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Objective Many studies on integration have reported conflicting results regarding the role of HPV integration in cervical cancer. We hypothesized that high viral load and disruption of E2 gene associated with integration of HPV were not the only pathway leading to cancer development. Methods This study analysed the viral load and integration status of HPV16, measured the HPV16 E6/E7 mRNA transcript levels, delineated the E2 and LCR sequence variation, and determined the methylation status of two E2 binding sites. Results The results showed that viral load was not associated with the physical status of HPV genome. Levels of the three E6/E7 mRNA transcripts in invasive cervical cancers containing purely episomal viral genome were found to be similar to those containing integrated viral genome, suggesting that cancers containing episomal viral genome were also mediated by an up-regulated E6/E7 mRNA expression, and more importantly, did not depend on integration and disruption of the E2 gene. Conclusions The alternative mechanism that up-regulated the expression of E6 and E7 in invasive cancers harbouring episomal viral genome was likely to be a consequence of methylation of the two E2 binding sites located at the promoter region of HPV16. These observations are in line with the hypothesis that HPV integration was not the only mechanism leading to the development of cervical cancer.

BACKGROUND A need for quadrivalent vaccines to cover both lineages of influenza B has been raised. Information on the circulation status of influenza B lineages and the associated hospitalization rates is important to assist evidence-based decision making. This retrospective study revealed the situation in a subtropical city over a 10-year period. METHODS Sequences of 268 influenza B isolates were analyzed to identify the circulating pool of virus lineages for each year. Hospital records and population census data were used to estimate annual age-specific hospitalization rates. RESULTS Cocirculation with 2 influenza B lineages was found in 9 of the 10 years. Only in 6 of the 10 years had the vaccine strain successfully matched with the lineage that was found in >50% of the circulating pool. Six years were predominated by one lineage (occupying >80% of the circulating pool), and these years had higher (average, 1.4-fold) hospitalization rates. Matching between vaccine and circulating lineage was achieved only in 2 of the 6 "predominated years." The Yamagata lineage accounted for most (5/6) of the predominated years. Overall, 24% of influenza admissions were due to influenza B, and influenza B contributed to a higher proportion (41.9%) among children and young teenagers (5-14 years old). CONCLUSIONS Cocirculation with 2 influenza B lineages is common in the subtropical region. To predict the next predominant lineage proves to be difficult. Influenza B accounts for a substantial fraction of influenza-associated hospitalizations, especially among children and young teenagers. Quadrivalent vaccines may improve the effectiveness of influenza vaccination programs.

The E6 and E7 proteins encoded by human papillomaviruses (HPV) are prime targets for therapeutic vaccine development. Ninety-five women with HPV 52 infection (33 transient infections, 17 cervical intraepithelial neoplasia grade II, 15 cervical intraepithelial neoplasia grade III, and 30 invasive cervical cancers) were examined for T-cell responses using interferon-γ enzyme-linked immunospot (IFN-γ ELISPOT) assay. Of the 29 peptides (13 L1, 10 E6, and 6 E7) screened positive by an in vitro peptide-binding assay, 14 were positive by the IFN-γ ELISPOT assay. Positive epitopes for HLA A11 were located at amino acid positions 103-111, 332-340, 342-350, and 373-381 of the L1 protein; and at 27-35 and 86-94 of the E6 protein; and at 1-9 and 27-35 of the E7 protein. A24-specific epitopes included 60-68 and 98-106 of the L1 protein, 42-50 and 59-67 of the E6 protein, and 24-32 of the E7 protein. Only one epitope (99-107) of the E6 protein showed positive responses for HLA A2 subjects. Overall, T-cell responses against L1 were observed mainly in subjects who had cleared infection; whereas responses against E6 and E7 were confined mainly to subjects who had developed cervical neoplasia. The proportion of subjects showing detectable T-cell responses was low across all grades of cervical neoplasia suggesting that immune evasion mechanisms had set on early in the course of disease progression. This study provides the first set of T-cell epitopes mapped for HPV 52, which can be considered for further evaluation as targets for immunotherapy.

Objective We undertook a prospective cohort study to ascertain the risk factors for the development of squamous intraepithelial lesions (SIL) in patients with systemic lupus erythematosus (SLE). Methods One hundred thirty-seven SLE patients with a normal Papanicolaou (Pap) smear at baseline were evaluated at 6-month intervals for up to 3 years. At each visit, a Pap smear, human papillomavirus (HPV) DNA test, and clinical assessment were performed. Results Among the 137 patients, there were 12 incident cases (8.8%) of SIL over a median followup duration of 30.7 months (interquartile range 25.5–31.7). Among the 30 patients with HPV infection detectable by DNA testing at baseline, 9 (30%) developed SIL. The independent risk factors for the incident SIL in this group of SLE patients included the use of cyclophosphamide (CYC) ever (odds ratio [OR] 5.6, 95% confidence interval [95% CI] 1.1–29.3; P = 0.041) and persistent high-risk HPV infection (OR 26.9, 95% CI 3.2–222.3; P = 0.002). The use of baseline HPV testing has a higher sensitivity than abnormal cytology (defined as atypical squamous cells of undetermined significance; 47.7% versus 33.3%) in predicting the development of SIL. Conclusion Independent risk factors associated with the development of SIL in SLE patients included persistent high-risk HPV infection and the use of CYC. Low-risk patients who receive negative test results on both cervical cytology screening and HPV DNA testing may not need to be rescreened within 3 years.

Human papillomavirus type 58 (HPV-58) exists in a relatively high prevalence in certain parts of the world, including East Asia. This study examined the T-cell response to HPV-58 L1, E6, and E7 peptides among women with cleared infection, cervical intraepithelial neoplasia grade 2 (CIN2) or CIN3, or invasive cervical cancer (ICC). Peptides found to be reactive in the in vitro peptide binding assay or mouse-stimulating study were tested with a gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay to detect peptide-specific responses from the peripheral blood mononuclear cells (PBMC) collected from 91 HPV-58-infected women (32 with cleared infection, 16 CIN2, 15 CIN3, and 28 ICC). Four HLA-A11-restricted HPV-58 L1 peptides, located at amino acid positions 296 to 304, 327 to 335, 101 to 109, and 469 to 477, showed positive IFN-γ ELISPOT results and were mainly from women with cleared infection. Two HLA-A11-restricted E6 peptides (amino acid positions 64 to 72 and 94 to 102) and three HLA-A11-restricted E7 peptides (amino acid positions 78 to 86, 74 to 82, and 88 to 96) showed a positive response. A response to E6 and E7 peptides was mainly observed from subjects with CIN2 or above. One HLA-A2-restricted E6 peptide, located at amino acid position 99 to 107, elicited a positive response in two CIN2 subjects. One HLA-A24-restricted L1 peptide, located at amino acid position 468 to 476, also elicited a positive response in two CIN2 subjects. In summary, this study has identified a few immunogenic epitopes for HPV-58 E6 and E7 proteins. It is worthwhile to further investigate whether responses to these epitopes have a role in clearing an established cervical lesion.

Human infection with the novel pandemic influenza A (H1N1) virus was first identified in April 2009. Two months later, the World Health Organization (WHO) had raised the pandemic level to phase 6. Rapid case identification is essential for prompt patient management and public health actions. This study developed real-time and conventional reverse transcription-polymerase chain reaction (rRT-PCR and cRT-PCR) assays for pandemic H1N1 detection, and compared their sensitivities with protocols developed by WHO reference centres. Altogether, three rRT-PCR and one cRT-PCR targeting the matrix gene for universal detection of influenza A; three rRT-PCR, one cRT-PCR targeting the hemagglutinin (HA) gene for specific detection of pandemic H1N1; and one multiplex cRT-PCR for differentiating co-circulating seasonal H1N1, H3N2, and pandemic H1N1 were examined. The lower detection limit ranged from 1.252 to 125.2 copy equivalents. In general, rRT-PCR assays were more sensitive than cRT-PCR assays. All assays showed 100% sensitivity for “optimal” specimens (nasopharyngeal samples collected within 4 days after illness onset). For the other 36 samples, cRT-PCR assays were less sensitive except that the new Protocol I-cRT-pdmH1 still retained 100% sensitivity. The new Protocol F-rRT-PCR-pdmH1 was the only pandemic virus-specific rRT-PCR assay with 100% sensitivity across all specimen categories. In conclusion, rRT-PCR assays are 10-fold more sensitive than cRT-PCR assays. The newly developed cRT-PCR assay targeting the HA gene allows rapid, specific, and sensitive screening of this novel agent, which can serve as an alternative for laboratories where a real-time PCR machine is not available. J. Med. Virol. 82:675–683, 2010. © 2010 Wiley-Liss, Inc.

This cross-sectional study examined the distribution of HPV 58 sequence variation in Korean women for the first time. Among 1,750 Korean women, 53 women were positive for HPV 58 single infection, of whom 26 were without disease, 20 were with cervical intraepithelial neoplasia (CIN) 1, and 7 with CIN 2 or 3. Altogether, 36 different nucleotide sequence variations were identified with the L1, 20 within E2, 5 within E6, and 10 within E7. Further studies on variants of oncogenic HPVs are necessary, particularly for the purpose of developing more predictive HPV detection methods.

Background A wide spectrum of human papillomavirus (HPV) types can infect the male genitalia. An HPV vaccine covering HPV6 and 11 is now available. Detailed data on the distribution of these two types in anogenital warts is needed to assess the potential benefits of the vaccine. Study design Anogenital wart specimens collected from 130 Chinese men were examined for HPV-type distribution by a method that covers a broad spectrum of high- and low-risk HPVs, and able to reveal multiple types from a single specimen. Results Forty-four (33.8%) of the 130 specimens had a coinfection with multiple HPV types. In 63.1% of cases, only HPV6 and/or HPV11 were/was found. In 26.2% of cases, HPV6 and/or HPV11 were/was found together with one or more other HPV types. In 10.8% of specimens, only non-6/11 HPV types were found. HPV16 and/or 18 were/was found in 12 (9.2%) specimens, with majority (8/12, 66.7%) of which existed as coinfections with HPV6/11. Other HPV types found included HPV39, 51, 52, 55, 59, 61, 62, 68, 58, 72, 81, 83, 84 and CP6108. Conclusions A substantial proportion of HPV6/11-positive male anogenital warts are coinfected with other HPV types. The efficacy of HPV6/11 vaccine for preventing these lesions needs to be defined before the benefits of vaccinating men can be precisely assessed.

BACKGROUND: Viral safety is of paramount importance for human plasma–derived therapeutic proteins. Recent reports of blood-associated transmission and continuous regional outbreaks of dengue fever have prompted a validation of clearance of dengue virus in the manufacture processes of the plasma-derived products. STUDY DESIGN AND METHODS: A high titer of cultured dengue virus serotype 2 was spiked into process samples before individual steps of albumin and immunoglobulin manufacture processes, including cold ethanol precipitation, cation-exchange chromatography, pasteurization, solvent/detergent treatment, and virus filtration. Clearance of dengue virus was quantified with TCID50 assays in the culture of Vero E6 cells and, when appropriate, real-time polymerase chain reaction (RT-PCR) assays. RESULTS: The individual process steps were all effective in the inactivation and/or removal of dengue virus, and the data obtained clearly demonstrate that the risk of dengue virus transmission was reduced cumulatively by at least 10.12 and at least 14.24 log in the albumin and immunoglobulin manufacture processes, respectively. CONCLUSION: The dedicated viral inactivation and/or removal approaches currently implemented in the manufacture of plasma-derived products provide a good safety margin with regard to the transmission of dengue virus.

Multiple determinants are involved in the progression of human papillomavirus (HPV)-infected cervical lesion to invasive cancer. Human leukocyte antigen (HLA) polymorphism seems to play a role. This study examined the association between HLA-DRB1 polymorphism, high-risk HPV infection, and the development of cervical neoplasia in southern Chinese. Three hundred and seventy women with cervical neoplasia (43 cervical intraepithelial neoplasia grade II, 154 grade III, and 173 invasive cancers) and 323 controls were recruited for HLA-DRB1 typing by a sequence-based approach. Cervical specimens were collected for HPV detection by a consensus primer-based polymerase chain reaction, and with the type of HPV identified by hybridization with type-specific oligonucleotide probes. A protective effect of HLA-DRB1*12 for cervical neoplasia was observed, and with stronger associations when subgroup analyses were carried out for patients infected with HPV16 and HPV58. The protective effect of HLA-DRB1*13 that had been reported from other populations was not observed. The data obtained in this study showed that HLA-DRB1*03 conferred a higher risk for HPV18-infected, but not for HPV16-, HPV52-, or HPV58-infected cervical lesions. Although, HPV52 was reported as uncommon worldwide, it was found to be the second most prevalent type in the southern Chinese population. However, no additional risk association was observed when subgroup analyses were performed for HPV52-infected patients. The current study shows that, among southern Chinese, the outcome of HPV-infected cervical lesions is associated with HLA-DRB1 polymorphism. These associations often vary with the type of HPV infection.

A 42-year-old male with stage IV mantle cell lymphoma received chemotherapy and autologous peripheral blood stem cell transplantation. He developed pancytopaenia, and bone marrow examination indicated a parvovirus B19 (PVB 19)-induced red cell aplasia, confirmed by virological tests. Multiple doses of intravenous immunoglobulin (IVIG) were given over the following months, with blood samples being taken after each dose for quantitative PVB 19 DNA and hematological testing to assess the response. Each dose of IVIG produced a 1–3 log10 drop in PVB 19 DNA levels. Eventually, after the fifth dose of IVIG, the PVB 19 DNA was reduced to

The severe acute respiratory syndrome–associated coronavirus (SARS-CoV) is reported to have deletions of various sizes. Recently, the large 386-nucleotide deletion (L386del) comprising nucleotide positions 27719-28104 and spanning open reading frames 9–11 has been reported in the genomes of some human isolates from Hong Kong. In this study, archived specimens from 71 patients with SARS who were admitted to the New Territory East Cluster Hospitals in Hong Kong were analyzed to determine whether the L386del variant of SARS-CoV was present. There was no clear relationship between the presence of the L386del variant and SARS clinical severity as defined either by the need for intensive-care therapy and/or ventilation or by death. One patient had evidence of both the L386del variant and the wild-type variant in the same clinical specimen, supporting the idea that SARS-CoV exists as a quasispecies in some patients, although the clinical significance of these quasispecies remains unclear

This study describes the distribution of hepatitis C genotypes among 106 intravenous drug users and 949 non-drug users in Hong Kong. Genotypes were identified by multiplex RT-PCR targeting the core region of viral genome. The accuracy of this typing system in identifying genotypes 1b and 6a was assessed by phylogenetic analysis. The distribution of hepatitis C virus (HCV) genotypes amongst non-drug users was 63.6% for genotype 1b, 23.6% for 6a, 4.5% for 1a, 3.9% for 3a, and 3.1% for 2a; whereas amongst the intravenous drug users, it was 58.5% for genotype 6a, 33.0% for 1b, 5.7% for 3a, 0.9% for 1a, and 0.9% for 2a. The proportion of genotype 6a was significantly higher (P

To examine the association between human leukocyte antigen-A (HLA-A) allele polymorphism human papillomavirus (HPV) infection and risk for cervical neoplasia in Chinese women 263 patients (155 with cervical intraepithelial neoplasia [CIN] II/III and 108 with invasive cervical cancer [ICC]) were compared with 572 controls. Overall regardless of HPV status a decreased risk for ICC was observed for patients with A*0207/ 0215N or A*2402 and an increased risk was observed for patients with A*1104. The protective association of A*0207/0215N was reproduced in HPV-16-positive patients with ICC but not in subgroups infected with other HPV types. The risk association between A*1104 and both HPV-16 and HPV-18 was reproduced in the subgroups with CIN III/ICC. The protective association between A*2402 and HPV-16 HPV-18 HPV-52 and HPV-58 was consistently observed in all subgroups with CIN III/ICC suggesting a linkage with a general antioncogenic genetic factor. The results of the present study indicate that HLA-A polymorphism is one of the host genetic factors that alter the risk for the development of cervical cancer in Chinese women. (authors)

The maturation of virus-specific immunoglobulin G avidity during severe acute respiratory syndrome-associated coronavirus infection was examined. The avidity indices were low (mean +/- SD, 30.8% +/- 11.6%) among serum samples collectedor =50 days after fever onset, intermediate (mean +/- SD, 52.1% +/- 14.1%) among samples collected between days 51 and 90, and high (mean +/- SD, 78.1% +/- 8.0%) among samples collected after day 90. Avidity indices of 40% and 55% could be considered as cutoff values for determination of recent (or =50 days) and past (65 days) infection, respectively. Measurement of antibody avidity can be used to differentiate primary infection from reexposure and to assess humoral responses to candidate vaccines.

Fifteen Chinese women with early stage cervical squamous cell carcinoma (14 stage IB, one stage IIA) were retrospectively analysed for the correlation between human papillomavirus (HPV) load in primary tumour and the presence of HPV DNA in histologically tumour-free pelvic lymph nodes. HPV16 DNA was detected from majority (12/15) of primary tumours, with a viral load ranging from 12 to 1800 copies per cell. Of the 156 histologically tumour-free pelvic lymph nodes, 41 (26.3%) were positive for HPV DNA. The levels of viral load detected in histologically tumour-free lymph nodes were low and most were not detectable by the less sensitive consensus PCR GP5+/6+. Among patients without histological evidence of nodal involvement, the presence of HPV DNA in lymph nodes was associated with a significantly higher viral load in primary tumour (mean [interquartile range]=800 [600–1450] versus 40 [19–70] copies per cell, P =0.016). Three of the four patients with recurrence had histological evidence of lymph node metastases. In contrast, none of the seven patients with HPV DNA-positive lymph nodes but without histologically evidence of nodal involvement developed recurrence. The results of this study suggest that the presence of HPV DNA in histologically tumour-free lymph nodes do not have prognostic significance. The HPV DNA detected from lymph nodes may have originated from circulating necrotic tumour cells or those internalized by scavengers, which was easier to be detected when the viral load per tumour cell was high.

Severe acute respiratory syndrome (SARS) is a new human infectious disease with significant morbidity and mortality. The disease has been shown to be associated with a new coronavirus (SARS-CoV). The clinical and epidemiological aspects of SARS have been described. Moreover, the viral genome of SARS-CoV has been fully sequenced. However, much of the biological behaviour of the virus is not known and data on the tissue and cellular tropism of SARS-CoV are limited. In this study, six fatal cases of SARS were investigated for the tissue and cellular tropism of SARS-CoV using an in-situ hybridization (ISH) technique. Among all the tissues studied, positive signals were seen in pneumocytes in the lungs and surface enterocytes in the small bowel. Infected pneumocytes were further confirmed by immunofluorescence-fluorescence in-situ hybridization (FISH) analysis. These results provide important information concerning the tissue tropism of SARS-CoV, which is distinct from previously identified human coronaviruses, and suggest the possible involvement of novel receptors in this infection. Whereas the lung pathology was dominated by diffuse alveolar damage, the gut was relatively intact. These findings indicated that tissue responses to SARS-CoV infection are distinct in different organs.

A high throughput accurate assay for anti‐SARS‐CoV IgG detection is needed for large‐scale epidemiological studies. The evaluation of a commercial recombinant nucleocapsid protein‐based microtitre plate enzyme immunoassay, ELISARS™ is described. The results on 150 sera from SARS patients and 450 sera from non‐SARS controls showed that this assay had a high level of sensitivity (96.2% for late serum samples) and specificity (97.8%). The performance and setup of this assay fulfills the requirement as a screening test for large‐scale studies. A vast majority of SARS patients developed antibodies against the nucleocapsid protein. In some patients (10/45), a high level of anti‐nucleocapsid antibody appeared very early in the course of the illness. In contrast, a minority (4 of 105 patients) never developed these antibodies. The implication of differences in antibody response to the nucleocapsid protein deserves further investigation. J. Med. Virol. 75:181–184, 2005. © 2004 Wiley‐Liss, Inc.

Glycoprotein B (gB) and glycoprotein H (gH) of human herpesvirus 7 (HHV-7) are believed to play an important role in virus entry and as targets for host immune response. This study examined the genetic diversity of these glycoproteins among 90 HHV-7 isolates collected from different individuals in Hong Kong. Overall, both the gB and gH genes were found to be highly conserved. Nucleotide polymorphism was detected only at four positions of the gB-encoding region, and all of these were synonymous substitutions. Most (97.8%) Hong Kong isolates were of gB allele group C. Two isolates collected from a Pakistani family showed a novel sequence pattern that did not match known gB allele groups. This sequence pattern was detected consistently from serial samples collected from the same individual, indicating a stable genetic entity. The gH-encoding region exhibited nucleotide polymorphism at six positions. Three of these were nonsynonymous substitutions (codon 271 Lys --> Gln, codon 308 Gly --> Glu, codon 397 Asn --> Tyr). Most (84.4%) Hong Kong isolates were of the gH allele group B, and all others were of the gH allele group C. These data indicate the possibility of using gB or gH alleles as markers for studying world-wide population movements and genetics.

Previous studies have suggested that a few uncommon human papillomavirus (HPV) genotypes are prevalent in Chinese cervical cancer patients. To elucidate the genotype spectrum of HPVs circulating among Hong Kong Chinese, a cross-sectional study was conducted on 553 women who attended a public sexually transmitted disease clinic. HPV DNA was detected from cervical samples using the polymerase chain reaction, followed by genotype identification based on restriction fragment length polymorphism and direct sequencing. The prevalence of HPV was 30.6% for all types combined, 14.8% for high-risk types, 10.8% for low-risk types, and 7.1% for unknown-risk types. Among the HPV-positive women, 89.9% had single type infections; whereas the other 10.1% harboured more than one HPV type. HPV11 was the most prevalent genotype, detected in 5.1% of subjects; followed by HPV16 (4.9%), HPV58 (4.3%), HPV6 (3.3%), and HPV53 and CP8304 (2.2% each). Other less common genotypes found were HPV18, 33, 39, 61, LVX160, MM4, MM7 (range: 0.7-1.6%); HPV26, 45, 54, 56, 59, and LVX100 (range: 0.4-0.5%); HPV35, 40, 52, 55, 68, MM8, and MM9 (0.2% each). This study shows that HPV58 is the second most common high-risk HPV genotype circulating among Chinese female sexually transmitted disease clinic patients in Hong Kong. This together with previous reports of the high prevalence of HPV58 among Chinese cervical cancer patients accentuate the importance of developing vaccines targeting at this otherwise uncommon genotype.

Controversies exist on the effect of pregnancy on human papillomavirus (HPV) infection. A cross-sectional section study was conducted to compare the prevalence and genotype distribution of cervical HPV infection between pregnant and non-pregnant women in Hong Kong. Cervical samples were collected from 308 pregnant women and from the same number of age-matched controls recruited from a cervical cancer screening center located at the same hospital. HPV was detected by the polymerase chain reaction, followed by genotype identification by restriction fragment length polymorphism and direct sequencing analyses. The prevalence of HPV for pregnant women was 10.1%, without significant variation with age, gestation, gravidity and parity. The prevalence of HPV for non-pregnant group was 11.4% and did not show significant difference when compared to the pregnant group either by overall or age-stratified subgroup analyses. When the analysis was stratified according to the risk-type of HPV infection, still no significant difference between pregnant and non-pregnant groups was observed (all types: 10.1 vs. 11.4%, P = 0.602; high-risk types: 5.8 vs. 7.8%, P = 0.338; low-risk types: 1.0 vs. 2.9%, P = 0.080; unknown-risk types: 3.2% vs. 1.3%, P = 0.105). The results of this study show no evidence for an influence of pregnancy on HPV prevalence, and a majority of HPV-infected pregnant women had normal cervical cytology. HPV positive results in pregnant women per se should be managed conservatively. J. Med. Virol. 67:583–588, 2002. © 2002 Wiley-Liss, Inc.

Peripheral blood mononuclear cells (PBMC), plasma, saliva and urine samples were collected from 50 thalassaemic patients for TT virus (TTV) detection by two sets of PCR. The set B nested PCR was more sensitive than the widely used NG hemi-nested PCR with TTV positive rates approximately PBMC: 98% vs. 70%; plasma: 92% vs. 66%; saliva: 62% vs. 22%; urine: 22% vs. 6%. All 50 patients had TTV detected in one or more specimens, with 16% of patients being positive in all four specimen types: 40% positive in PBMC, plasma and saliva; 30% positive in PBMC and plasma. In 82 NG hemi-nested PCR-positive samples TTV genotype was identified, 68.3% had a single genotype, 25.6% had multiple genotypes and 6.1% were uncharacterized. The positive rates for genotypes by specimen were: G1 (36/82), G2 (49/82), G3 (2/82), G4 (7/82), G5 (1/82) and G6 (3/82). Among the 42 patients for whom the genotype was examined, 42.9% had single-type infection, 45.2% had co-infections and 11.9% had uncharacterized genotypes. Sixteen of them had TTV detected both in PBMC and plasma with seven having identical genotypes in both samples. Eight patients had TTV detected in PBMC, plasma and saliva; two of them harboured identical genotypes in all three samples. The results indicate that, apart from hepatocytes, PBMC is a major cell type for TTV infection occurs. Shedding of TTV in urine and saliva is common and may have a significant role in nonblood-borne transmission among the general population. TTV-infected patients often harbour multiple genotypes suggesting infection with one genotype does not necessarily confer protection against the others. No correlation between TTV infection and liver dysfunction was observed.

Although it has been recognised that human herpesvirus 7 (HHV-7) establishes latent infection in CD4+ T lymphocytes and productive infection in salivary glands, recent data suggest that its in vivo tropism may be more widespread. In this study, the prevalence and distribution of HHV-7 in brain tissues of 30 consecutive post-mortems were examined by nested polymerase chain reaction. For each post-mortem, 10 fresh autopsy tissue samples were collected respectively from the cerebellum, frontal, temporal, parietal, and occipital lobes of both cerebral hemispheres. These patients were aged from 20-95 years (mean = 61.4, SD = 20.2) with a male:female ratio of 2:1. Three patients died of intracranial haemorrhage, the others died of causes unrelated to the central nervous system. Overall, 5% (15/300) of the brain tissue samples were positive for HHV-7 DNA. The positive rates with respect to anatomical positions were similar (0-3/30). When analysed by patient, 36.7% (11/30) were HHV-7 DNA positive. The viral DNA-positive and -negative groups did not show a significant difference in gender or age distribution. The majority (81.8%) of viral DNA-positive patients had HHV-7 DNA detected at only one anatomical position; only two patients had viral DNA detected simultaneously at three anatomical sites. These results suggest that HHV-7 persists in brain tissues of a substantial proportion of the adult population, and in most individuals, its distribution is probably confined to one site rather than pervasive. Further studies to elucidate the role of this ubiquitous virus in neuropathology are warranted.

The three novel human herpesviruses (HHV) 6, 7, and 8 are predominantly, but not exclusively, lymphotropic. In an attempt to elucidate their neurotropism in vivo, viral DNA sequences present in fresh autopsy cortical brain tissues obtained from 84 consecutive Chinese subjects (mean age, 66.9 years; range, 21-98 years) were detected by a nested polymerase chain reaction. These patients were apparently immunocompetent and free of clinical signs of viral diseases. HHV-6 DNA was detected in 36 of 84 (42.9%) patients, and the DNA-positive and -negative groups did not show a significant difference in age or sex distribution. Of the 36 HHV-6 DNA-positive cases, 9 (25%) were variant A and 27 (75%) were variant B. In view of the lower prevalence of variant A than variant B in the adult population, the two variants may share a comparable neuroinvasive potential. HHV-7 and HHV-8 DNA were detected respectively in three and two patients. The low positive rates of HHV-7 and HHV-8 may represent a relatively lower neuroinvasive potential of the viruses. Alternatively, the localization of HHV-7 and HHV-8 may be more restricted and the sampled cortical tissues may not represent the most abundant site of persistence in the nervous system. The results provide molecular evidence of the presence of the three newly identified herpesviruses in brain tissue. The pathogenic role for HHV-7 and HHV-8, as with HHV-6, in neurological diseases should not be overlooked.

The prevalence of human papillomavirus (HPV) among 332 Hong Kong Chinese women with abnormal Papanicolaou smears were determined by polymerase chain reaction and restriction fragment length polymorphism analysis. The overall HPV positive rate was 44.3% with 18.6% (16/86) for normal/inflamed cervices, 36.4% (32/88) for condyloma, 64.7% (33/51) for cervical intraepithelial neoplasia grade 1 (CIN 1), 37.9% (11/29) for CIN 2, 68.3 (41/60) for CIN 3, and 77.8% (14/18) for carcinoma. Double HPV infection was detected in 17 of the 147 positive samples, with a significantly higher proportion in patients with normal or inflamed cervices than those with CIN or carcinoma (31.3% vs 10.5%, P = .029). The six most commonly identified genotypes were HPV 16 (33.3%), HPV 58 (23.8%), HPV 11, 18, 31 (8.8% each), and HPV 33 (6.8%). The worldwide uncommon genotype HPV 58 was found to be the second most common genotype detected in patients with cervical carcinoma (6 of 18 patients). HPV 58 infection showed a significant association with CIN/carcinoma (odds ratio [OR] = 3.98; 95% confidence interval [CI] = 1.22–14.35) and a significant trend of increase in prevalence with increasing severity of cervical lesion (χ2 = 5.84; P = .016). Among Hong Kong Chinese women with abnormal cervical cytology, the detection of HPV 58 carried a positive predictive value of 68.6% for a cervical lesion of CIN 1 or higher severity. The high prevalence of HPV 58 among Chinese women, particularly in patients with carcinoma, has an implication on the design of HPV detection methods and the development of vaccines. J. Med. Virol. 59:232–238, 1999. © 1999 Wiley-Liss, Inc.

Consensus primers targeting human papillomaviruses (HPVs) have biases in sensitivity toward certain HPV types. We applied 3 primer sets (GP5+/6+, MY09/11, PGMY09/11) in parallel on 120 Chinese cervical cancer specimens. GP5+/6+ exhibited a poor sensitivity for HPV52, for which the prevalence among squamous cell cervical cancer was underestimated from 14.6% to 0%. The fact that HPV52 should rank second in prevalence among squamous cell cervical carcinoma in Hong Kong could be missed if GP5+/6+, a worldwide commonly used primer set, was selected for HPV detection. Biases in HPV type-specific sensitivity may result in misprioritization of vaccine candidates.

The seroprevalence of antibody to severe acute respiratory syndrome (SARS)–associated coronavirus (SARS-CoV) in cohorts of health care workers (HCWs) with subclinical infection in SARS and non-SARS medical wards was 2.3% (3 of 131 HCWs) and 0% (0 of 192 HCWs), respectively. Rates for clinical SARS-CoV infection among 742 HCWs on these wards were highest among nurses (11.6%) and health care assistants (11.8%), indicating that these occupations are associated with the highest risks for exposure.

Background. Human papillomavirus type 58 (HPV-58) accounts for a much higher proportion of cervical cancers in East Asia than other types. A classification system of HPV-58, which is essential for molecular epidemiological study, is lacking. Methods and results. This study analyzed the sequences of 401 isolates collected from 15 countries and cities. The 268 unique concatenated E6-E7-E2-E5-L1-LCR sequences that comprised 57% of the whole HPV-58 genome showed 4 distinct clusters. L1 and LCR produced tree topologies that best resembled the concatenated sequences and thus are the most appropriate surrogate regions for lineage classification. Moreover, short fragments from L1 (nucleotides 6014–6539) and LCR (nucleotides 7257–7429 and 7540–52) were found to contain sequence signatures informative for lineage identification. Lineage A was the most prevalent lineage across all regions. Lineage C was more frequent in Africa than elsewhere, whereas lineage D was more prevalent in Africa than in Asia. Among lineage A variants, sublineage A2 dominated in Africa, the Americas, and Europe, but not in Asia. Sublineage A1, which represents the prototype that originated from a patient with cancer, was rare worldwide except in Asia. Conclusions. HPV-58 can be classified into 4 lineages that show some degree of ethnogeographic predilection in distribution. The evolutionary, epidemiological, and pathological characteristics of these lineages warrant further study.

Background Aspartic acid to glycine substitution (D222G) of haemagglutinin subunit (HA1) was associated with adverse outcomes in 2009 pandemic influenza A (H1N1) infections. Objectives To characterize the virological profile and antiviral response of patients infected with the HA1 D222G mutant. Study design Sixty-three adults admitted for pandemic influenza in Hong Kong were tested for D222G mutation by direct sequencing. Nasopharyngeal viral concentration on presentation was measured by real-time PCR to evaluate shedding from the upper respiratory tract. Serial upper and lower respiratory tract specimens were monitored to determine preferential tropism and document virological response to treatment. Results The frequency of D222G infection was 17.4% among cases with severe pneumonia, and 26.7% among cases requiring intensive care. Altogether, four sporadic D222G cases spread across the first and second waves in Hong Kong were detected. A significant association between D222G infection with severe pneumonia (100% vs. 32.2%, P = 0.015) and intensive care admission (100% vs. 18.6%, P = 0.002) was observed. D222G was associated with lower concentrations of virus in the upper respiratory tract compared to wildtype, but persisted in the lower respiratory tract at high concentrations, despite clearance from the upper respiratory tract following antiviral treatment. Conclusions These observations suggest that D222G can arise de novo , sheds less virus from the upper respiratory tract and may be less transmissible, but more pneumotropic and more resistant to antiviral treatment. D222G is associated with a higher chance of developing critical disease. Lower respiratory tract specimen is needed for a reliable detection of this mutant.

The clinical value of viral load and integration testing for human papillomavirus (HPV) remains unclear. Data on HPV type 18 (HPV18) is limited. We examined the HPV18 viral load and integration status of 78 women with normal cervix or neoplasia. While the crude viral load appeared to increase with lesion severity, the association was not significant after normalization with sample cellularity. Unlike reports for HPV16, the amino-terminal 1 region of HPV18 E2 was most frequently (71.0%) disrupted, representing the best marker for integration. A substantial proportion (57.1%) of invasive cancers harbored only the episomal genome, thus jeopardizing the clinical value of integration testing. A large proportion (41.7%) of normal/low-grade lesions showed viral integration, suggesting that integration of HPV18 starts early and is unlikely to be a sole determinant for progression. Interpretation of viral load should take into account the form of HPV infection as single infections had significantly higher viral loads than coinfections ( P = 0.046). More data generated from routinely collected samples are warranted to verify the clinical value of viral load and integration testing. Viral load quantitation for HPV18 is premature for clinical use at this stage.

Infection load and the integration of human papillomaviruses (HPV) have been implicated as determinants for oncogenesis, but whether variation among different HPV types exists remains unclear. We investigated 91 women infected with HPV type 52 (HPV-52), a type that is rare worldwide but common in East Asia. The median viral load increased with the severity of the lesion (248 copies/cell equivalent for normal/cervical intraepithelial neoplasia [CIN] grade 1, 402 copies/cell equivalent for CIN 2, 523 copies/cell equivalent for CIN 3, and 1,435 copies/cell equivalent for invasive cancer). The proportion of specimens with integration increased significantly with the severity of the lesion ( P < 0.001). The viral load was associated with the physical status of the viral genome, with higher levels for the pure episomal form ( P = 0.001). Infection status should be considered when interpreting viral load data for HPV-52, as single infections with this HPV type were found to have marginally higher viral loads than coinfections ( P = 0.051). All except one sample had E2 disruption restricted to only a part of the gene. Integration is a critical step in HPV-52-induced carcinogenesis. The profile of E2 disruption was different from that described for HPV-16, with the amino-terminal region being most frequently involved. Selecting the appropriate E2 region for amplification is critical in studying the integration of HPV-52. In summary, the HPV-52 viral load and the integrated proportion increased with the severity of the cervical lesions but had a different pattern than that of HPV-16.

The clinical utility of viral-load and integration status of human papillomavirus (HPV) infection remains uncertain. We examined 75 women infected with HPV58, a worldwide rare type found to be prevalent in cervical cancers in eastern Asia. Viral load was significantly higher for cervical intraepithelial neoplasia (CIN) 1/2, but those for a normal control group and for CIN 3 or cancer overlapped substantially. A pure integrated genome was found for all lesion grades, giving a poor positive predictive value (23.1%) for cancer. The pure episomal form's negative predictive value for cancer was only 76.3%. Mixed patterns of E2 gene disruption were common and often involved the amino-terminal and hinge regions. Disruption of the whole E2 gene was rare and was restricted to high-grade lesions. The HPV58 variant E67-HK-2 was more likely to exist in the pure episomal form. Routinely collected cervical samples contain a heterogeneous population of viruses, hampering the application of viral load and integration testing in clinical settings.

Objectives and methods. HLA II DQB1 polymorphisms have been shown to associate with cervical cancer risk, but results varied among different populations. In this study, the HLA DQB1 alleles among 221 southern Chinese women with cervical intraepithelial neoplasia grade III (CIN III)/invasive cervical carcinoma (ICC) were compared to 191 controls. Results. The frequency of DQB1*03 was significantly lower among ICC overall as compared to controls (65.4% vs. 79.1%, odds ratio [95% confidence interval]: 0.50 [0.28–0.88], corrected p -value: 0.04). The protective association of DQB1*03 remained significant for human papillomavirus (HPV) 16-positive ICC, but not for HPV16-negative cases. This is in contrast to studies on European populations where DQB1*03 was associated with an increased risk for ICC. In the current study, 70.1% of the HPV16 isolates were Asian variants, and 28.0% were European variants. However, no significant association between HPV16 variant and DQB1*03 distribution was observed. HPV52 and HPV58 were found respectively in 16.3% and 10.0% of CIN III/ICC, which were higher compared to that of Europe and North America. Further analyses revealed a positive risk association between DQB1*06 and HPV58-positive CIN III/ICC (3.68 [1.37–9.92], corrected p -value: 0.012). Conclusion. The host genetics and the distribution of HPV types/variants may account for the observed differences among southern Chinese and other populations.

The clinical utility of human papillomavirus (HPV) load and integration status remains unclear. We applied refined methods to delineate the viral load, integration status, and lineage of 104 women with HPV-16 monotype infection, including 19 with normal cervices, 9 with histologically proven cervical intraepithelial neoplasia (CIN) 1, 24 with CIN 2, 27 with CIN 3, and 25 with squamous cell carcinoma (SCC). Higher crude viral load, as determined by real-time polymerase chain reaction (PCR) targeting the E7 gene, was observed for SCC but became insignificant after normalization for cell content. Integration was located and quantified by real-time PCRs targeting, respectively, the carboxyl, amino, and hinge domains of the E2 gene. Pure episomal, integrated, and mixed forms were observed in all disease groups. Most E2 gene disruptions involved the amino-terminal, but sparing the hinge region that has been frequently used as a surrogate marker of integration. Large-fragment disruption involving all 3 E2 regions was observed only in the CIN 3 and SCC groups. Altogether, 33.3% of the CIN 3 group and 28.0% of the SCC group harbored pure episomal genomes. The Asian lineage was associated with a higher risk for CIN 3/SCC than the European lineage, and 6 of the 7 large-fragment E2 disruptions were from Asian lineage. The link between viral lineage, integration pattern, and oncogenesis deserves further study.

Background: The spike glycoprotein (S) gene of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) has been useful in analyzing the molecular epidemiology of the 2003 SARS outbreaks. Objectives: To characterize complete SARS-CoV S-gene sequences from Hong Kong. Study design: Fifty-six SARS-CoV S-gene sequences, obtained from patients who presented with SARS to the Prince of Wales Hospital during March–May 2003, were analysed using a maximum likelihood (ML) approach, together with 138 other (both human and animal) S-gene sequences downloaded from GenBank. Results: The maximum-likelihood (ML) trees showed little evolution occurring within these 56 sequences. Analysis with the other sequences, showed three distinct SARS clusters, closely correlated to previously defined early, middle and late phases of the 2003 international SARS outbreaks. In addition, two new single nucleotide variations (SNVs), T21615A and T21901A, were discovered, not previously reported elsewhere. Conclusions: The ML approach to the reconstruction of tree phylogenies is known to be superior to the more popular, less computationally and time-demanding neighbour-joining (NJ) approach. The ML analysis in this study confirms the previously reported SARS epidemiology analysed mostly using the NJ approach. The two new SNVs reported here are most likely due to the tissue-culture passaging of the clinical samples.

A population-based study was conducted on 256 southern Chinese with cervical intraepithelial neoplasia grade III (CIN III) or invasive cervical cancer (ICC) and on 258 controls to examine the associations between HLA-B alleles, infection with high-risk human papillomaviruses (HPVs) and the development of cervical neoplasia. HLA-B15 was found to be protective for CIN III/ICC overall (pcorrected = 0.003), and for HPV52-positive CIN III/ICC (pcorrected = 0.003). A marginal protective effect of B15 was observed for HPV16-positive CIN III/ICC, but no significant associations were revealed for HPV18- or HPV58-positive cases. None of the HLA-B alleles were found to confer an increased risk for cervical neoplasia. HLA-B15 is common among Asian for whom HPV52, a worldwide uncommon HPV type, also exists in a relatively high prevalence. It would also be worthwhile to assess the association between HLA-B15, HPV52 and cervical cancer in other Asian populations. © 2005 Wiley-Liss, Inc.

Severe acute respiratory syndrome coronavirus (SARS‐CoV) can produce gastrointestinal symptoms. The intestinal tract is the only extrapulmonary site where viable viruses have been detected. This study examined seven established human intestinal cell lines, DLD‐1, HCT‐116, HT‐29, LoVo, LS‐180, SW‐480 and SW‐620, for their permissiveness to SARS‐CoV infection. The results showed that only LoVo cells were permissive to SARS‐CoV infection as evident by positive findings from indirect immunofluorescence staining for intracellular viral antigens, in situ hybridization for intracellular viral RNA, and electron microscopy for intracellular viral particles. In contrast to Vero cells, SARS‐CoV did not produce cytopathic effects on LoVo cells. However, LoVo cells were found to be highly permissive for productive infection with a high viral titre (>3 × 107 viral copies/ml) produced in culture supernatant following a few days of incubation. SARS‐CoV established a stable persistent chronic infection that could be maintained after multiple passages. Being a cell line of human origin, LoVo cells could be a useful in vitro model for studying the biology and persistent infection of SARS‐CoV. Our results on the expression of angiotensin‐converting enzyme 2 (ACE2), a recently identified cellular receptor for SARS‐CoV, in these cell lines indicated that it might not be the sole determinant for cells to be susceptible to SARS‐CoV infection. J. Med. Virol. 74:1–7, 2004. © 2004 Wiley‐Liss, Inc.

Human herpesvirus 7 (HHV-7) is the least studied beta-herpesvirus in transplant settings. This prospective study examined the activity of HHV-7 during the first 12 weeks post-stem cell transplant in 59 paediatric patients. The presence of HHV-7, human cytomegalovirus (HCMV) and human herpesvirus 6 (HHV-6) in blood was monitored weekly by a multiplex nested polymerase chain reaction. Overall, 33 (55.9%) patients had one or more surveillance blood sample(s) positive for HHV-7. In contrast to HCMV and HHV-6, no obvious peak time of reactivation was observed for HHV-7. The occurrence of HHV-7 DNAaemia showed a significant negative association with HHV-6 (P=0.022), but with no association with HCMV. A significant higher positive rate for HHV-7 was found in autologous versus allogeneic (P=0.002), and in peripheral blood versus umbilical cord/marrow (P

Human papillomavirus (HPV) type 58 has been found to be prevalent among Chinese patients with cervical cancer. This study examined the oncogenic risk of HPV58 variants in Hong Kong, a southern part of China. Altogether, 1924 women were studied: 42.8% with a normal cervix, 16.2% with cervical intraepithelial neoplasia (CIN) I, 12.7% with CIN II, 20.8% with CIN III, and 7.6% with invasive cervical cancer (ICC). The overall prevalence of HPV58 was 11.4% (220) and increased statistically significantly with the severity of neoplasia (P(trend).001, chi(2) test for trend). Among HPV58-positive women, the occurrence of E7 632C--T (T20I) and E7 760G--A (G63S) variants (T20I/G63S) showed a positive trend of association with the severity of neoplasia (P(trend).001, chi(2) test for trend). HPV58 variants carrying these two substitutions showed an odds ratio (OR) for ICC of 26.79 (95% confidence interval = 10.14 to 74.72), and this OR was 6.9-fold higher than the ORs of variants without these substitutions. Patients with CIN III or ICC who were also infected with T20I/G63S variants had a statistically significant younger age at diagnosis than those infected with other variants (median age = 37 years versus 48 years; P =.038, two-sided Mann-Whitney U test). Thus, HPV58 variants carrying E7 T20I/G63S substitutions may be associated with an increased risk for cervical cancer.

A case-control study was conducted on 1986 Hong Kong women to assess the risk of human papillomavirus (HPV) type 16 variants for cervical neoplasia. In total, 255 women were HPV-16 positive and were analyzed for E6 and E7 sequence variation. Two novel substitutions at E6 (T86I and Q116E) and 1 at E7 (R66W) were found. Most HPV-16 variants were of Asian (50.6%) or European (44.3%) lineage, and both lineages showed similar risk associations for high-grade and invasive cervical neoplasia. No increased risk was observed for the subclasses European variant and European 350G, which carry a higher risk for invasive cancer in some Western populations. The E7 N29S substitution, reported to have a higher risk in Korean women, was found equally distributed among normal and various degrees of neoplasia. The epidemiology and risk implication of HPV-16 variant infection in Hong Kong differ markedly from other parts of the world.

This cross-sectional survey assessed the determinants of human papillomavirus (HPV) infections among 2080 women who participated in cervical cancer screening. HPVs were typed by restriction and sequencing analyses. The prevalence of HPV was 7.3% (4.2% for high-risk, 1.9% for low-risk, and 2.1% for unknown-risk types). High-risk HPV prevalence decreased with age, whereas low- and unknown-risk HPVs had a second peak in older women. Young age was the only common variable associated with the 3 groups of HPV infections. Lifetime number of sex partners was associated with high- and low-risk types but not with unknown-risk HPVs. Previous Pap smear, treatment for cervical lesions, induced abortion, smoking and having smoker(s) in the family were risk factors for high-risk HPVs. Barrier contraception was protective for low-risk HPVs; current vaginal discharge had a negative association with unknown-risk HPVs. The results indicate that different risk profiles exist for infections with different HPV groups.

Introduction Prevalence of an abnormal Papanicolaou smear was significantly increased in lupus patients in cross-sectional studies, associated with a higher prevalence of high-risk human papillomavirus (HPV) infection. The nucleic acid-specific Toll-like receptors (TLRs) locate at the endolysosomal compartments and trigger the induction of cytokines for the innate immune response. This study evaluated whether abnormal host innate immune response in lupus patients may enhance HPV persistence. Methods Protein levels of TLRs 3, 7, 8 and 9 in cervical epithelial cells of lupus patients and controls with or without HPV infection were assessed using flow cytometry. Characteristics associated with the differential expression of TLRs in systemic lupus erythematosus (SLE) were elucidated. The effect and interferon-stimulated genes (ISGs) (ISG15 and Mx-1) gene expressions were then measured in oncogenic HeLa (HPV18), CaSki (HPV) and C33A (HPV negative) cell lines using flow cytometry and quantitative real-time PCR. Ex vivo productions of cytokines and interferon-gamma (IFN-γ) upon TLR ligands stimulations were subsequently measured using cytometric bead array and ELISA. Results For subjects with HPV infection, levels of TLR3 and TLR7 were significantly lower in lupus patients compared with controls. Significantly decreased TLRs 7, 8 and 9 levels were observed in HPV-negative SLE compared to healthy controls. For SLE with and without HPV infection, TLR7 and 9 levels were significantly lower in infected SLE than those in HPV-negative patients. Independent explanatory variables associated with down-regulation of TLR7 level included HPV infection and a higher cumulative dose of prednisolone; while a higher cumulative dose of hydroxychloroquine and HPV infection were associated with down-regulation of TLR9 level. In cervical cell lines, TLRs 3, 7, 8, 9 protein levels and antiviral ISG15 and Mx-1 gene expressions were inhibited in two oncogenic HPV types. Functional data showed that the induction of pro-inflammatory cytokines by TLR ligands (R837, ssRNA and ODN2395) was greatly impaired in CaSki and HeLa than C33A cells. Conclusions In conclusion, prednisolone and TLR antagonist (hydroxychloroquine) may down-regulate protein levels of TLR7 and TLR9 in lupus patients, thereby decreasing the innate immune response against HPV infection. Upon infection, HPV further down-regulate TLR7 and 9 levels for viral persistence. Furthermore, reduction of nucleic acid-sensing TLRs 7, 8 and 9 in carcinogenic HPVs ensures that the expression of inducible pro-inflammatory cytokines is minimized to prevent the expression of antiviral ISGs (ISG15 and Mx-1) on a biologically relevant antiviral response.

Prevalence studies of the recently identified TT virus (TTV) have suggested that parenteral transmission is a common route of infection, but other routes also appear likely. In this study, a high rate of cervical carriage (66%) of TTV DNA was found by polymerase chain reaction, which suggests that perinatal and sexual transmission is possible.

Brain tissues from both sides of the cerebellum and frontal, temporal, parietal, and occipital lobes were collected postmortem from 30 patients for human herpesvirus 8 (HHV-8) detection by PCR. Overall, 42 of 300 (14.0%) samples were positive, with similar rates for each position. Nineteen patients (63.3%) were positive and showed a significant increase in positivity with age ( P = 0.007). The results indicate a neuroinvasive and neuropersistent potential of HHV-8.

BackgroundThe Severe Acute Respiratory Syndrome (SARS) was a newly emerged infectious disease which caused a global epidemic in 2002–2003. Sequence analysis of SARS-coronavirus isolates revealed that specific genotypes predominated at different periods of the epidemic. This information can be used as a footprint for tracing the epidemiology of infections and monitor viral evolution. However, direct sequencing analysis of a large number of clinical samples is cumbersome and time consuming. We present here a simple and rapid assay for the screening of SARS-coronavirus genotypes based on the use of fluorogenic oligonucleotide probes for allelic discrimination.MethodsThirty SARS patients were recruited. Allelic discrimination assays were developed based on the use of fluorogenic oligonucleotide probes (TaqMan). Genotyping of the SARS-coronavirus isolates obtained from these patients were carried out by the allelic discrimination assays and confirmed by direct sequencing.ResultsGenotyping based on the allelic discrimination assays were fully concordant with direct sequencing. All of the 30 SARS-coronavirus genotypes studied were characteristic of genotypes previously documented to be associated with the latter part of the epidemic. Seven of the isolates contained a previously reported major deletion but in patients not epidemiologically related to the previously studied cohort.ConclusionWe have developed a simple and accurate method for the characterization and screening of SARS-coronavirus genotypes. It is a promising tool for the study of epidemiological relationships between documented cases during an outbreak.