Molecular Epidemiology of Cytomegalovirus UL97 and UL54 variants in Taiwan

BackgroundThe antiviral resistance of cytomegalovirus (CMV) infections is associated with mutations in the CMV UL54 and UL97 gene regions and is a serious problem in immunocompromised patients. However, the molecular epidemiology of UL54 and UL97 in Taiwan is unclear.MethodsWe conducted a retrospective study of patients with CMV infections between January and December 2016 in two tertiary hospitals, one regional hospital in Taiwan. CMV DNAemia was confirmed by elevated CMV DNA titers. Then the regions of the UL54 and UL97 mutations were amplified by PCR and sequenced.ResultsOf 729 patients with CMV syndrome, 112 CMV DNAemia patients were enrolled. Twelve novel variants in UL54 (P342S, S384F, K434R, S673F, T754M, R778H, C814S, M827I, G878E, S880L, E888K, and S976N) and one novel variant in UL97 (M615T) were discovered. UL97 antiviral resistance mutations (L595S, M460I, and M460V) were found in four patients (3.6%). In the drug resistance strains, the mutation events occurred after 83–150 days of therapy, and drug resistance was also observed in these patients. The following high frequency variants were observed: D605E in UL97 and A885T, N898D, V355A, N685S, and A688V in UL54.ConclusionThe results demonstrate that the positive rate of CMV DNAemia was 15.3% (112/729) among the patients with clinical CMV infection symptoms. The proportion of antiviral resistance CMV strains within CMV DNAemia patients was 3.6%. With the information of polymorphism incidence in the UL54 and UL97 patients from our study, determination of the genetic profile of UL54 and UL97 among immunocompromised populations with refractory CMV infection is recommended.     

人巨细胞病毒UL148A、UL148B、UL148C、UL148D基因在临床株中的多态性研究

目的研究人巨细胞病毒（human cytomegalovirus，HCMV）UL148A、UL148B、UL148C和UL148D基因序列在临床低传代分离株中的多态性。方法对22株经荧光定量PCR方法（Q-PCR）检测HCMV-DNA为阳性的临床株进行HCMV UL148A、UL148B、UL148C和UL148D基因全序列PCR扩增，并对PCR扩增产物进行序列测定及分析。结果22株临床株序列与Merlin株相比，临床分离株UL148A基因的核苷酸变异率为0．5％-8．3％，氨基酸变异率为1．3％-6．3％；UL148B基因核苷酸变异率为0．5％-4．6％，氨基酸变异率为1．3％-5．0％；UL148C基因核苷酸变异率为0．5％-3．0％，氨基酸变异率为1．3％-3．9％；UL148D基因核苷酸变异率为0．6％-8．5％，氨基酸变异率为1．7％-8．1％。22株HCMV UL148A和UL148D序列均分为3个型。与Merlin株比较，除U96株外，来自未经传代的尿标本中的UL148A、UL148B、UL148C和UL148D基因编码产物含有的翻译后修饰位点均保守。结论UL148C基因无论在核苷酸序列还是在蛋白的氨基酸结构上均较为保守。统计学分析显示HCMV UL148A和UL148D基因呈现一定的相关性。     

Polymorphisms of human cytomegalovirus UL148A, UL148B, UL148C, UL148D genes in clinical strains.

BACKGROUND: Clinical isolates of human cytomegalovirus (HCMV) display polymorphisms in multiple genes. Some authors have suggested that polymorphisms are implicated in HCMV-induced immunopathogenesis, as well as in strain-specific behaviors, such as tissue-tropism and the ability to establish persistent or latent infections. OBJECTIVE: To describe the features of HCMV UL148A, UL148B, UL148C and UL148D open reading frames (ORFs) and the variable sites within the frames in clinical strains. STUDY DESIGN: PCR was performed to amplify these ORFs in 22 clinical strains. PCR amplification products were sequenced directly and analyzed. RESULTS: The nucleotide diversity of UL148A, UL148B, UL148C and UL148D ORFs in studied strains is 0.5-8.3%, 0.5-4.6%, 0.5-3% and 1.7-8.1%, respectively; the amino acid diversity of their putative proteins is 1.3-6.3%, 1.3-5.0%, 1.3-3.9% and 1.7-8.1%, respectively, related to the Merlin strain. The modification sites of UL148A, UL148B, UL148C and UL148D predicted proteins from strains in unpassaged urine samples were conserved, except for strain U96, compared with that of the Merlin strain. By phylogenetic and statistical analysis, the UL148A and UL148D sequences of clinical strains were classified into three groups. CONCLUSION: Compared to the UL148A, UL148B and UL148D ORFs, the UL148C ORF was relatively conserved, as was the amino acid sequence of the UL148C putative protein. Isolates that have been passaged several times in human embryonic lung fibroblasts (HELF) showed some changes of modification sites, however. A discrete linkage was found between the groups of UL148A gene and those of UL148D gene.     

先天性巨结肠患者人类巨细胞病毒UL144基因多态性的研究

目的研究人类巨细胞病毒（human cytomegalovirus,HCMV）UL144基因在先天性巨结肠（Hirschsprung＇s disease,HD）临床株中的多态性,探讨HCMV UL144基因多态性与致病性之间的关系.方法随机选取53个先天性巨结肠患儿痉挛段结肠手术标本及经荧光定量PCR方法检测HCMV DNA为阳性的4个HD患儿的尿标本,对照组为无症状或仅有皮肤轻度黄疸的6个尿标本.应用巢式聚合酶链反应的方法,扩增HCMV UL144基因开放阅读框架（ORF）,扩增阳性的临床株进行双向DNA测序,最后通过DNAclub、Bioedit、DNAstar、GeneDoc等软件进行分析.结果23份HD痉挛段肠组织（46%）及4份尿标本HCMV UL144基因扩增阳性,并且完成测序.种系进化树分析结果显示25个HD患儿的DNA序列分为3个基因型,G1A型64.0%,G2型24%,G3型12%.与对照组比较,经χ^2检验,χ^2=10.93,P为0.012;其中HD临床株G1A和G3型基因经Fisher检验,P为0.015,差异具有统计学意义.全结肠型、长段型及普通型HD分散分布于UL144各个基因型中.结论HD与HCMV感染有关,HCMV可能是HD的病因之一;在HD患儿中,HCMV感染以UL144基因G1A型为主;HD的临床分型与HCMV UL144基因分型无关.     

Human cytomegalovirus UL131A, UL130 and UL128 genes are highly conserved among field isolates

Summary. Coding sequences of the UL131A, UL130, and UL128 genes of human cytomegalovirus (HCMV) were found to be highly conserved among 34 field isolates from pregnant women with primary HCMV infection and their fetuses or newborns, as well as from solid organ transplant recipients and patients with AIDS. No strain clustering was observed. In contrast, sequencing of UL55 (gB coding gene) allowed the 34 isolates to be clustered into 4 genotypes. The conservation of the UL131A-UL128 locus is consistent with the conclusion that the three encoded proteins are all essential for growth of HCMV in endothelial cells and virus transfer to leukocytes.     

The virulence-determining genomic BamHI fragment 4 of pseudorabies virus contains genes corresponding to the UL15 (partial), UL18, UL19, UL20, and UL21 genes of herpes simplex virus and a putative origin of replication.

The genomic BamHI-fragment 4 of pseudorabies virus (PrV) has previously been shown to encode functions necessary for expression of PrV neurovirulence (B. Lomniczi, S. Watanabe, T. Ben-Porat, and A. S. Kaplan, 1984, J. Virol. 52, 198-205). To identify proteins that might be involved in the neurotropism of PrV we sequenced the complete 9382-bp fragment BamHI-4, the longest contiguous sequence determined in the UL region of PrV so far, and analyzed its coding capacity. In an arrangement similar to that found in herpes simplex virus type 1 we identified complete open reading frames encoding proteins with strong homology to the UL18 (50% homology), UL19 (60% homology), UL20 (33% homology), and UL21 (36% homology) polypeptides and the 3'-part of a gene homologous to UL15 (67% homology) of HSV-1. In addition, a consensus sequence for an alphaherpesviral origin of replication was found at the left terminus of the fragment.     

Cell lines that support replication of a novel herpes simplex virus 1 UL31 deletion mutant can properly target UL34 protein to the nuclear rim in the absence of UL31

Previous results indicated that the herpes simplex virus 1 (HSV-1) U(L)31 gene is necessary and sufficient for localization of the U(L)34 protein exclusively to the nuclear membrane of infected Hep2 cells. In the current studies, a bacterial artificial chromosome containing the entire HSV-1 strain F genome was used to construct a recombinant viral genome in which a gene encoding kanamycin resistance was inserted in place of 262 codons of the 306 codon U(L)31 open reading frame. The deletion virus produced virus titers approximately 10- to 50-fold lower in rabbit skin cells, more than 2000-fold lower in Vero cells, and more than 1500-fold lower in CV1 cells, compared to a virus bearing a restored U(L)31 gene. The replication of the U(L)31 deletion virus was restored on U(L)31-complementing cell lines derived either from rabbit skin cells or CV1 cells. Confocal microscopy indicated that the majority of U(L)34 protein localized aberrantly in the cytoplasm and nucleoplasm of Vero cells and CV1 cells, whereas U(L)34 protein localized at the nuclear membrane in rabbit skin cells, and U(L)31 complementing CV1 cells infected with the U(L)31 deletion virus. We conclude that rabbit skin cells encode a function that allows proper localization of U(L)34 protein to the nuclear membrane. We speculate that this function partially complements that of U(L)31 and may explain why U(L)31 is less critical for replication in rabbit skin cells as opposed to Vero and CV1 cells.     

The equine herpesvirus 1 UL34 gene product is involved in an early step in virus egress and can be efficiently replaced by a UL34-GFP fusion protein

The structure and function of the equine herpesvirus type 1 (EHV-1) UL34 homologous protein were characterized. A UL34 protein-specific antiserum reacted with an M(r)28,000 protein that could not be detected in purified extracellular virions. Confocal laser scanning microscopy demonstrated that UL34 reactivity mainly concentrated at the nuclear rim, which changed into a punctuate and filamentous pattern at late times after infection. These changes in UL34 distribution were especially prominent when analyzing the distribution of a GFP-UL34 fusion protein. A UL34-negative EHV-1 was generated by mutagenesis of a recently established BAC clone of EHV-1 strain RacH (pRacH). Release of extracellular infectious virus was severely impaired after infection of Rk13 cells with HDelta34. Electron microscopy revealed a virtual absence of virus particles in the cytoplasm of infected cells, whereas nucleocapsid formation and maturation within the nucleus appeared unaffected. A UL34-GFP fusion protein with GFP linked to the C-terminus of UL34 was able to complement for the UL34 deletion in trans, while a GFP-UL34-fusion protein with GFP linked to the N-terminus of UL34 was able to only partially restore virus growth. It was concluded that the EHV-1 UL34 product is essential for an early step in virus egress, i.e., release of capsids from infected-cell nuclei.     

Variability of UL18, UL40, UL111a and US3 immunomodulatory genes among human cytomegalovirus clinical isolates from renal transplant recipients.

BACKGROUND: Variability of human cytomegalovirus (HCMV) genes counteracting immune responses is poorly investigated in non-cultured clinical strains. OBJECTIVES: In HCMV-infected renal graft recipients, we aimed to (i) investigate the variability of four HCMV immunomodulatory genes, without any culture-related viral selection, (ii) provide evolutionary sequence data, and (iii) study co-existing HCMV variants and their evolution. STUDY DESIGN: UL18, UL40, UL111a and US3 were sequenced in 31 blood samples from 17 patients (8 with sequential samples). Cloning of UL40 PCR products was performed in one donor-positive/recipient-positive (D+/R+) patient's samples. RESULTS: Each patient harboured a unique strain (combination of four genes), however single identical genes were demonstrated among various patients, suggesting recombination events. Sequencing showed in D+/R- recipients, either complete gene stability (four patients) or significant variability (one patient); in three D+/R+ patients, multiple gene variations, possibly linked to super- or co-infections. Cloning evidenced different variants at each time point with an increasing variability over time, illustrating possibly viral reactivations and the subsequent evolution of the variants mixture. CONCLUSION: A noticeable HCMV natural polymorphism was shown, with different evolutive patterns. Moreover, we described the co-evolution of variants mixtures in one patient. Consequences on HCMV infection and graft function deserve further studying.     

The role of protein kinase activity expressed by the UL13 gene of herpes simplex virus 1: the activity is not essential for optimal expression of UL41 and ICP0

Herpes simplex virus 1 (HSV-1) UL13 is a viral protein kinase that is packaged into virions and regulates optimal expression of ICP0 and a subset of late (gamma) proteins, including UL41 in infected cells. In the present study, we investigated the role(s) of the protein kinase activity of UL13 in viral replication using a recombinant virus expressing enzymatically inactive UL13 after an amino acid substitution in the invariant lysine of UL13. The recombinant virus carrying this mutation formed smaller plaques yielded 10-fold less progeny than wild-type virus but could not be differentiated from wild-type virus with respect to accumulation of UL41 and ICP0 in infected cells. These results indicate that the protein kinase activity of UL13 plays a role in viral replication in cell culture, but the activity is not essential for the optimal expression of UL41 and ICP0.     

Association of Two Membrane Proteins Encoded by Herpes Simplex Virus Type 2, UL11 and UL56

Herpes simplex virus (HSV) acquires envelope by budding into trans-Golgi network (TGN)-derived vesicles. Previous studies showed that the UL11 gene product enables efficient virion envelopment and export from infected cells and is incorporated into virions as tegument protein. At its N-terminus, UL11 is dually acylated by myristoic and palmitoic acids. Fatty acylation of UL11 provides both membrane binding strength and Golgi-targeting specificity. We show here that UL11 interacts with UL56 protein, a tail-anchored type II membrane protein encoded by HSV, which associated with the Golgi apparatus and cytoplasmic vesicles. We previously showed that UL56 is involved in vesicular transport in infected cells. The UL11–UL56 complex localized to the perinuclear region of the cytoplasm in infected cells. Fatty acylation of UL11 was important for the formation of the UL11–UL56 protein complex. Taken together, our results identify a novel interaction between two HSV proteins facilitated by mutual interactions with Golgi-derived vesicles.     

Distribution of UL144, US28 and UL55 genotypes in Polish newborns with congenital cytomegalovirus infections

Human cytomegalovirus (HCMV) is the most common congenital infection. HCMV strains display genetic variability in different regions. Distribution of HCMV genotypes in the population of congenitally infected newborns from Central Poland and viral load in newborns' blood is described and discussed. HCMV isolates were analysed by sequencing at three sites on the genome: the UL144 tumour necrosis factor-alpha (TNFα)-like receptor gene, the US28 beta-chemokine receptor gene and the UL55 envelope glycoprotein B (gB) gene. The newborns' blood was examined for HCMV DNA with a nested (UL144, UL55) or heminested (US28) polymerase chain reaction, and the genotypes were determined by sequence analysis. HCMV DNA was detectable in 25 out of 55 examined newborns born by HCMV-infected mothers (45.5%). The blood viral load in mother-infant pairs was determined. Most of the newborns had identical virus genotype, gB2 (96%), UL144 B1 (88%) and US28 A2 (84%). These genotypes were detected in all newborns with asymptomatic congenital infection. The occurrence of UL144 B1 or US28 A2 genotypes in the babies examined was significant in comparison to other genotypes (p=0.0002 and p=0.040 respectively). There was no association between specific gB subtypes in all patients groups (p=0.463). There was no correlation between HCMV genotypes and the outcome.     

Genetic Complexity of EHV-1 Defective Interfering Particles and Identification of Novel IR4/UL5 Hybrid Proteins Produced During Persistent Infection

This study examined the genetic complexity of three equine herpesvirus 1 (EHV-1) defective interfering particles (DIP) and found the DIP genomes to range from 5.9 kbp to 7.3 kbp in total size. Each DIP contains an identical 5′ end (∼1.9 kb) that harbors UL3 and UL4 genes that are 100% identical to those of the infectious virus. DIP2 and DIP3 contain a previously described unique IR4/UL5 (EICP22/EICP27) hybrid gene (Hyb1.0). The DIP1 genome, however, appears to be generated from a different recombination event which results in the formation of a new distinct hybrid ORF. The new ORF (Hyb2.0) is comprised of 684 bp from the 5′ end of IR4 fused to 45 bp from the 3′ terminus of UL5. In contrast to Hyb1.0, the UL5 sequences present in Hyb2.0 are not in-frame. Thus, the Hyb2.0 protein is comprised of 228 residues from IR4 linked to a sequence of 15 amino acids that result from a frameshifted reading of UL5 sequences. Western blot analysis confirmed that the Hyb2.0 ORF is expressed during persistent infection to produce a family of proteins that migrate at 36–42 kDa. Fluorescence microscopy revealed that both Hyb proteins display diffuse cytoplasmic localization patterns dissimilar to the nuclear localization patterns of both IR4 and UL5. Neither Hyb protein, however, disrupts the nuclear entry of the EHV-1 immediate-early, IR4, or UL5 proteins or cellular TATA box binding protein (TBP) previously shown to interact with both IR4 or UL5 in productive infection. DIP genomic segments (∼3.5–5.0 kbp) downstream of the 100% conserved origin of replication are highly variable among the three DIP genomes and contain large areas of repetitive sequences. The possibility that the non-coding sequences play a role in viral interference and/or persistent infection remains to be determined.     

UL16 binding proteins

According to present concepts, innate immunity plays an important role in tumor surveillance and immune modulation. The state of NK cells depends on the balance between inhibitory and activating signals from corresponding receptors. As one of the activating receptors, NKG2D recognises some self ligands such as MICA/B in human and Rae1 in mice, which is dissimilar to those toll-like receptors that recognise some pathogen-derived ligands. NKG2D is expressed not only on NK cells, but on gammadelta T cells, CD8+ alphabeta T cells in normal individuals and CD4+ alphabeta T cells in rheumatoid arthritis patients and plays a different role on respective cells. Whereas NKG2D can only function as a costimulatory receptor on CD8+ alphabeta T cells under the domination of alphabeta TCR in spite of a deficiency of costimulatory molecule CD28, NKG2D can directly activate NK cells even in the presence of inhibitory signals from MHC-I and corresponding receptor complexes. Experiments in mice have identified that alternative splicing produces two distinct NKG2D polypeptides that associate differentially with the DAP10 and DAP12 signaling subunits and that differential expression of these isoforms and of signaling proteins determines whether NKG2D only functions as a costimulatory receptor in the adaptive immune system (CD8+ T cells) or as both a primary recognition unit and a costimulatory receptor in the innate immune system (natural killer cells and macrophages). This review summarizes the research achievements in a new ligand family (UL16 binding proteins) of NKG2D in human and shows the possible prospects of ULBP function and application.     

The equine herpesvirus 1 UL11 gene product localizes to the trans-golgi network and is involved in cell-to-cell spread

Experiments were conducted to identify and characterize the equine herpesvirus type 1 (EHV-1) UL11 homologous protein. At early-late times after EHV-1 infection of Rk13 cells several proteins at an M(r) of 8000 to 12,000 were detected using a UL11 protein-specific antiserum. Particularly, an M(r) of 11,000 protein was found abundantly in purified virions and could be assigned to the tegument fraction. As demonstrated by confocal laser scanning microscopy, UL11 reactivity localized predominantly to the trans-Golgi network of infected cells, but was also noted at the plasma membrane, specifically of transfected cells. Deletion of UL11 sequences in EHV-1 vaccine strain RacH (Hdelta11) and in the virulent isolate RacL22 (Ldelta11) resulted in viruses that were able to replicate on noncomplementing cells. It was shown in one-step growth kinetics on Rk13 cells that the reduction of intracellular and of extracellular virus titers caused by the absence of UL11 expression in either virus was somewhat variable, but approximately 10- to 20-fold. In contrast, a marked influence on the plaque phenotype was noted, as mean maximal diameters of plaques were reduced to 23.2% (RacL22) or 34.7% (RacH) of parental virus plaques and as an effect on the ability of RacH to cause syncytia upon infection was noted. It was therefore concluded that the EHV-1 UL11 product is not essential for virus replication in Rk13 cells but is involved in cell-to-cell spread.     

男性不育症患者血清IL-6、T、LH、FSH、PRL检测的临床意义

目的：探讨了男性不育症患者血清IL-6、T、LH、FSH和PRL水平的变化及临床意义。方法：应用放射免疫分析对68例男性不育症患者进行了血清IL-6、T、LH、FSH和PRL检测,并与35名男性正常人作比较。结果：男性不育症患者血清T水平非常显著地低于男性正常人组（P〈0.01）,而IL-6、FSH、LH和PRL水平又非常显著地高于男性正常人组（P〈0.01）。血清IL-6水平与T水平呈负相关（r=-0.4186,P〈0.01,与FSH、LH和PRL水平呈正相关（r=0.6018、0.5912、0.6214,P〈0.01）。结论：检测男性不育症患者血清IL-6、T、LH、FSH和PRL水平的变化对诊断、预后判断及治疗均有较大的实用价值。     

Characterization of the guinea pig cytomegalovirus genome locus that encodes homologs of human cytomegalovirus major immediate-early genes, UL128, and UL130

We reported previously that the guinea pig cytomegalovirus (CMV) stock purchased from the American Type Culture Collection contained two types of strains, one containing and the other lacking a 1.6 kb locus, and that the 1.6 kb locus was required for efficient viral growth in animals but not in cell culture. In this study, we characterized the genetic contents of the locus, and found that i) the 1.6 kb locus encodes homologs of human CMV UL128 and UL130, GP129 and GP131, respectively, ii) these genes are expressed with late gene kinetics, iii) GP131 protein (pGP131) localized to cell surface only in the presence of glycoproteins H and L, and iv) pGP131 is a virion component. Therefore, it is plausible that pGP131 forms a complex with glycoproteins H and L and becomes a virion component as does UL130 protein (pUL130). Since pUL130 is one of the glycoproteins essential for infection of endothelial and epithelial cells in human and primates, functional and immunological analyses of this GPCMV homolog of pUL130 may help to illuminate the in vivo role of pUL130.