Successful valganciclovir treatment of post‐transplant cytomegalovirus infection in the presence of UL97 mutation N597D

Mutations in the human cytomegalovirus (CMV) UL97 protein kinase are the most common mechanism of ganciclovir (GCV) resistance in the clinical setting. A CMV strain with a previously unrecognized UL97 mutation N597D was identified in the blood of a heart transplant recipient who experienced a persistent CMV infection with high viral loads accompanying pain and fever while receiving valganciclovir (valGCV) therapy. The N597D mutation was transferred by mutagenesis to an antiviral sensitive CMV strain for analysis of antiviral susceptibility by standardized phenotypic assay. Recombinant phenotyping showed N597D conferred a less than twofold increase in GCV IC₅₀ compared to the sensitive control strain. Despite the presence of this mutation, valGCV eventually resolved the infection after 6 weeks of therapy. A subsequent CMV reactivation was also responsive to valganciclovir. This case illustrates the diversity of UL97 mutations in the codon segment 590–607 usually associated with GCV resistance, with some mutations producing minimal levels of resistance that do not preclude a therapeutic response to the drug. Accurate interpretation of genotypic test results ultimately requires experimental determination of the level of resistance conferred by newly discovered UL97 mutations. J. Med. Virol. 81:507–510, 2009. © 2009 Wiley‐Liss, Inc.     

Evidence that the human cytomegalovirus 46-kDa UL72 protein is not an active dUTPase but a late protein dispensable for replication in fibroblasts

The Human Cytomegalovirus (HCMV) UL72 gene is considered to be the equivalent of the dUTPase gene of the Alpha- and Gamma-herpesviruses. To characterize its function, the expression profiles of UL72 at both the RNA and the protein level were determined. The gene is expressed with a late kinetics and the corresponding UL72 46-kDa protein accumulates late during infection in the cytoplasm of infected cells. The pUL72 was expressed in E. coli and the purified recombinant protein did not display a detectable dUTPase activity. The viral yields of reconstituted HCMV RVDeltaUL72 viruses carrying a deletion within the UL72 ORF demonstrated a moderate growth defect following low MOI infections, whereas their DNA synthesis profiles were not significantly different from those of the parental HCMV RVAD169. These results demonstrate that the UL72 gene product is not a dUTPase and is not essential for replication in human fibroblasts.     

应用非标记探针高分辨率熔解曲线技术检测人巨细胞病毒UL97基因突变

 目的 建立非标记探针高分辨率熔解曲线分析技术（HRMA）检测人巨细胞病毒（HCMV）UL97基因突变。方法 构建HCMV UL97野生型以及H520Q突变质粒，非标记探针HRMA分型技术检测H520Q突变。结果 直接从PCR产物得到的熔解曲线峰型无法区分野生型及H520Q突变型，应用非标记探针HRM A技术可以明显增加实验的敏感性及准确性，可以成功区分从野生型到含不同量的H520Q突变的基因型。结论 非标记探针HRMA 技术是一个敏感、经济的检测H520Q突变的基因分型方法。     

Restriction to human cytomegalovirus replication in vitro removed by physiological levels of cortisol

Since viral infection is in most cases contrary to the survival of the host cell, it is reasonable to assume that cells possess innate viral replication inhibitory mechanisms. Even between strains of permissive cells, degrees of permissiveness are observed. Restriction to human cytomegalovirus (CMV) replication in vitro is well known, especially in epithelioid cells or cells derived from certain organs. We have studied restriction in a fibroblastic strain of human embryonic kidney cells. By treatment of cell cultures with maximum physiologic concentration of the hormone cortisol (25 micrograms%) both pre and post virus inoculation, susceptibility to laboratory strain Ad169 CMV and low-passaged clinical isolate JSS CMV was enhanced by factors of 6.4 +/- 0.7 and 11.1 +/- 0.4, respectively; effectively converting these cells to a totally permissive state. A linear dose response, which peaks at 25 micrograms% and declines thereafter up to 300 micrograms%, is evident for both virus strains in this enhancement system. Breakdown of restriction increases in linear fashion with increasing time of cortisol pretreatment of cells. The characterization of cortisol effects converting restrictive human fetal cells in vitro to the permissive state further indicates that human hormones may play a significant role in CMV susceptibility in vivo.     

Quantitative changes in cytomegalovirus DNAemia and genetic analysis of the UL97 and UL54 genes in AIDS patients receiving cidofovir following ganciclovir therapy.

Five AIDS patients with cytomegalovirus (CMV) retinitis who had received ganciclovir (GCV) therapy were followed with serial blood sampling to detectchanges both in CMV load and in the genetic composition of genes UL97 and UL54 whilst receiving cidofovir (CDV) therapy. CDV neither reduced CMV load in blood nor prevented its quantitative resurgence during therapy. These effects were not explained by the initial presence or development of CDV-associated drug resistance mutations in UL54. In two patients, UL97 genotypic resistance to GCV involving either a L595S mutation or a deletion of amino acids 590-603 were present at the initiation of CDV and, in both patients, repopulation of CMV strains with wild-type UL97 sequences occurred during CDV therapy. These data are consistent with GCV-resistant strains containing UL97 mutations being less fit than their wild-type counterparts and so being able to persist only with the selective pressure of GCV.     

Rapid detection by reverse hybridization of mutations in the UL97 gene of human cytomegalovirus conferring resistance to ganciclovir.

BACKGROUND OF STUDY: Diseases due to human cytomegalovirus (HCMV) infection constitute a major threat in marrow and solid organ transplant recipients. Ganciclovir (GCV) is widely used in prophylaxis and pre-emptive therapy of active HCMV infection. Resistance to ganciclovir (GCV) may arise at variable frequency under GCV therapy and is conferred by mutations (i) in the UL97 gene (codons 460, 520, and 591-607) encoding a phosphotransferase which is essential for monophosphorylation of GCV and, to a lesser extent, (ii) in the UL54 gene coding for the DNA polymerase of HCMV. OBJECTIVE: The purpose was to develop a rapid assay to screen for emerging GCV resistance mutations in the UL97 gene of HCMV whereby avoiding virus isolation and nucleotide sequencing procedures. STUDY DESIGN: A nested PCR (nPCR) amplifying UL97 codons 450-672 was developed. Nested amplicons were subsequently sequenced directly. Oligonucleotides for use in a reverse hybridization assay were designed to detect relevant non-synonymous mutations at codons UL97 460, 520, 603 and 607. Strain AD169 served as a wild-type control. RESULTS: UL97-specific nPCR amplicons were obtained from 18 EDTA blood samples of ten transplant recipients receiving GCV for more than 30 days. In three consecutive samples from a single patient a GCV resistance mutation at codon 603 (C-->W) was detected. In addition, two out of four cell culture-adapted HCMV isolates known to exhibit GCV resistance in vitro revealed mutations at codons 460 (M-->V) and 607 (C-->Y), respectively. By reverse hybridization a discrimination of single nucleotide changes at codons 460, 520, 603 and 607 was possible whereby matching exactly the results of the nucleotide sequence analysis for all 23 amplicons examined. CONCLUSIONS: Reverse hybridization appeared to be a rapid and convenient alternative to nucleotide sequencing when screening the UL97 gene of HCMV for selected markers of GCV resistance.     

Analysis of cytomegalovirus DNA polymerase (UL54) mutations in solid organ transplant patients receiving valganciclovir or ganciclovir prophylaxis

We previously reported the absence of CMV UL97 (kinase) gene resistance mutations up to 12 months post-transplant following 100 days of valganciclovir prophylaxis, and a low incidence of resistance mutations following 100 days of oral ganciclovir prophylaxis in a prospective multicenter study in solid organ transplant recipients excluding lung transplants. Herein, we report UL54 (DNA polymerase) gene sequencing results for all patients with previous UL97 PCR-positive samples (n = 99) in our study. One UL54 resistance mutation (L545S known to confer ganciclovir and cidofovir resistance) was detected in a routine day-100 sample from an asymptomatic patient who received oral ganciclovir. Notably, this CMV UL54 mutation occurred in the absence of a UL97 mutation. Additionally, new UL54 variants were observed. Thus, emergence of CMV UL54 mutations in the absence of UL97 mutations is a rare but possible event that is not necessarily associated with detrimental clinical outcome in solid organ transplant recipients.     

Confirmation of the low clinical effect of human herpesvirus-6 and -7 infections after renal transplantation

Human herpesvirus‐6 and ‐7 (HHV‐6 and HHV‐7) may lead to pathological manifestations in renal transplant recipients. The aim of this study was to investigate beta‐herpesvirus infections in 50 adult kidney transplant recipients after transplantation to examine the effect, interactions, and pathogenic consequences of infection and the effect of immunosuppressive regimens and Human cytomegalovirus (HCMV) prophylaxis with VACV. Beta‐herpesviruses loads in the blood of 50 adult kidney transplant recipients over a 6‐month period after transplantation and 198 blood donors were determined using polymerase chain reaction. The rate of HHV‐6 detection in peripheral mononuclear cells (PBMCs) was higher in patients with end‐stage renal disease and during the post‐transplantation follow‐up than in healthy subjects (33% and 68% vs. 12%, respectively). The detection rate of HHV‐7 in PBMCs was similar between patients, both before grafting and during the follow‐up for transplant recipients (69% and 88%, respectively), and healthy subjects (78%), and correlated with the number of lymphocytes. HCMV in plasma was detected only in patients during the post‐transplant period (24%). VACV prophylaxis had no negative effect on the replication of HHV‐6 or HHV‐7, and univariate analyses demonstrated associations between HHV‐6 infection and acute graft rejection [Odds ratio (OR) = 2.94, 95% confidence interval (CI), 1.05–8.2, P = 0.04], and between HHV‐7 infection and cholestasis [OR = 2.61 (95% CI, 1.08–6.3), P = 0.03]. Immunosuppressive regimens had no effect on beta‐herpesviruses infections. This study revealed the differing behavior of HCMV, HHV‐6, and HHV‐7 in kidney transplant recipients, and confirmed the association of HHV‐6 with graft rejection. J. Med. Virol. 84:450–456, 2012. © 2011 Wiley Periodicals, Inc.     

The HLA-G 14 bp insertion/deletion polymorphism is a putative susceptible factor for active human cytomegalovirus infection in children

Human leukocyte antigen-G (HLA-G) expression is a potential factor for the pathogenesis of virus infection. A 14 bp insertion/deletion polymorphism (rs16375) in the 3'-untranslated region of the HLA-G gene is involved in the stability of HLA-G mRNA and HLA-G protein expression. Therefore, the HLA-G 14 bp polymorphism might be involved in human cytomegalovirus (hCMV) infection. To test a possible association between the HLA-G 14 bp deletion/insertion polymorphism and the active hCMV infection, in this study, a total of 54 patients with active hCMV infection and 165 age- and sex-matched, unrelated, normal Chinese Han population were genotyped for the 14 bp insertion/deletion polymorphism. Association of 14 bp polymorphism with hCMV urine DNA copies and the odds ratio (OR) of the polymorphism as a risk factor for active hCMV infection were analyzed. Our results showed that the prevalence of −14 bp/ −14 bp genotype in active hCMV patients was markedly increased [ P _c = 0.00034, OR = 3.31, 95% confidence interval (CI): 1.77–6.18], and similar significance was also observed for the frequency of −14 bp allele ( P c = 0.0023, OR = 2.24, 95% CI: 1.38–3.64) when compared with that of healthy controls. Furthermore, urine hCMV DNA copies in patients with the −14 bp/ −14 bp genotype were significantly higher than those in patients with the +14 bp/ +14 bp genotype ( P = 0.041). Our findings support a potential role of HLA-G 14 bp insertion/deletion polymorphism as a susceptible factor for the active hCMV infection.     

Selective intracellular retention of virally induced NKG2D ligands by the human cytomegalovirus UL16 glycoprotein

Human cytomegalovirus (HCMV) has evolved a multitude of molecular mechanisms to evade the antiviral immune defense of the host. Recently, using soluble recombinant molecules, the HCMV UL16 glycoprotein was shown to interact with some ligands of the activating immunoreceptor NKG2D and, therefore, may also function as a viral immunomodulator. However, the role of UL16 during the course of HCMV infection remained unclear. Here, we demonstrate that HCMV infection of fibroblasts induces expression of all known NKG2D ligands (NKG2DL). However, solely MICA and ULBP3 reach the cellular surface to engage NKG2D, whereas MICB, ULBP1 and ULBP2 are selectively retained in the endoplasmic reticulum by UL16. UL16-mediated reduction of NKG2DL cell surface density diminished NK cytotoxicity. Thus, UL16 functions by capturing activating ligands for cytotoxic lymphocytes that are synthesized in response to HCMV infection.     

RNase P对人巨细胞病毒mRNA的体外靶定切割研究

目的探讨棱酶P对HCMV UL97 mRNA的体外切割能力.方法以人巨细胞病毒(human cytomegalovirus,HCMV)磷酸转移酶mRNA序列为靶设计extemal guide sequences(EGS),共价结合到大肠杆菌来源M1 RNA中,构建成M1GS-T5核酶.对其进行UL97基因亚克隆片段转录产物的体外切割实验.结果该核酶在一定离子浓度下具对UL 97mRNA片段产生特异切割能力.结论新构建得到的核酶MIGS-T5具特异性切割活性,可发展为一种抗病毒试剂.     

Mapping ganciclovir resistance in the human herpesvirus-6 U69 protein kinase

Human herpesvirus-6 (HHV-6) is a growing concern in immunocompromised individuals, such as in the transplant setting. Alone, or in concert with human cytomegalovirus (HCMV), infections with HHV-6 are often severe enough to require antiviral therapy, generally in the form of ganciclovir (GCV). GCV resistance in HCMV is well documented, both clinically and in the laboratory, and has been shown to result from mutations in the UL97 protein kinase and/or UL54 DNA polymerase. GCV resistance in HHV-6 has been documented. However, to date, it has only been investigated to a limited extent. The baculovirus system has previously been shown to be useful in studying GCV resistance with respect to herpesvirus protein kinase mutations. Using the baculovirus system, we created recombinant baculoviruses expressing either a wild-type HHV-6 U69 protein kinase or a mutated form containing homologous mutations to those documented in the UL97 protein kinase of GCV resistant HCMV isolates. The recombinant baculoviruses were used to infect Sf-9 cells and cultured in the presence of GCV to determine the effect of the HHV-6 U69 protein kinase mutations on GCV susceptibility. Mutations in the HHV-6 U69 protein kinase, homologous to those in the HCMV UL97 protein kinase documented to cause GCV resistance, result in GCV resistance in the recombinant baculoviruses.     

Simultaneous genotyping for all three known structural mutations in the human mannose-binding lectin gene

We describe a rapid and simple method for genotyping the three known structural mutations within exon 1 of the mannan-binding lectin (MBL) gene. A PCR-amplifiable synthetic DNA (Universal Heteroduplex Generator) was annealed to genomic PCR product from exon 1 to generate unique DNA heteroduplexes for each mutation. Heteroduplexes were then resolved by non-denaturing polyacrylamide gel electrophoresis. The technique was initially validated with previously typed samples and then applied to previously untyped samples with the results confirmed by DNA sequencing. © 1997 Wiley-Liss, Inc.