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Background: Cytomegalovirus (CMV) is a major pathogen in lung transplant recipients (LTRs). The emergence of different CMV strains in lung and blood after transplantation has not yet been analyzed., Methods: In total, 75 serum and 91 broncheoalveolar lavage (BAL) samples obtained from 25 LTRs in the follow-up after transplantation were tested for the presence of different CMV strains. The gB, gN, and gO genes of the CMV isolates were analyzed by subtype-specific PCR, restriction fragment length polymorphism (RFLP), sequencing, and phylogenetic analysis., Results: Mixed CMV-strain populations were detected after cessation of antiviral prophylaxis in up to 80% and 90% of the patients' BAL and serum, respectively, and this was independent of the CMV serostatus of donor and recipient. In five patients, the same single CMV strain was consistently detectable over at least 1 year in lung and blood, although in two of these cases donor and recipient had both been CMV-seropositive. Most CMV strains were distributed in the lung and blood compartment. Symptomatic CMV infection within the first year after transplantation was observed only in patients with mixed CMV-strain populations (P<0.05)., Conclusion: Most LTRs harbor more than one CMV strain in their lung and blood compartment after cessation of prophylaxis, but the CMV strain distribution within and between the compartments varies between individuals and is not associated with the donor/recipient serostatus. The data further show that compartmentalization of CMV strains in lung versus blood seems to be a rare event and that the presence of mixed CMV-strain infections within the first year after transplantation may be disadvantageous for LTRs.

Background: Although the sequence of the human cytomegalovirus (HCMV) genome is generally conserved among unrelated clinical strains, some open reading frames (ORFs) are highly variable. UL146 and UL147, which encode CXC chemokine homologues are among these variable ORFs., Results: The region of the HCMV genome from UL146 through UL147A was analyzed in clinical strains for sequence variability, genotypic stability, and transcriptional expression. The UL146 sequences in clinical strains from two geographically distant sites were assigned to 12 sequence groups that differ by over 60% at the amino acid level. The same groups were generated by sequences from the UL146-UL147 intergenic region and the UL147 ORF. In contrast to the high level of sequence variability among unrelated clinical strains, the sequences of UL146 through UL147A from isolates of the same strain were highly stable after repeated passage both in vitro and in vivo. Riboprobes homologous to these ORFs detected multiple overlapping transcripts differing in temporal expression. UL146 sequences are present only on the largest transcript, which also contains all of the downstream ORFs including UL148 and UL132. The sizes and hybridization patterns of the transcripts are consistent with a common 3'-terminus downstream of the UL132 ORF. Early-late expression of the transcripts associated with UL146 and UL147 is compatible with the potential role of CXC chemokines in pathogenesis associated with viral replication., Conclusion: Clinical isolates from two different geographic sites cluster in the same groups based on the hypervariability of the UL146, UL147, or the intergenic sequences, which provides strong evidence for linkage and no evidence for interstrain recombination within this region. The sequence of individual strains was absolutely stable in vitro and in vivo, which indicates that sequence drift is not a mechanism for the observed sequence hypervariability. There is also no evidence of transcriptional splicing, although multiple overlapping transcripts extending into the adjacent UL148 and UL132 open reading frames were detected using gene-specific probes.

Women with sexually transmitted diseases (STDs) and bacterial vaginosis (BV) have increased rates of cytomegalovirus (CMV) seroprevalence and CMV seroconversion. To characterize the association between genital tract CMV infection and BV, vaginal wash specimens from 52 women attending an STD clinic were analyzed. Significantly more women with BV shed CMV in the lower genital tract than did women without BV. In addition, most of the women who were shedding CMV were infected with >1 virus strain. These results suggest that local CMV replication and infection with multiple CMV strains is facilitated by the presence of BV.

Background: The herpesviruses, ancient pathogens which have co-evoluted with human, are etiologically associated with a number of diseases, from asymptomatic to oncogenic and mortal diseases. It seems that some of them have also an important role in the pathogenesis of human periodontal disease., Objective: This study aimed to determine the prevalence of Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), human herpesvirus 8 (HHV-8) and human cytomegalovirus (HCMV) in gingival crevicular fluid (GCF) and, eventually, to find the correlation between specific virus types and clinical parameters which are important in periodontitis, like plaque index (PI), gingival index (GI) and probing depth (PD)., Study Design: A polymerase chain reaction (PCR) and digestion of PCR products with restriction endonuclease were employed to identify the presence of EBV, HHV-6, HHV-8 and HCMV., Results: Out of 66 samples of GCF taken from the patients with periodontal disease, EBV was found in 29 (43.9%), HHV-6 in 16 (24.2%) and HCMV in 2 (3%) samples, while in the samples of healthy persons, these viruses were not found. HHV-8 was detected neither in the patients with periodontitis nor in healthy control group. More positive results were found in clinical samples taken from people with higher PI and GI and in the samples taken from the patients with medium PD (PD=3-6mm). In all HHV-6 positive samples, we found only variant A; as for EBV positive samples, type A and type B were identified and also co-infection with the two types. It seems that there is a correlation between PI, PD and EBV types, but no correlation was found between EBV types and GI or HHV-6 types and PI, PD, GI., Conclusions: The present findings confirm some association between herpesviruses and human periodontitis.

Real-time quantitative PCR systems (Q-PCR) for the rapid detection and quantification of microorganisms in clinical specimens employ oligodeoxyribonucleotide primers and probes for specificity, which makes them vulnerable to false negatives caused by sequence diversity in the template. Schaade et al. (J. Clin. Microbiol. 39:3809, 2001) reported a sequence variant (C630T) in the cytomegalovirus (CMV) glycoprotein B (gB) gene that, although detectable in their Q-PCR assay, could not be accurately quantified. In an effort to evaluate the impact of CMV sequence variants in our patient population by use of a similar Q-PCR assay, we surveyed 54 isolates of CMV, each from a different patient. We detected evidence for the C630T variant in 4 of 54 (7.4%) patients. Furthermore, isolates from two additional patients were completely negative in the test. Sequencing of these false-negative isolates revealed multiple mutations within the probe hybridization sites. A Q-PCR that targeted the CMV polymerase gene instead of gB detected all 54 isolates. We suggest that Q-PCR assays for viral load be rigorously tested on large panels of viral isolates to assess the impact of sequence diversity on detection as well as quantification.

Human CMV (hCMV) encodes several captured chemokine ligand and chemokine receptor genes that may play a role in immune evasion. The adjacent viral alpha-chemokine genes UL146 and UL147 appear to have duplicated subsequent to a recent gene capture event. Sequence data from multiple hCMV isolates suggest accelerated protein evolution in one of the paralogues, UL146. Extensive sequence variation was noted throughout the more rapidly evolving paralogue, although significant variation was also observed within the more slowly evolving gene, especially within a region corresponding to a possible signal peptide. In contrast to the haplotype structure observed for other hCMV genes, the distribution of nucleotide variants indicates a marked loss of linkage disequilibrium within UL146 and to a lesser extent UL147. Despite evidence of accelerated protein evolution, the rate of nonsynonymous to synonymous substitutions (d(N)/d(S)) in the more rapidly evolving paralogue was not indicative of neutral evolution, but of moderate purifying selection. The data presented here provides a unique opportunity to study the mechanisms by which a recently duplicated pair of genes has diverged and suggests a role for recombination.

The genetic variability of 74 human cytomegalovirus (HCMV) clinical isolates from 60 Japanese infants and children during 1983-2003 was investigated, and the relevance to their clinical course was studied. The patients consisted of 10 asymptomatic congenitally infected babies, 45 infected perinatally or postnatally resulting in HCMV mononucleosis/hepatitis and 5 immunocompromised hosts. The hypervariable region of the HCMV genome, that is the a sequence and UL144 region was analyzed using the polymerase chain reaction (PCR) and unrooted phylogenetic trees. HCMV glycoprotein B (gB) polymorphism was also studied. Unrooted phylogenetic trees of a sequence and UL144 allowed the isolates to be grouped to 5 and 3 clades, respectively. Three gB genotypes were also determined. However, there was no correlation between specific genotypes of these three genes and clinical forms, except for congenital infection which fell into one of three clades of the UL144 gene. In addition, the variability of the three genes had no correlation with each other. This implies that study of a single gene is insufficient for investigating the molecular epidemiology of HCMV. This study provides basic data on the genetic variability of HCMV in an Asian population and should help to determine the strains for vaccine candidates., (Copyright (c) 2005 Wiley-Liss, Inc.)

We have cloned and sequenced the glycoprotein B genes from five strains of BaCMV, isolated from three subspecies of cynocephalus baboons (olive, yellow and chacma). Primers were designed using conserved DNA regions of the gB gene to allow DNA amplification from all strains of BaCMV. These regions differ sufficiently from human CMV that HCMV strains are not amplified, thus allowing differentiation of BaCMV from HCMV. These diagnostic primers were used to test crude nucleic acid extracts from 27 strains of BaCMV and detected 26 of them. Overall, the sensitivity and specificity of this assay are 96.7 and 100%, respectively. BaCMV strains isolated from yellow and olive baboons were very similar and could be discriminated from strains isolated from chacma baboons using a second set of PCR primers. Phylogenetic analysis of the gB genes supported the inferred close relationship of strains isolated from olive and yellow baboons.

Background: Information on strain types of human cytomegalovirus (HCMV) isolates from Saudi Arabian patients is lacking., Materials and Methods: 80 clinical isolates of HCMV from Saudi Arabian patients were analyzed by PCR amplification of three regions (DNA polymerase, glycoprotein B, and glycoprotein H) of the virus genome. The resultant amplicons (2.0-2.7 kb) were further studied by restriction fragment length polymorphism (RFLP) using four enzymes (HaeIII, HhaI, MspI, and RsaI)., Results: Combined analysis of the cleavage patterns generated by the enzymes identified five strains, S1-S5, and several mixed and unique strains. 18 isolates belonged to S1 strain and were similar to laboratory strain AD169. Eight isolates were present in each of S2 and S3 strains. Six isolates and four isolates were found in S4 and S5 strains, respectively. 12 isolates contained a mixture of S3 and S5, which may have resulted from a dual infection. Each of the 24 remaining isolates had a different strain pattern., Conclusion: Our findings show that 80 HCMV clinical isolates were distributed into 30 different strains using PCRRFLP analysis of multiple viral subgenomic regions. However, the number of isolates is not uniformly distributed among strains (p < 0.02).

Sequence polymorphisms in the gN and gO genes of cytomegalovirus (CMV) amplified from mouth rinse and urine samples of 19 Malawian patients with Kaposi's sarcoma (KS) and 58 of their first-degree relatives were investigated. CMV-DNA was amplified from 41 people (53%) from either the gN or gO region in at least one sample, from 14 people (18%) in both domains in at least one sample, and from 13 (17%) in either domain in both samples. Twenty-one (51%) were seropositive for human immunodeficiency virus-1 (HIV). Identical gN sequences were recovered from eight families and non-identical sequences in six, while identical gO sequences were found in three families and non-identical sequences in five. Five people, four of whom were children, each carried multitypic gN sequences or gO sequences. The findings are consistent with CMV spread along intra- and extra-household routes, and with multiple intra-host CMV infection., ((c) 2005 Wiley-Liss, Inc.)

Objective: To compare cytomegalovirus (CMV) strains found in cerebrospinal fluid (CSF) of patients with HIV infection and CMV encephalitis with those present in the general population with respect to genetic variation in the N terminus of the glycoprotein B (gBn)-gene., Design and Methods: We sequenced gBn, which is a major target of the antiviral immune response, of CMV strains present in CSF of nine HIV-infected patients with acute encephalitis, in serum of 18 immunocompetent patients with primary CMV infection, and in serum of nine HIV-infected patients without neurological illness. Sequences were compared to prototype strains and analysed by use of phylogeny., Results: Fourty-four percent (4/9) of gBn-sequences present in CSF did not cluster with any of the four gBn-prototype strains. Phylogenetic analysis revealed that these sequences represented two further, distinct genotypes and comparison of sequences was highly suggestive for intragenic recombination. In immunocompetent patients and HIV-infected patients without neurological illness, genotype gBn1 was the predominant strain (4/9, 44% and 8/18, 42%, respectively). Genotypes distinct from prototype strains were found in none of the immunocompetent patients and 22% (2/9) of HIV- infected patients without neurological illness., Conclusions: CMV strains present in CSF of HIV-infected patients with encephalitis differ significantly from those present in the general population. Intragenic recombination of CMV may be common in patients with advanced HIV infection and a source of new CMV strains with altered biological properties.

Infection with cytomegalovirus (CMV) remains a significant cause of morbidity and mortality among hematopoietic cell transplant (HCT) recipients. We describe two pediatric HCT recipients who developed persistent and severe drug-resistant CMV infections. CMV resistance to foscarnet and ganciclovir was detected after only 6 and 11 weeks of therapy, respectively. Viral pol mutations associated with drug resistance in these patients included T838A (a novel mutation) and D588N, which were shown by marker transfer to confer foscarnet and multidrug resistance, respectively. Each of these mutations significantly reduced in vitro replication of CMV, suggesting that they may decrease viral fitness. This finding was further supported by the disappearance of mutations upon withdrawal of antiviral pressure in one patient. Novel antivirals or combination therapy may be required for the treatment of drug-resistant CMV in HCT recipients and perhaps in other severely immunocompromised patients.

To explore a possible role for viral genes as determinants of virulence, portions of the UL144 tumor necrosis factor-like receptor gene and the UL55 envelope glycoprotein B gene from 42 patients with congenital human cytomegalovirus (HCMV) infection or other diseases were sequenced. Of the 42 patients, 16 (38%) had UL144 group 1 [group 1A, 15 of 16 (94%); group 1B, 1 of 16 (6%); group 1C, 0 of 16 (0%)], 5 patients (12%) had UL144 group 2, and 21 patients (50%) had UL144 group 3. Although group 1C was not found in Japan strains (0%), it was found in USA strains (22%). Other HCMV polymorphisms should be further evaluated for their potential relevance to neonatal infection, and acquired immunodeficiency syndrome-associated HCMV diseases., (Copyright (c) 2005 S. Karger AG, Basel.)

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that infects a variety of cell types in vivo. A region (referred to as UL/b') present in the Toledo strain of HCMV and low passage clinical isolates contains 22 additional genes, which are absent in the highly passaged laboratory strain AD169. One of these genes, UL146, encodes an alpha-chemokine. PCR amplification and sequencing of this gene from serial samples obtained from transplant recipients and samples from infants with suspected congenital HCMV infection, revealed that UL146 is a hypervariable gene in vivo. However, genetic changes were highly conserved in individuals and in renal transplant recipients multiple genotypes of UL146 were present. The majority of strains characterized maintained the conserved ELRCXC motif present in the Toledo strain of HCMV. These results provide further evidence that AD169 does not represent the authentic virus in vivo and although Towne and Toledo are more representative, major genetic differences still exist. Mixed populations of HCMV strains occur in vivo so cloning of these strains is essential if an authentic genotype is to be defined., (2004 Wiley-Liss, Inc.)

Human cytomegalovirus (HCMV) clinical isolates display genetic polymorphisms in multiple genes. Some authors have suggested that those polymorphisms may be implicated in HCMV-induced immunopathogenesis, as well as in strain-specific behaviours, such as tissue-tropism and ability to establish persistent or latent infections. This review summarises the features of the main clustered HCMV polymorphic open reading frames and also briefly cites other variable loci within the viral genome. The implications of gene polymorphisms are discussed in terms of potentially advantageous higher fitness obtained by the strain, but also taking into account that the published data are often speculative. The last section of this review summarises and critically analyses the main literature reports about the linkage of strain specific genotypes with clinical manifestations of HCMV disease in different patient populations affected by severe cytomegalovirus infections, namely immunocompromised subjects and congenitally infected newborns., (2004 John Wiley & Sons, Ltd.)

Two different betaherpesviruses, the English and Maastricht species of rat cytomegalovirus (CMV), have previously been isolated from Rattus norvegicus. CMVs were isolated from both the brown rat, R. norvegicus, and the black rat, R. rattus, within Australia. The viruses isolated from R. norvegicus appeared to be genetically related to the English species of rat CMV by PCR, RFLP, and sequencing, but the viruses isolated from R. rattus were distinct from both prototype virus species, although more closely genetically related to the Maastricht virus. This is the first genetic characterization of cytomegaloviruses from R. rattus, and the first isolation of CMVs from Australian rats.

Background: Herpesviruses are implicated in the pathogenesis of human periodontitis. However, the quantity of herpesviruses in periodontal sites remains unknown., Objective: The aim of this study was to compare levels of subgingival human cytomegalovirus (HCMV) in aggressive periodontitis patients and in periodontally healthy subjects., Methods: A total of 16 consecutive subjects with aggressive periodontitis and 15 healthy control subjects were included in the study. Subgingival specimens were collected by a periodontal curette. TaqMan real-time polymerase chain reaction (PCR) assay was used to quantify HCMV., Results: HCMV was detected in 68.8% of aggressive periodontitis lesions but not in any of the periodontally healthy study sites. HCMV viral load in positive subgingival specimens ranged from 5 x 10(2) to 7.4 x 10(3) copies/ml., Conclusions: The TaqMan real-time PCR technology seems to provide a rapid and sensitive method for quantifying HCMV in periodontal pockets. The present findings confirm the frequent presence of HCMV in aggressive periodontitis lesions. Determining HCMV levels in different types of periodontitis may help elucidate the periodontopathic role of the virus and advance our understanding of the disease pathogenesis.

Human cytomegalovirus (CMV) messenger (m) RNA expression in circulating leukocytes reflects directly viral activity in the human host. In this study, sixty-nine patients were monitored prospectively for CMV infection and mRNA expression during the first year after renal transplantation. Of the 69 recipients, 58 (84%) recipients were positive for CMV immediate early 1 (IE1) mRNA as detected by nucleic acid sequence-based amplification. The median onset of IE1 expression started at day 22 after transplantation and continued for a median duration of 82 days. IE1 mRNA expression started significantly earlier in recipients who developed an active CMV infection (P = 0.001) and in mycophenolate mofetil (MMF) treated recipients (P = 0.002). The duration of IE1 mRNA expression was significantly longer in recipients that had previously an early onset of IE1 mRNA expression (P = 0.001) and in recipients with active CMV infection (P = 0.007). Remarkably, longer prednisolone intake was correlated with a significantly (P = 0.02) shorter duration of IE1 expression compared to a longer duration of IE1 expression in recipients with only a short prednisolone intake. In recipients infected with glycoprotein B (gB) type 1 CMV strains, the duration of IE1 expression was significantly (P = 0.04) shorter compared to recipients infected with non-gB type 1 CMV strains (64 days vs. 150 days). The study indicates that multiple factors play a role in the onset and/or duration of CMV IE1 mRNA expression, for example, MMF treatment, prednisolone intake, and gB type of the specific CMV strain. The clinical significance of these correlations remains to be studied in more detail., (Copyright 2004 Wiley-Liss, Inc.)

The development of a multiplex polymerase chain reaction (PCR) method for rapid and accurate detection and typing of herpes simplex virus type 1 (HSV-1), and type-2 (HSV-2), cytomegalovirus (CMV) and Epstein-Barr virus (EBV) is very important for clinical diagnosis to allow the deliver of therapy as early as possible. Large scale amplifications by multiplex PCR of viral DNA can lower the cost and time for viral diagnosis. In this study, therefore sensitive quadruplex PCR was achieved by optimizing parameters such as primers, and 1.5 mM magnesium and 200 uM dNTPs concentrations. The concentrations of HSV-1, HSV-2, CMV and EBV primers were 0.5, 0.3, 0.25 and 0.25 pmoles, respectively. Optimal annealing temperature was 54 degrees C. Employing these conditions, we could detect 10 copies of reconstructed template plasmid DNA, which were cloned to vectors containing target sequences of viral DNA. PCR products of 271 bp for HSV-1, 231 bp for HSV-2, 368 bp for CMV, and 326 bp for EBV were separated on 5.0% polyacrylamide gel electrophoresis and confirmed by direct sequencing. The present study showed that the quadruplex PCR assay described herein has potential application in clinical diagnosis, when rapid, accurate detection and typing of viruses HSV-1, HSV-2, CMV or EBV are necessary.

To investigate any association between cytomegalovirus glycoprotein B (CMV gB) subtypes and central nervous system (CNS) disease in AIDS patients, proportions of different gB genotypes detected in AIDS patients with CNS disease were compared with the gB genotypes detected in AIDS patients with no neurological disorder. The patients were matched by CD4+ cell counts. CMV was detected by PCR in cerebrospinal fluid (CSF) samples obtained from AIDS patients with CNS disease and from urine and saliva samples obtained from AIDS patients without CNS disease. CMV strains obtained were digested by restriction enzymes HinffI and RsaI to classify the genotypes. The CMV gB genotype was determined in 26 CSF samples. Of these, 11/26 (42.3%) typed as gB group 1, seven (26.9%) as gB2, four (15.4%) as gB3, and four (15.4%) as gB4. The CMV gB genotype frequency distribution in the 42 AIDS patients without CNS disease showed that 18/42 (42.8%) were classified as gB group 1, 10 (23.8%) as gB2, seven (16.6%) as gB3, and seven (16.6%) as gB4. In the present study, no association was found between CMV gB genotypes and CMV-related central nervous system disease., (Copyright 2003 Wiley-Liss, Inc.)

Background: Recent studies have identified human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) in symptomatic periapical lesions. Little information exists on HCMV and EBV in asymptomatic periapical lesions., Aim: To compare the presence of late transcripts of HCMV, EBV and herpes simplex virus (HSV) in symptomatic and asymptomatic periapical lesions., Methods: Periapical samples were collected from seven symptomatic and seven asymptomatic periapical lesions at the time of apicoectomy. HCMV, EBV and HSV late mRNAs were identified by reverse transcription polymerase chain reaction., Results: HCMV mRNA was detected in all seven symptomatic periapical lesions and in one asymptomatic lesion (Chi-squared test, Yates'P-value = 0.007). EBV mRNA was detected in six symptomatic lesions and in one asymptomatic lesion (P = 0.04). One asymptomatic lesion yielded HSV mRNA., Conclusions: HCMV and EBV active infections are associated with acute exacerbation of apical periodontitis. HSV seems to be unimportant in periapical pathosis.

Background: Human cytomegalovirus (HCMV) clinical isolates display genetic polymorphisms, supposed to be related with strain-specific tissue-tropism and HCMV-induced immunopathogenesis. One recently discovered polymorphic gene is ORF UL73, encoding for the envelope glycoprotein gN. Among HCMV clinical strains, it shows four distinct genomic variants denoted as gN-1, gN-2, gN-3 and gN-4., Objectives: Aims of this study were to assess the prevalence of the different gN types in the populations examined and to investigate the possible relationship between genotypes and severity of congenital CMV disease., Study Design: The gN genotyping was carried out by sequencing analysis of the HCMV ORF UL73. Comparisons were made by chi-square test and contingency tables., Results: All the four gN genotypes can cause congenital infections and the overall distribution was as follows: gN-1, 23.6%; gN-2, 1.1%; gN-3, 12.9%; gN-4, 62.4%. None of them seems to be preferentially associated with vertical transmission or with acute outcome of congenital infection. However, considering the chronic outcome and long-term sequelae, there was a statistically significant (P<0.05) difference between congenitally infected infants with or without adverse chronic outcome., Conclusions: HCMV congenital infections, which displayed a prevalence of the gN-1 variants, seem to be associated with favorable chronic outcome.

Certain mutations in the viral DNA polymerase (pol) gene are known to confer drug resistance when transferred to susceptible human cytomegalovirus (CMV) strains, whereas other putative resistance mutations remain unproven. A new marker-transfer technique was used to produce recombinant CMV strains, to determine the drug susceptibility phenotypes conferred by 10 pol mutations (9 observed in clinical isolates). Various degrees of resistance to ganciclovir and cidofovir were conferred by mutations D301N, N410K, D413E, T503I, and L516R, which are located within exonuclease functional domains where D301N and D413E affect highly conserved residues. Mutations A692S, E756K, and E756D, which are not located within recognized functional domains, each conferred foscarnet resistance. This study significantly increases the number of confirmed CMV pol resistance mutations, at both conserved and nonconserved loci, with implications for molecular mechanisms and the genotypic diagnosis of antiviral resistance.

A novel and highly sensitive multiplex nested PCR assay has been developed for the simultaneous glycoprotein B (gB) typing of four gB genotypes of human cytomegalovirus (CMV) directly from clinical specimens. Specifically, a pair of primers to conserved regions of all gB genotypes within the gB gene (gB and gpUL55) was used for primary amplification. A mixture of nested primers to specific and conserved regions of each gB genotype was used for a secondary PCR amplification, yielding amplicons of different sizes for each gB genotype that could easily be differentiated by agarose gel electrophoresis. A total of 40 of 44 serum specimens and 26 of 26 cerebrospinal fluid (CSF) samples, which had previously tested positive for CMV in 66 of 70 AIDS patients with different CMV disease conditions, were gB genotyped by this novel assay. Significant differences in glycoprotein B genotype distribution between serum and CSF specimens were found (P = 0.001). gB genotype 3 (gB3) and gB2 were the most prevalent genotypes in sera (42.5%) and CSF (38.5%), respectively. A different distribution was also observed when only patients with CMV retinitis were studied (P = 0.005), suggesting a gB2 neuron tropism. Neither CMV disease nor any particular clinical manifestation of CMV disease was associated with gB genotypes (P > 0.05). A high incidence of mixed infection with the gB1 and gB3 genotypes (27.5%) was detected in serum specimens, indicating that reinfection and reactivation are common traits in advanced AIDS patients.

The incidence of human cytomegalovirus (CMV) genotype gB2 (UL55) is high in patients with human immunodeficiency virus (HIV) infection in the San Francisco Bay area of California. Virus neutralizing antibody (NAb) to human CMV strain Ad169, a gB2 laboratory strain, was measured prospectively in HIV-infected patients, with CD4 T-lymphocyte counts <200, who were at risk for CMV-associated disease. Patients were grouped according to CMV DNA copy number, as quantified by PCR, and presence or absence of CMV-induced retinitis. Mean NAb titres were similar in all patient groups and unrelated to either virus load or outcome of CMV infection. Both gB2 and mixtures of gB2 with other gB genotypes were represented in isolates from blood and/or urine, even in the presence of high titres of antibody to the gB2 genotype challenge virus.

Genetic variation in the glycoprotein B (gB) gene may play a role in human cytomegaloviruses (HCMVs) pathogenesis. Using restriction analysis of the gB gene product (PCR-RFLP), amplified by the nested polymerase chain reaction, the HCMV strains can be compared and classified into at least four HCMV groups. PCR single-strand conformation polymorphism (PCR-SSCP) is one of the techniques used to identify a mutant sequence or a polymorphism in a known gene. SSCP analysis has the advantage over RFLP analysis on detection of DNA polymorphisms and point mutations at a variety of positions in DNA fragments. However, the original SSCP protocols using the incorporation of radioactive label and polyacrylamide gel electrophoresis for detection are labour intensive and time-consuming. A simplified SSCP protocol is described to identify HCMV strains and the gB genotype, allowing the detection of sequence variations not residing in the endonuclease recognition sites.

Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality in immunocompromised patients, such as renal transplant recipients. Analysis of the gene encoding the envelope glycoprotein B (gB) showed that clinical isolates adopted one of the sequence configurations, permitting the isolates to be assigned a gB genotype of 1-4. It has been suggested that HCMV gB genotypes could be correlated with tropism and pathogenesis. A number of reports in the literature refer to shedding of different gB strains, permitting follow-up of renal transplant recipients. Considering that a single strain might be responsible for the clinical expression of the disease in multiply exposed individuals, the frequency distribution of gB genotypes was examined by nested polymerase chain reaction and restriction fragment length polymorphism in 20 renal transplant recipients at the time of diagnosis. The association between gB genotypes and cellular tropism was determined using blood, saliva, and urine for each patient. HCMV gB genotype 2 was found more frequently than other genotypes (gB2, 40%; gB1, 30%; gB3, 25%; and gB4, 5%) in renal transplant recipients. The gB type did not correlate with tropism for different body sites. All the patients with HCMV infections presumably harbored a single HCMV strain at the time of diagnosis. In multiply exposed patients, the immunomodulation provided by acute HCMV infection could favor later shedding of different strains., (Copyright 2003 Wiley-Liss, Inc.)

The 86-kDa major immediate-early protein, IEP86 (IE2, IE2(579aa), or ppUL122a), from the Towne and AD169 strains of human cytomegalovirus show four amino acid variations, namely, R68Q, K455E, T541A, and seven consecutive serines beginning at position 258 in Towne and eight serines in AD169. A commonly utilized IEP86 cDNA expression clone (herein called the original cDNA) (E. Baracchini, E. Glezer, K. Fish, R. M. Stenberg, J. A. Nelson, and P. Ghazal, Virology 188:518-529, 1992) shows the Towne R68 and seven serines but contains the AD169 E455 and A541 plus two amino acid mutations, M242I and A463T. In transcriptional activation analyses using several promoters, the IEP86 produced by the original cDNA was 40 to 60% less active than wild-type (WT) Towne IEP86, whereas AD169 IEP86 was two to three times more active than WT Towne IEP86. To determine which amino acid variations or mutations accounted for the differences in transcriptional activation, they were individually tested in the WT Towne IEP86 background. K455E, M242I, and the eighth serine had little effect on transcriptional activation or sumoylation when inserted into the Towne background. T541A significantly increased transcriptional activation on all promoters tested and showed increased sumoylation; T541A is the primary reason that WT AD169 IEP86 has increased activity over WT Towne IEP86. The increased sumoylation seen with T541A was quantitatively reduced to WT Towne levels when the K455E alteration was present, suggesting that K455 may be a sumoylation site or that E455 may cause alterations in the IEP86 structure which affect overall sumoylation. A463T was very deleterious to transcriptional activation and caused reduced sumoylation. The A436T mutation in the original cDNA is partially compensated by the presence of the T541A variation. Phosphopeptide mapping suggests that a threonine at 463 or 541 does not introduce a phosphorylation site. However, the A463T mutation does affect phosphorylation at a distant site, suggesting that it alters the conformation of the protein. Promoter-specific effects were noted with some of the amino acid variations, particularly T541A. Structural modeling is presented which suggests how A463T and T541A alter the functional structure of WT Towne IEP86. A hydrophobic core containing A463 is predicted to be responsible for the functional integrity of the carboxy-terminal region of IEP86 between amino acids 344 and 579.

Studies with animal cytomegaloviruses, epidemiological data from humans as well as in vitro studies suggest the involvement of the human cytomegalovirus (HCMV) in the development of atherosclerosis. Cell culture systems are insufficient for examination of the entire pathogenetic process and a satisfactory animal model for HCMV is not available. An organ culture model was established for HCMV infection of human renal arteries in vitro. After infection with three representative HCMV strains, infectious virus was recovered from supernatants until 144 days post-infection with a peak around day 30 due to a long-lasting productive HCMV infection in still vital cells. Differences in cell tropism and kinetics of infection were identified between the HCMV strains. Specifically, differences in infecting endothelial cells and virus penetration into the lamina media were observed. In infected artery segments, but also in some non-infected arteries from seropositive donors, HCMV DNA could be localized by in situ PCR. Nevertheless, HCMV early antigen was detected by immunohistochemistry exclusively in artery segments infected in vitro. The new organ culture model will permit the study of functional and molecular consequences of HCMV infection in a more physiological micro-environment.

The possible correlation between cytomegalovirus, human herpesvirus types 6, 7 and cytomegalovirus-related clinical symptoms was studied in kidney transplant patients in Kuwait. Cytomegalovirus infection was diagnosed using the pp65 antigenemia assay. DNA of cytomegalovirus was detected by nested polymerase chain reaction (nested-PCR). PCR was also used to amplify the genes coding for structural proteins of human herpesvirus-6 (240 bp) and human herpesvirus-7 (186 bp). Glycoprotein B genotypes of cytomegalovirus were determined by restriction fragment length polymorphism. The average number of cells positive for cytomegalovirus pp65 antigen showed a steady increase with the severity of the cytomegalovirus-related symptoms. Furthermore, cytomegalovirus pp65 antigen positivity was significantly more frequent among recipients of cadaver kidney (45.5%) than among those who received live related kidneys (22.6%). Cytomegalovirus gB genotype 1 was detected more frequently (P<0.036) in recipients with live related donor kidney (38%) than in patients of cadaver kidney (13%). The genome of human herpesvirus-6 was detected at the same rate in patients with or without cytomegalovirus-related symptoms. However, the genome of human herpesvirus-7 was detected significantly more frequently (P<0.0001) in asymptomatic patients (41.7%) than in recipients with symptomatic cytomegalovirus infection (17%). We conclude that cytomegalovirus gB genotypes are not associated with the outcome of a cytomegalovirus infection in kidney transplant patients, that human herpesvirus-6 does not play a role in cytomegalovirus pathogenesis and that the role of human herpesvirus-7 in cytomegalovirus-related morbidity in kidney recipients remains unclear.

Human cytomegalovirus isolates were analyzed, both by restriction fragment-length polymorphism typing and by sequencing for intra- and intergenic variability at 9 sites on the genome, to determine whether genetic variation influenced disease outcome and whether linkage among genes could be identified. Variation at the UL55 (glycoprotein B [gB]), UL74 (gO), UL75 (gH), UL115 (gL), US9, and US28 gene open-reading frames was studied in relationship to outcome of cytomegalovirus disease. Major findings were that (1) on the basis of analysis of only 9 genomic sites, it is apparent that an almost infinite number of genetic combinations are theoretically possible; (2) genetic linkages are rare; (3) intragenic variability may be a complicating factor in molecular epidemiologic studies; and (4) analysis of only a single gene from a clinical isolate may not reveal the presence of either intragenic variants or mixtures of genotypes.

The 230-kb human cytomegalovirus genome is among the largest of the known viruses. Experiments to determine the genetic determinants of attenuation, pathogenesis, and tissue tropism are underway; however, a lack of complete sequence data for multiple strains and substantial problems with genetic instability during in vitro propagation create serious complications for such studies. For example, recent findings suggest that common laboratory strains Towne and AD169 passaged in cultured human fibroblasts are missing up to 15 kb of genetic information relative to clinical isolates. To establish standard, genetically stable genomes that can be sequenced, disseminated, and repeatedly reconstituted to produce virus stocks, we have undertaken to clone two variants of Towne, designated Towne(long) and Towne(short) (referred to as TownevarRIT3) (A., Proc. Natl. Acad. Sci. USA 98, 7829-7834), and the pathogenic strain Toledo into bacterial artificial chromosomes (BACs). We further demonstrate the ease with which mutagenesis can be achieved by deleting 13.5 kb from the Toledo genome using a PCR-based technique.

Human cytomegalvirus (HCMV) ORF UL73 is a polymorphic locus, encoding the viral glycoprotein gpUL73-gN, a component of the gC-II envelope complex. The previously identified gN genomic variants, denoted gN-1, gN-2, gN-3 and gN-4, were further investigated in this work by analysing a large panel of HCMV clinical isolates collected from all over the world (223 samples). Sequencing and phylogenetic analysis confirmed the existence of the four gN genotypes, but also allowed the identification of a novel subgroup belonging to the gN-3 genotype, which was designated gN-3b. The number of non-synonymous (d(N)) and synonymous (d(S)) nucleotide substitutions and their ratio (d(N)/d(S)) were estimated among the gN genotypes to evaluate the possibility of positive selection. Results showed that the four variants evolved by neutral (random) selection, but that the gN-3 and gN-4 genotypes are maintained by positive selective pressure. The 223 HCMV clinical isolates were subdivided according to their geographical origin, and four main regions of gN prevalence were identified: Europe, China, Australia and Northern America. The gN variants were found to be widespread and represented within the regions analysed without any significant difference, and no new genotype was detected. Finally, for clinical and epidemiological purposes, a rapid and low-cost method for genetic grouping of the HCMV clinical isolates was developed based on the RFLP revealed by SacI, ScaI and SalI digestion of the PCR-amplified UL73 sequence. This technique enabled us to distinguish all four gN genomic variants and also their subtypes.

Human cytomegalovirus (HCMV) strains, obtained from immunocompetent and immunocompromised Italian hosts, were typed with glycoprotein B (gB) gene restriction analysis. A predominant circulation of HCMV strains with gB type 2 and 3 was detected in both the immunocompetent host with a primary HCMV infection and the immunocompromised host with or without HCMV disease. No association between gB types and subjects with different risks of developing HCMV disease was found. All four gB genotypes were capable of causing congenital infection in Italian babies, with gB type 1 accounting for 50% of the strains examined in symptomatic infants and a remarkable incidence of gB type 4 viruses.

Preemptive ganciclovir therapy has reduced the occurrence of early cytomegalovirus (CMV) disease after hematopoietic stem cell (HSC) transplantation. However, late disease is increasingly reported. We describe 2 patients who developed late CMV central nervous system (CNS) disease after haploidentical HSC transplantation. Direct genotypic analysis was used to examine the presence of ganciclovir resistance. One patient had a mixed viral population in the cerebrospinal fluid (CSF), with coexistent wild-type and mutant UL97 sequences. The presence of 2 different strains was confirmed by subclone sequencing of the UL54 gene. One of the strains was different from the concurrent blood strain. The second patient had resistant variant in the lungs. These cases raise concern about the changing natural history of CMV disease in HSC transplantation, with emergence of previously uncommon manifestations following prolonged prophylaxis. Under these circumstances the CNS may be a sanctuary site, where viral persistence and antiviral drug resistance could result from limited drug penetration.

Non-human primate herpesviruses establish and maintain a lifelong persistent infection in immunocompetent hosts in the absence of clinical signs of disease. A fundamental issue for understanding the natural history of non-human primate herpesviruses is whether the viruses are maintained in a truly latent state or one characterized by a low level of chronic expression. To address this issue, a real-time PCR assay was developed to quantify Cercopithecine herpesvirus type 1 (B virus) DNA in mucosal fluids of rhesus macaques. This assay was rapid, sensitive (10 genome copies) and specific for B virus obtained from multiple species of macaques. The shedding profile of B virus was compared to another endemic herpesvirus, rhesus cytomegalovirus (RhCMV), in colony-reared monkeys. Mucosal swabs or saliva samples were taken daily from two groups of seropositive monkeys undergoing either a stressful relocation (group 1) or daily chair restraint (group 2). B virus DNA was detected in mucosal fluids from four animals relocated during the breeding season (group 1) but not from 10 animals moved at other times of the year. No B virus DNA was detected in any group 2 monkey. In contrast, RhCMV DNA was detected in the majority of animals of both groups 1 and 2. Detection of B virus DNA shedding is a relatively rare event associated with the breeding season, while RhCMV DNA is persistently detected in mucosal fluids of most monkeys.

To investigate the role of cytomegalovirus (CMV) gB genotypes, we examined the relationship between gB genotypes and clinical features in 19 patients with CMV infection. We analyzed 9 immunocompetent patients (1A) and 5 patients (1B) with various basic disorders in the symptomatic group. Five patients were enrolled in the asymptomatic group (2). To investigate gB genotypes, PCR was performed with oligonucleotide primers using clinical specimens and PCR products were digested with HinfI and RsaI We determined the PCR genotype by using restriction fragment length polymorphism (RFLP) analysis patterns. In the symptomatic group (1A and 1B), all clinical specimens were identified as gB genotype 1. In the asymptomatic group (2), only 2 specimens were identified as gB genotype 3. The distribution of CMV gB genotypes in clinical specimens was as follows: gB 1-17 out of 19 (89.5%) and gB 3-2 out of 19 (10.5%). Our data revealed that gB genotype 3 CMV did not show symptomatic infection.

The UL74 (glycoprotein O [gO])-UL75 (gH)-UL115 (gL) complex of human cytomegalovirus (CMV), known as the gCIII complex, is likely to play an important role in the life cycle of the virus. The gH and gL proteins have been associated with biological activities, such as the induction of virus-neutralizing antibody, cell-virus fusion, and cell-to-cell spread of the virus. The sequences of the two gH gene variants, readily recognizable by restriction endonuclease polymorphism, are well conserved among clinical isolates, but nothing is known about the sequence variability of the gL and gO genes. Sequencing of the full-length gL and gO genes was performed with 22 to 39 clinical isolates, as well as with laboratory strains AD169, Towne, and Toledo, to determine phylogenetically based variants of the genes. The sequence information provided the basis for identifying gL and gO variants by restriction endonuclease polymorphism. The predicted gL amino acid sequences varied less than 2% among the isolates, but the variability of gO among the isolates approached 45%. The variants of the genes coding for gCIII in laboratory strains Towne, AD169, and Toledo were different from those in most clinical isolates. When clinical isolates from different patient populations with various degrees of symptomatic CMV disease were surveyed, the gO1 variant occurred almost exclusively with the gH1 variant. The gL2 variant occurred with a significantly lower frequency in the gH1 variant group. There were no configurations of the gCIII complex that were specifically associated with symptomatic CMV disease or human immunodeficiency virus serologic status. The potential for the gCIII complex to exist in diverse genetic combinations in clinical isolates points to a new aspect that must be considered in studies of the significance of CMV strain variability.

To facilitate identification of rhesus cytomegalovirus (RhCMV)-infected cells, a recombinant virus expressing enhanced green fluorescent protein (EGFP), designated RhCMV-EGFP, was constructed. An expression cassette for EGFP under the control of the simian virus 40 (SV40) early promoter was inserted into the intergenic region between unique short 1 (US1) and US2 of the RhCMV genome by homologous recombination. RhCMV-EGFP exhibited comparable growth kinetics to that of wild-type virus in rhesus fibroblast cultures and retained its pathogenicity in monkey fetuses. Typical neurologic syndromes caused by CMV infection were observed in all fetuses experimentally inoculated with RhCMV-EGFP, as evidenced by sonographic and gross examinations. Systemic RhCMV infections were established in all fetuses, as viral antigen was detected in multiple organs and virus was isolated from fetal blood samples. The engineered viral genome was stable following rapid serial passages in vitro and multiple rounds of replication in vivo. Infected cells could be readily distinguished by green fluorescence both in tissue cultures and in the fetuses. In addition, EGFP expression was detected in various cell types that were permissive to RhCMV infection, consistent with a broad tissue tropism of the SV40 promoter. These results demonstrate that RhCMV can be successfully engineered without loss of wild-type replication and pathogenic potential. Further, the spectrum of cortical anomalies and the distribution of infected cells in the brain tissues indicated that RhCMV may have preferentially targeted immature neuronal cells. The pattern of RhCMV infection in the central nervous system may offer an explanation for the severe developmental outcomes associated with congenital human CMV infection early in gestation.

The Towne strain of human cytomegalovirus (HCMV), originally recovered from the urine of a congenitally infected newborn, was attenuated through 125 passages in human embryonic lung fibroblast cell cultures. Although reliable markers of attenuation were not identified, the virus was shown to be attenuated by inoculation of both healthy human volunteers and immunocompromised patients. More recently, Towne (like other laboratory-adapted strains) was shown not to have two biological properties typical of recent clinical isolates: endothelial cell tropism and polymorphonuclear leukocyte tropism. These markers of attenuation are lost by all clinical isolates on extensive propagation in cell cultures and are apparently associated with one another. Here, we show that Towne may reacquire both endothelial cell tropism and leuko- (polymorphonuclear- and monocyte-) tropism on adaptation to growth in endothelial cell cultures. However, reversion to endothelial cell tropism is dissociated from reversion to leukotropism, since the latter was reacquired 10-20 passages later. Thus, these two biological properties, which were considered to be encoded by the same viral gene(s), appear to be distinct. Both restriction fragment length polymorphism and Southern blot analysis demonstrated the identity of the attenuated and endothelial cell tropic variants of Towne, thus suggesting that only minor variations (mutations) of the viral genome may be responsible for loss or reacquisition of the two biological properties. Viral genes involved in endothelial cell tropism and leukotropism remain to be identified. However, reversion of attenuated strains to pathogenicity in vivo cannot be excluded a priori.

Background & Objectives: Since cytomegalovirus (CMV) is heterogenous and exhibits genomic polymorphism, polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) was applied to identify the glycoprotein B subtypes in patients diagnosed to have CMV disease., Methods: CMV standard strain (AD 169) and 55 clinical specimens from 50 patients (35 males; 15 females) positive for CMV by PCR coding for the morphological transforming region II gene were subjected to PCR coding for gp 55 region of CMV gB gene. PCR amplified products were subjected to RFLP using Hinf I., Results: Of 50 patients, 26 (52.0%) (20 males; 6 females) were positive by PCR (gB gene). Upon RFLP, AD 169 and CMV strains of 14 (53.8%) patients (14 males) were typed as subtype II, and 12 (46.1%) (6 males; 6 females) as subtype III. Of 11 paediatric patients, 7 (63.6%) were infected with CMV subtype II and 4 (36.4%) with subtype III. CMV strains of the dual specimens from 3 PCR positive patients belonged to the same subtype., Interpretation & Conclusion: In this study on CMV genotypes, CMV gB subtypes II and III were found in 53.8 and 46.1 per cent patients studied respectively, while I and IV were not present.

A polymerase chain reaction (PCR) based assay for detection and species identification of four human herpesviruses, including herpes simplex virus types 1 and 2, Epstein-Barr virus, and cytomegalovirus was developed. The detection of the herpesviruses was achieved by seminested PCR with three primers targeting well-conserved regions within the DNA-polymerase gene. Virus species were identified by simple restriction enzyme digestion of the amplified products with TaqI or RsaI. In comparison with mono-specific nested PCR assays the tetra-specific assay demonstrated similar specificity and sensitivity with reference and clinical samples. The tetra-specific assay is sensitive, cost effective, and can be used for examination of clinical samples of different origin.

Porcine cytomegalovirus (PCMV) is one of the pathogens that should be eliminated from pigs intended for use as organ donors in xenotransplantation. For this purpose, reliable diagnostic test systems are needed. To provide a basis for this goal and to analyse the evolutionary relationships of PCMV within the herpesvirus family, the putative glycoprotein B (gB) gene of PCMV was identified by assuming gene colinearity and a relative conservation of nucleotide sequences in comparison with closely related herpesviruses. Using this approach the complete nucleotide sequence of the PCMV gB gene was determined. A protein of 860 amino acids was deduced and a putative cleavage site, conserved cysteine residues, as well as potential N-terminal glycosylation motifs were identified. In a comparison of PCMV gB with the corresponding region of other herpesviruses, the highest identities were found with human herpesviruses 6 and 7 (HHV-6 and 7; 43.4% and 42.6%, respectively). Also in phylogenetic analysis, the PCMV gB clustered with HHV-6 and HHV-7. Between the complete gB sequences of five different PCMV strains and isolates from the United Kingdom, Germany, Spain, Japan and Sweden, differences of 3.4% were found, indicating a considerable intra-species variation. The characterisation of the protein deduced from the identified gene provides further evidence that this is indeed the gB gene of PCMV and provides important taxonomical information regarding PCMV. The identification of the gB gene should facilitate the development of sensitive and robust diagnostic methods for the PCMV screening of pigs.

On the basis of the sequence variation of the glycoprotein B (gB) gene, human cytomegalovirus (HCMV) can be classified into four gB genotypes. Genotyping of HCMV from congenital infections was carried out on the assumption that the envelope gB may influence the outcome of prenatal infection. Sixty-three pregnant women were included in the study: 40 pregnant women whose fetuses were strongly suspected of having viral infection, and 23 women with normal pregnancies, from whom amniotic fluid was taken for fetal karyotype assessment. The amniotic fluid, fetal blood, blood, and urine of the newborns were examined for HCMV DNA by a nested polymerase chain reaction, and the gB genotype was determined by restriction fragment length polymorphism. HCMV DNA was detected in 12 cases in which the fetuses were suspected of having a viral infection and in 3 of the normal pregnancies. All the HCMV DNA had identical genotype, gB1. These data clearly indicate the dominance of the gB1 genotype in congenital HCMV infections. The clinical outcome of these pregnancies, however, cannot be predicted on the basis of the involvement of this genotype., (Copyright 2001 Wiley-Liss, Inc.)

Clinical isolates of human cytomegalovirus (HCMV) show differences in tissue tropism, severity of clinical manifestations and ability to establish persistent or latent infections, characteristics that are thought to be related to genomic variation among strains. This work analysed the genomic variants of a new HCMV polymorphic locus, open reading frame (ORF) UL73. This ORF encodes the envelope glycoprotein gpUL73 (gN), which associates in a high molecular mass complex with its counterpart, gM, and induces a neutralizing antibody response in the host. Detailed sequence analysis of ORF UL73 and its gene product from clinical isolates and laboratory-adapted strains shows that this glycoprotein is highly polymorphic, in the N-terminal region in particular. gpUL73 hypervariability is not randomly distributed, but the identified genomic variants are clearly clustered into four distinct genotypes (gN-1, gN-2, gN-3 and gN-4), which are not associated with the gB subtype.

The term 'genotyping' describes the genetic characterization of a genome. The genotype analysis is performed to identify mutations that differentiate one individual or strain from another. The mutations may confer resistance to specific antiviral drugs or they may simply allow classification of a strain as to 'type' and 'subtype'. There are four human viruses for which genotype information is clinically useful. Hepatitis B virus (HBV) infections are being treated with antiretroviral drugs and resistance after prolonged treatment is common. Since HBV cannot be cultured, the only method of detecting resistance-conferring mutations in the genome is a genotypic analysis. Hepatitis C virus (HCV) infection can be cured by treatment with the combination of interferon and ribavirin but certain strains of virus are more resistant to treatment than others. The current recommendations are that all HCV type 1 infections be treated for 12 months whereas other types may be successfully treated in 6 months. Since interferon treatment may have significant side effects, the determination of HCV genotype is an important aspect of this therapeutic regimen. Treatment of cytomegalovirus (CMV) disease with nucleoside analogues occasionally results in resistant virus with mutations in the phosphotransferase gene (UL97) and/or the DNA polymerase gene (UL54) that can be tested with phenotypic or genotypic assays. Since CMV grows very slowly, it may be more clinically useful to perform a rapid genotypic assay although only the UL97 gene can be efficiently genotyped. Finally, the virus for which genotyping has become the standard of care, human immunodeficiency virus type 1 (HIV-1) can now be genotyped routinely by many clinical virology labs experienced with molecular amplification methods and automated DNA sequencing technology. All currently-available antiretroviral drugs are directed against either the protease or reverse transcriptase genes of HIV-1 and the mutations within these genes that confer resistance have been well described. Sequence-based genotyping methods are not necessarily the best approach for routine genotyping of these four viruses, but sequencing is the gold standard from which other methods are developed and against which they are compared.