonsillar tissue blocks with either EV-71 or HHV-6B

Determine viral load in eachtissue block (Real-Time PCR)

Expected Results

Figure 2. Example of LC-MS/MS (Liquid Chromatography Tandem Mass Spectrometry) plot showing both the presence of ACV-MP and ACV-TP. These peaks are only expected in HHV-6B cultures and the coinfected cultures because ACV must be monophosphorylated by a viral thymidine kinase. Cellular enzymes take over after initial phosphorylation.