The study of antiviral drug resistance is focused on two HCMV genes: the viral protein kinase gene encoded by the UL97 open reading frame (ORF) and the DNA polymerase gene encoded by the UL54 ORF. These gene products are the targets of all of the licensed systemic antiviral drugs: ganciclovir (and the valine ester valganciclovir), foscarnet, and cidofovir. We have an ongoing study of clinical isolates to define all potential resistance mutations in these two genes, and to determine the conditions under which these mutations arise. There are new antiviral agents, in particular maribavir, which are not yet approved by the FDA, which have different mechanisms of action from the approved drugs. The targets of these new drugs will have to be determined. Resistance mutations will undoubtedly arise and will have to be identified. We intend to apply our knowledge of the HCMV genome and sequencing capabilities to pursue the targets of these new antiviral agents.

Our studies of HCMV pathogenesis have focused on ORFs, which encode potential immunomodulatory functions including UL144, UL146, and UL147, but which have highly variable sequences among clinical strains. Our large repository of clinical strains has provided material for our initial studies. We also receive direct patient buffy coat specimens, which allow us to look at these sequences in viral genomes that have not been subject to cell culture. The absence of cell culture eliminates the possibility of selection for viral subpopulations that have altered cell tropism.

Our most recent work related to pathogenesis is the development of a cervical tissue explant model, which allows us to study HCMV in intact tissue containing cells of the immune system captured at the time of tissue acquisition. In addition to studying HCMV infection and replication in this model, it is possible to study co-infection with other viruses such as HIV-1. Quantitation of viral genomes in the explants and supernatant fluid demonstrate the relative effects on replication of each virus.

From our need for quantitative assays, we have developed expertise in real-time PCR assays. We have developed an in-house assay for HCMV. As an extension of this work, we are also involved in collaborative work with the VQA Lab. Commercial and in-house assays for quantitation and genotyping of other HIV-1-related co-pathogens such as hepatitis B virus (HBV) and hepatitis C virus (HCV) are the focus of these comparative studies.

Tatarowicz, W.A., N.S. Lurain, and K.D. Thompson. In situ ELISA for antiviral susceptibility testing of human cytomegalovirus. 1991. J. Virol. Methods 35:207-215.

Tatarowicz, W.A., N.S. Lurain, and K.D. Thompson. 1992. A ganciclovir-resistant clinical isolate of human cytomegalovirus exhibiting cross-resistance to other DNA polymerase inhibitors. J. Infect. Dis.166:904-907.

Lurain, N.S., K.D. Thompson, E.W. Holmes, and G.S. Read. 1992. Point mutations in the DNA polymerase gene of human cytomegalovirus resulting in resistance to antiviral agents. J. Virol. 66:7146-7152.

Lurain, N.S., L.E. Spafford, and K.D. Thompson. 1994. A mutation in the UL97 open reading frame of human cytomegalovirus strains resistant to ganciclovir. J. Virol. 68:4427-4431.

Spear, G.T., N.S. Lurain, C.J. Parker, M. Ghassemi, G.H. Payne and M. Saifuddin. 1995. Host cell-derived complement control proteins CD55 and CD59 are incorporated into the virions of two unrelated enveloped viruses; Human T cell leukemia/lymphoma virus type-I (HTLV-I) and human cytomegalovirus (HCMV). J. Immunol. 155:4376-4381.

Lurain, N.S., H.C. Ammons, K.S. Kapell, V.V. Yeldandi, E.R. Garrity, and J.P. O'Keefe. 1996. Molecular analysis of human cytomegalovirus strains from two lung transplant recipients with the same donor. Transplantation 62:497-502.

McSharry, J.J., N. Lurain, A. Landay, J. Manischewitz, M. Nokta, M. O'Gorman, H. Shapiro, A. Weinberg, P. Reichelderfer, and C. Crumpacker. 1998. Flow cytometric determination of ganciclovir susceptibility of human cytomegalovirus (HCMV) clinical isolates. J. Clin. Microbiol. 36:958-64.

McSharry, J.J., N.S. Lurain, G.L. Drusano, A. Landay, M. Nokta, M. O'Gorman, A. Weinberg, H.M. Shapiro, P. Reichelderfer, and C. Crumpacker, C. 1998. A rapid flow cytometric drug susceptibility assay. Antimicrob. Agents Chermother 42:2326-2361.

Chou, S., N.S. Lurain, A. Weinberg, G.-Y. Cai, P.C. Sharma, and C.S. Crumpacker. 1999. Analysis of interstrain variation in the human cytomegalovirus DNA polymerase sequence and its effect on the genotypic diagnosis of antiviral drug resistance. Antimicrob. Agents Chemother. 43:1500-1502.

Benedict, C.A., K.D. Butrovich, N.S. Lurain, J. Corbeil, I. Rooney, P. Schneider, J. Tschopp, and C.F. Ware. 1999. A novel viral TNF receptor superfamily member in virulent strains of human cytomegalovirus. J. Immunol. 162:6967-6970.

Waldman, W.J., D.A. Knight, N.S. Lurain, D.M. Miller, D.D. Sedmak, J.W.Williams, A. S-F. Chong. 1999. Novel mechanism of inhibition of human cytomegalovirus by the experimental immunosuppressive agent leflunomide. Transplantation. 68:814-825.

Lurain, N.S., K.S. Kapell, D.D. Huang, J.A. Short, J. Paintsil, E. Winkfield, C.A. Benedict, C.F. Ware, and J.W. Bremer. 1999. The human cytomegalovirus UL144 open reading frame: sequence hypervariability in low-passage clinical isolates. J. Virol. 73:10040-10050.

Landry, M.L., S. Stanat, K. Biron, D. Brambilla, W. Britt, J. Jokela, S. Chou, W.L. Drew, A. Erice, B. Gilliam, N. Lurain, J. Manischewitz, R. Miner, R. Pollard, P. Reichelderfer, S. Spector, A. Weinberg, B. Yen-Lieberman, and C. Crumpacker. 2000. Standardized plaque reduction assay for determination of drug susceptibilities of cytomegalovirus clinical isolates. Antimicrob. Agents Chemother. 44:688-692.

Waldman, W.J., D.A. Knight, L. Blinder, J. Shen, N.S. Lurain, D.M. Miller, D.D. Sedmak, J.W. Williams, and A. S.-F. Chong. 2000. Inhibition of cytomegalovirus in vitro and in vivo by the experimental immunosuppressive agent leflunomide. Intervirology. 42:412-418.

Caliendo, A., R. Schuurman, B. Yen-Lieberman, S.A. Spector, J. Anderson, R. Manjiry, C. Crumpacker, N. Lurain, and A. Erice. 2001. Comparison of quantitative and qualitative PCR assays for CMV DNA in plasma. J. Clin. Micro. 39:1334-1338.

Lurain, N.S., A. Weinberg, C.S. Crumpacker, and S. Chou. 2001. Sequencing of the cytomeglaovirus UL97 gene for genotypic antiviral resistance testing. Antimicrob. Agents Chemother. 45:2775-2780.

Bhorade, S.M., N.S. Lurain, A. Jordan, J. Leischner, J. Villanueva, R. Durazo, W.T. Vigneswaran, and E.R. Garrity. 2002. Emergence of ganciclovir resistant cytomegalovirus in lung transplant recipients. J. Heart Lung Transplant 12:1274-1282.

Isada, C.M., B. Yen-Lieberman, N.S. Lurain, R. Schilz, D. Kohn, D.L. Longworth, A.J. Taege, S.B. Mossad, J. Maurer, S.M. Flechner, S.D., Mawhorter, W. Braun, S.M. Gordon, S.K. Schmitt, M. Goldman, J. Long, M. Haug, R.K. Avery. 2002. Clinical characteristics of 13 solid organ transplant recipients with ganciclovir-resistant cytomegalovirus infection. Transpl. Infect. Dis. 4:189-194.

Lurain, N.S., S.M. Bhorade, K.J. Pursell, R.K Avery, V.V. Yeldandi, C.M. Isada, E.S. Robert, D.J. Kohn, M.Q. Arens, E.R. Garrity, A.J. Taege, M.G. Mullen, K.M. Todd, J.W. Bremer, and B. Yen-Lieberman. 2002. Analysis and characterization of antiviral drug-resistant cytomegalovirus isolates from solid organ transplant recipients. J. Infect. Dis. 186:760-768.

Wolf, D.G., N.S. Lurain, J. Satinger, A. Honigman, N. Saleh, Z. Kra-Oz. 2003. Emergence of late cytomegalovirus central nervous system disease in hematopoietic stem cell transplant recipients. Blood 101:463-465

Weinberg, A., D.A. Jabs, S. Chou, B.K. Martin, N.S. Lurain, M.S. Forman, C. Crumpacker. 2003. Mutations conferring foscarnet resistance in a cohort of patients with acquired immunodeficiency syndrome and cytomegalovirus retinitis. J. Infect. Dis. 186:777-784.

Chou, S., N.S. Lurain, K.D. Thompson, R.C. Miner, W. Lawrence Drew. 2003. Viral DNA polymerase mutations associated with drug resistance in human cytomegalovirus. J. Infect. Dis. 188:32-39.

Caliendo, A.M., B. Yen-Lieberman, J. Baptista, J. Andersen, C. Crumpacker, R. Schuurman, S.A. Spector, J. Bremer, and N.S. Lurain. 2003. Comparison of molecular tests for the detection and quantification of cell-associated CMV DNA. J. Clin. Microbiol. 41:3509-3513.

Baldanti, F., N. Lurain, and G. Gerna. 2004. Human cytomegalovirus resistance to antiviral drugs. Human Immunology 65:403-409.

Blackman, S.C., N.S. Lurain, D.P. Witte, A.H. Filipovich, P. Groen, and M.R. Schleiss. 2004. Emergence and compartmentalization of fatal multi-drug-resistant cytomegalovirus infection in a patient with autosomal-recessive severe combined immune deficiency. J. Pediatr. Hematol. Oncol. 26:601-605.

Lurain, N.S., E.S. Robert, J. Xu, M. Camarca, A. Landay, A.A. Kovacs, and P.S. Reichelderfer. 2004. Human immunodeficiency virus type 1 and cytomegalovirus co-infection in the female genital tract. J. Infect. Dis. 190:619-623.

Avery, R.K., B. Bolwell, B. Yen-Lieberman, N. Lurain, W.J. Waldman, D.L. Longworth, A.J. Taege, S.B. Mossad, D. Kohn, J. Long, J. Curtis, M. Kalaycio, B. Pohlman, J. Williams. 2004. Use of leflunomide in an allogeneic bone marrow transplant recipient with refractory cytomegalovirus infection. Bone Marrow Transplant. 34:1071-1075.

Zariffard, M.R., R. Novak, N. Lurain, B.E. Sha, P. Graham, and G .T. Spear. 2005. Induction of TNF-α secretion and expression of toll-like receptors 2 and 4 by genital tract secretions from women with bacterial vaginosis. J. Infect. Dis. 191:1913-21.

Cheung, T.C., I.R. Humphreys, K.G. Potter, P.S. Norris, H.M. Shumway, B.R. Tran, G. Patterson, R. Jean-Jacques, M. Yoon, P.G. Spear, K.M. Murphy, N.S. Lurain, C.A. Benedict, C.F. Ware. 2005. Evolutionarily divergent herpesviruses modulate T cell activation by targeting the herpesvirus entry mediator cosignaling pathway. Proc. Natl. Acad. Sci. 102:13218-13223.

Lurain, N.S., A.M. Fox, H.S. Lichy, S.M. Bhorade, C.F Ware, D.D. Huang, S.-P. Kwan, E.R. Garrity, and S. Chou. 2006. Analysis of the human cytomegalovirus genomic region from UL146 through UL147A reveals sequence hypervariability, genotypic stability, and overlapping transcripts. Virol. J. Jan 12;3:4 .

Caliendo, A.M., A. Valsamakis, Y. Zhou, B. Yen-Lieberman, J. Andersen, S. Young, A. Ferreira-Gonzalez, G.J. Tsongalis, R. Pyles, J.W. Bremer, and N.S. Lurain. 2006. Multilaboratory comparison of hepatitis C virus viral load assays. J. Clin. Microbiol. 44:1726-1732.

Chapters:
Caliendo, A.M. and N.S. Lurain. Cytomegalovirus. 2005. In: Molecular Diagnostics for the Clinical Laboratorian, 2^nd edition. W.B. Coleman and G.J. Tsongalis, ed. Humana Press, Totawa, NJ.

Lurain, N.S. and K.D. Thompson. Antiviral Agents. In: Manual of Clinical Microbiology, 9^th Edition. (In press, 2006, ASM Press)