A panel of human sera exhibited a ¢128-fold higher neutralizing potency against a human cytomegalovirus (HCMV) clinical isolate propagated and tested in endothelial (or epithelial) cells than against the same virus infecting human fibroblasts. In a group of 18 primary infections, the reverse geometric mean titre was in the range of 10-15 in human fibroblasts within the first 3 months after the onset of infection, whereas the endothelial cell infection-neutralizing activity was already present within the first 10 days, reaching median levels of 122, 320 and 545 at respectively 30, 60 and 90 days after onset, then declining slowly. This difference was also confirmed in the majority of reactivated and remote HCMV infections, as well as in a hyperimmune globulin preparation. The antibody response to HCMV pUL131A, pUL130 and pUL128 locus products, which are required for endothelial/epithelial cell infection, provided a potential molecular basis for such a differential neutralizing activity. In addition, monoclonal/monospecific antibodies raised against the pUL131A, pUL130 and pUL128 proteins were found to display an inhibitory activity on HCMV plaque formation and HCMV leukocyte transfer from HCMV-infected cells. Hence, conventional determination of the neutralizing activity of human sera in fibroblasts is misleading. Antibodies to pUL131A, pUL130 and pUL128 appear to display a major HCMV-neutralizing and dissemination-inhibiting activity.

BackgroundDirect-acting antiviral (DAA) agents target HCV proteins; some of these have already been approved for the treatment of HCV infection, while others are in development. However, selection of DAA-resistant viral variants may hamper treatment. The aim of this study was to illustrate potential natural DAA-resistance mutations in the HCV NS5A and NS5B regions of HCV genotypes 1a and 1b from DAA-naïve patients.MethodsDirect sequencing of HCV NS5A and NS5B regions was performed in 32 patients infected with HCV genotype 1a and 30 patients infected with HCV genotype 1b; all subjects were naïve to DAAs.ResultsIn genotype 1a strains, resistance mutations in NS5A (M28V, L31M and H58P) were observed in 4/32 (12.5%) patients, and resistance mutations in NS5B (V321I, M426L, Y448H, Y452H) were observed in 4/32 (12.5%) patients. In genotype 1b, resistance mutations in NS5A (L28V, L31M, Q54H, Y93H and I280V) were observed in 16/30 (53.3%) patients, while resistance mutations in NS5B (L159F, V321I, C316N, M426L, Y452H, R465G and V499A) were observed in 27/30 (90%) patients.ConclusionsMutations conferring DAA resistance were detected in NS5A and NS5B of HCV genotypes 1a and 1b from DAA-naïve patients. Although some mutations confer only a low level of resistance, the presence at baseline of mutated HCV variants should be taken into consideration in the context of DAA therapy.

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BackgroundProtease inhibitors (PIs) to treat hepatitis C (HCV) virus infection have been approved and others are under development.ResultsThe aims of this study were to illustrate natural polymorphisms in the HCV protease and measure the frequency of PI resistance mutations in different HCV genotypes from PI-naïve patients.Direct sequencing of HCV NS3/4A protease was performed in 156 HCV patients naïve to PIs who were infected with genotype 1a (n = 31), 1b (n = 39), 2 (n = 30), 3 (n = 33) and 4 (n = 23).Amino acid (aa) substitutions associated with HCV PI resistance were found in 17/156 (10.8%) sequences. Mutations V36L, T54S, V55A/I, and Q80K/L were observed in 29% of patients with genotype 1a, and V55F, Q80L/N and M175L in 10% of patients with genotype 1b. The mutation V158M was found in 3% of patients with genotype 2, D168Q was present in 100% of patients with genotype 3 and D168E was observed in 13% of patients with genotype 4. In addition, multiple aa polymorphisms not associated with PI resistance were detected in patients with genotypes 1a, 1b and 4.ConclusionsAlthough major PI resistance mutations were not detected, other resistance mutations conferring low level resistance to PIs together with a number of natural polymorphisms were observed in proteases of PI naïve HCV patients. A more extensive analysis is needed to better evaluate the impact of baseline resistance and compensatory mutations in the efficacy of HCV PI treatment.

Human cytomegalovirus (HCMV) growth in endothelial cells (EC) requires the expression of the UL131A-128 locus proteins. In this study, the UL130 protein (pUL130), the product of the largest gene of the locus, is shown to be a luminal glycoprotein that is inefficiently secreted from infected cells but is incorporated into the virion envelope as a Golgi-matured form. To investigate the mechanism of the UL130-mediated promotion of viral growth in EC, we performed a complementation analysis of a UL130 mutant strain. To provide UL130 in trans to viral infections, we constructed human embryonic lung fibroblast (HELF) and human umbilical vein endothelial cell (HUVEC) derivative cell lines that express UL130 via a retroviral vector. When the UL130-negative virus was grown in UL130-complementing HELF, the infectivity of progeny virions for HUVEC was restored to the wild-type level. In contrast, the infectivity of the UL130-negative virus for UL130-complementing HUVEC was low and similar to that of the same virus infecting control noncomplementing HUVEC. The UL130-negative virus, regardless of whether or not it had been complemented in the prior cycle, could form plaques only on UL130-complementing HUVEC, not control HUVEC. Because (i) both wild-type and UL130-transcomplemented virions maintained their infectivity for HUVEC after purification, (ii) UL130 failed to complement in trans the UL130-negative virus when it was synthesized in a cell separate from the one that produced the virions, and (iii) pUL130 is a virion protein, models are favored in which pUL130 acquisition in the producer cell renders HCMV virions competent for a subsequent infection of EC.Human cytomegalovirus (HCMV) is a betaherpesvirus that establishes life-long, subclinical infections ubiquitously in human populations (5). HCMV causes serious morbidity in settings of immune system immaturity or depression: it is the leading viral cause of defects at birth and generates a potentially life-threatening disease in immunocompromised patients. In patients with HCMV disease, the virus can be demonstrated in a variety of cells, including hematopoietic cells (monocytes-macrophages, dendritic cells, and neutrophils), endothelial cells (EC), epithelial cells, fibroblasts, neurons, smooth muscle cells, and hepatocytes (5, 38). Much recent work has focused on HCMV infections of EC, for several reasons: (i) arterial endothelia, along with CD34 ϩ myeloid progenitors, have been proposed as sites of HCMV persistence and latency (20); (ii) a bidirectional transmission of HCMV between EC and leukocytes can be demonstrated in vitro and may reflect a mechanism of dissemination in vivo (11,14,34,44); (iii) circulating giant EC may similarly contribute directly to dissemination (32); (iv) HCMV infections of the uterine microvasculature and cytotrophoblasts may underlie motherto-fetus transmission and compromise the placental trophic function in affected pregnancies (25, 47); and (v) HCMV may play a role in atherogenesis, postangioplasty restenosis, posttransplantation endot...

BackgroundEnvironmental factors may play a role in colon cancer. In this view, several studies investigated tumor samples for the presence of various viral DNA with conflicting results.FindingsWe undertook a systematic DNA analysis of 44 consecutive, prospectively collected primary tumor samples by real time and qualitative PCR for viruses of known or potential oncogenic role in humans, including polyomavirus (JCV, BKV, Merkel cell polyomavirus), HPV, HTLV, HHV-8 and EBV. Negative controls consisted of surgical resection margins. No evidence of genomic DNA fragments from tested virus were detected, except for EBV, which was found in a significant portion of tumors (23/44, 52%). Real-time PCR showed that EBV DNA was present at a highly variable content (median 258 copies in 105 cells, range 15–4837). Presence of EBV DNA had a trend to be associated with high lymphocyte infiltration (p = 0.06, χ2 test), and in situ hybridization with EBER1-2 probes revealed latency in a fraction of these lymphoid cells, with just a few scattered plasma cells positive for BZLF-1, an immediate early protein expressed during lytic replication. LMP-1 expression was undetectable by immunohistochemistry.ConclusionsThese results argue against a significant involvement of the tested oncogenic viruses in established colon cancer.

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Human cytomegalovirus (HCMV) encodes a protein related to the large (R1) subunit of ribonucleotide reductase (RR), but does not encode the corresponding small (R2) subunit. The R1 homologue, UL45, lacks many catalytic residues, and its impact on deoxyribonucleotide (dNTP) production remains unknown. Here, UL45 is shown to accumulate at late stages of infection and to be a virion tegument protein. To study UL45 function in its genome context, UL45 was disrupted by transposon insertion. The UL45-knockout (UL45-KO) mutant exhibited a growth defect in fibroblasts at a low m.o.i. and also a cell-to-cell spread defect. This did not result from a reduced dNTP supply because dNTP pools were unchanged in resting cells infected with the mutant virus. Irrespective of UL45 expression, all cellular RR subunits -S-phase RR subunits, and the p53-dependent p53R2 -were induced by infection. p53R2 was targeted to the infected cell nucleus, suggesting that HCMV diverts a mechanism normally activated by DNA damage response. Cells infected with the UL45-KO mutant were moderately sensitized to Fas-induced apoptosis relative to those infected with the parental virus. Together with the report on the UL45-KO endotheliotropic HCMV mutant (Hahn et al., J Virol 76, 9551-9555, 2002), these data suggest that UL45 does not share the prominent antiapototic role attributed to the mouse cytomegalovirus homologue M45 (Brune et al., Science 291, 303-305, 2001). INTRODUCTIONHuman cytomegalovirus (HCMV), a ubiquitous b-herpesvirus that causes severe disease in immunocompromised individuals and in the newborn, replicates in differentiated cells (fibroblasts, microglial, epithelial, endothelial and smooth muscle cells, and monocyte-derived macrophages; Britt & Alford, 1996; Sinzger et al., 1995), and is transported in the bloodstream by abortively infected neutrophils (Gerna et al., 2000). Latent infection is detected in macrophagegranulocyte precursors in the bone marrow and in circulating monocytes (Maciejewski et al., 1992; SoderbergNaucler et al., 1997;Hahn et al., 1998).The class I ribonucleoside diphosphate (ribonucleotide) reductases (RR), which catalyse a limiting step in de novo deoxyribonucleotide (dNTP) synthesis, are essential for DNA replication of both eukaryotic cells and the DNA viruses that infect them (Jordan & Reichard, 1998;Stubbe et al., 2001). RR holoenzymes comprise a catalytic large (R1) subunit, and a small (R2) subunit required for enzyme activation (Jordan & Reichard, 1998). Like many large DNA viruses, the a-and c-herpesviruses encode both subunits of a viral RR, which is important for virus replication in resting or post-mitotic cells (Jacobson et al., 1989;Idowu et al., 1992;Heineman & Cohen, 1994;Aurelian, 1998) because these have minute dNTP pools and virtually no endogenous RR (Engstrom et al., 1985;Jordan & Reichard, 1998;Chabes & Thelander, 2000). In addition, the herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) R1 proteins bear an N-terminal extension (see supplementary data Fig. 1 at JGV Online, http://vir.sg...