bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytotoxic Factors Released by Dengue Virus‐Infected Human Blood Monocytes
Authors: Shaio Men‐Fang , Cheng Shin‐Nan , Yuh Yeong‐Seng , Yang Kuender D.
Published: March 2007

Human monocyte‐derived cytotoxic factors (CF) induced by dengue virus were studied. Using several human leukemia cell lines as precursors, the biological activities of CF in conditioned medium from dengue virus‐infected monocytes were demonstrated through the measurement of tumor cell growth inhibition. The conditioned medium from dengue virus infected monocytes suppressed significantly growth of CEM, HL60, K562, and U937 cells. In the presence of 10% conditioned medium (v/v) from dengue virus infected monocytes, DNA synthesis of U937 cells, as measured by [3H]thymidine incorporation, decreased by 99% in contrast to their synthesis in conditioned medium from noninfected control monocytes, which did not have any suppressive effect. Partial characterization of CF showed that it is a proteinase‐K‐sensitive and heat‐labile protein with a molecular mass over 100 kDa. Employing a flow cytometric analysis of the cell cycle, it was found that U937 cells, treated either with conditioned medium from dengue virus infected monocytes or with CF, but not treated with conditioned medium from noninfected monocytes, showed cell‐cycle arrest in G1 phase by 48 hr. This suppressive effect of CF on U937 growth was dose‐ and time‐dependent. These results suggest that dengue virus‐infected monocytes may produce CF to target myeloid cells, resulting in the hematological changes observed in patients with dengue fever. © 1995 Wiley‐Liss, Inc.
Serial density analysis of hepatitis C virus particle populations in chronic hepatitis C patients treated with interferon‐α
Authors: Kanto Tatsuya , Hayashi Norio , Takehara Tetsuo , Hagiwara Hideki , Mita Eiji , Oshita Masahide , Katayama Kazuhiro , Kasahara Akinori , Fusamoto Hideyuki , Kamada Takenobu
Published: March 2007

In interferon treatment of chronic hepatitis C patients, the biochemical and virological responses mostly parallel each other. However, some patients who show persistent ALT normalization display continued viremia after cessation of therapy. High‐density hepatitis C virus (HCV) particles, which are immune complex forms, are reported to be less infectious both in vitro and in vivo. To assess whether high‐density HCV contributes to the response discrepancies and to clarify the association with patient outcome, sera were examined from chronic hepatitis C patients who were treated with interferon‐α. This study included 10 sustained responders with viremia (SR + ve), 5 SR without viremia, 3 transient responders (TR), and 3 nonresponders (NR). The SR + ve patients were defined as those with continued ALT normalization and serum HCV‐RNA positivity at 24 weeks after therapy completion. Serum samples obtained before and 24 weeks after therapy were ultracentrifuged on 35% sucrose. The ratio between high‐density and low density HCV was determined by quantification of HCV‐RNA titers in the bottom and top fractions by competitive reverse transcription and by the polymerase chain reaction, and expressed as the bottom/top (B/T) ratio. The B/T ratios before therapy were 1:l in all groups of patients, and 1:l after therapy in TR and NR groups. Five out of 6 SR + ve patients who showed 1:1 ratio after the apy relapsed within 1 year. In contrast, all SR + ve patients whose ratios were 10‐100:1 continued to show ALT normalization. These findings demonstrate that patients who have high‐density HCV dominance after therapy show persistent ALT normalization despite viremia, which can be explained by predominance of the neutralized immune complex. © 1995 Wiley‐Liss, Inc.
Reverse transcriptase mutations in sequential HIV‐1 isolates in a patient with AIDS
Authors: Gurusinghe A. D. , Land S. A. , Birch C. , McGavin C. , Hooker D. J. , Tachedjian G. , Doherty R. , Deacon N. J.
Published: March 2007

Sequential human immunodeficiency virus type 1 (HIV‐1) isolates were obtained over a 29‐month period from a person before, during, and after AZT therapy. DNA sequence analysis of polymerase chain‐amplified reverse‐transcriptase gene showed a gradual accumulation of mutations to peak resistance (lC50 2.13 μM AZT) in association with mutations at codons 44, 210, and 369, as well as at 41, 67, 70, and 215. Eight months after cessation of AZT therapy, when an HIV‐1 isolate from the patient was again sensitive to AZT, these mutations had all returned to the pretherapy sequence. © 1995 Wiley‐Liss, Inc.
Injection with nondisposable needles as an important route for transmission of acute community‐acquired hepatitis C virus infection in Taiwan
Authors: Chen Trong‐Zong , Wu Jaw‐Ching , Yen Fu‐Shun , Sheng Wen‐Yung , Hwang Shinn‐Jang , Huo Teh‐Ia , Lee Shou‐Dong
Published: March 2007

A prospective case‐controlled study was conducted in order to determine the transmission route of community‐acquired hepatitis C virus (HCV) infection in Taiwan. Thirty‐eight consecutive patients (25 men and 13 women) with acute community‐acquired HCV infection and 76 age (within 3 years)‐ and sex‐matched healthy control subjects without HCV infection were enrolled. Serum anti‐HCV was tested by second generation immunoassay. The sera of 26 family members from 12 families of index patients were also tested for anti‐HCV. A questionnaire covering the history of blood transfusion, surgery, intravenous drug abuse, prostitute contact, dental procedures, injection, acupuncture, tattooing, and ear‐piercing was conducted among patients and control subjects. Univariate analysis revealed injection with nondisposable needles was an independent risk factor (P = 0.02, odds ratio = 4.17, 95% confidence interval = 1.24‐14.47) associated with HCV infection. Other risk factors were not significant. Only 2 (7.7%) family members of index patients had an anti‐HCV. In conclusion, more vigorous effort to prohibit the use of nondisposable needles should be promoted to interrupt the spread of community‐acquired HCV infection in Taiwan. Of note, a significant number of patients (34.2%) contracted HCV infection without identifiable risk factors. Unidentified routes need to be investigated. © 1995 Wiley‐Liss, Inc.
Long‐term follow‐up of hepatitis B virus and hepatitis C virus replicative levels in chronic hepatitis patients coinfected with both viruses
Authors: Ohkawa Kazuyoshi , Hayashi Norio , Yuki Nobukazu , Masuzawa Manabu , Kato Michio , Yamamoto Keiji , Hosotsubo Hideo , Deguchi Matsuo , Katayama Kazuhiro , Kasahara Akinori , Fusamoto Hideyuki , Kamada Takenobu
Published: March 2007

Dual infection with hepatitis B and C viruses is often encountered in endemic areas of both viruses. However, understanding of the clinical and virological implications is limited. The aim of this study was to investigate the role of each virus in liver injury and the interaction between the two viruses in dual infection with hepatitis B and C viruses. Three patients who had chronic infection with both hepatitis B and C viruses were examined, and a longitudinal study of both serum hepatitis B virus DNA and hepatitis C virus RNA levels over 4 years was undertaken. The results were correlated with serum alanine aminotransferase levels. Serum alanine aminotransferase values showed a relationship with hepatitis B virus replicative levels, but not with hepatitis C virus replicative levels in all 3 patients. Serial changes of replicative levels of both viruses were studied, and it was found that hepatitis C virus replicative levels were enhanced after the decline of hepatitis B virus replication in 1 of the 3 patients. In the remaining 2 patients, a transient rise of hepatitis C virus replicative levels in association with a decrease of hepatitis B virus replication was also observed during part of the follow‐up period. These findings indicate that hepatitis B virus may play a dominant etiological role in liver injury, and that a suppressive action between hepatitis B and C viruses may occur in dual infection with both viruses. © 1995 Wiley‐Liss, Inc.
Rapid HCV RNA detection by PCR followed by a new non‐radioactive liquid hybridisation assay and comparison with RIBA
Authors: Schacker Ulrike , Bermayer Hans‐Peter , Boehler Valerie , Ionescu Doina , Zapata Manuel , Grauer Hasan , Rai Kamal
Published: March 2007

A one‐stage polymerase chain reaction (PCR) followed by an automated liquid hybridisation assay was used to examine anti‐HCV‐positive patients. The presence of HCV‐RNA in 251 randomly selected enzyme immunoassay (EIA) positive clinical specimens was compared to their RIBA pattern. An association of a RIBA pattern with presence or absence of HCV‐RNA was not detected. One hundred of these samples were also evaluated after dividing them into normal and elevated serum alanin aminotransferase (ALT) levels. PCR results were obtained on the basis of amplification products from two different gene regions (5′‐NC region and NS 3). To prove the specificity of the PCR products, a commercially available digoxigenin‐based liquid hybridisation assay was evaluated. The sensitivity was comparable to the results obtained after nested PCR. Based on the results of the study, the two‐stage PCR can be changed in favour of the easier one‐step PCR which offers the advantage of fewer contamination problems. © 1995 Wiley‐Liss, Inc.
HCV replication in mononuclear cells stimulates anti‐HCV‐secreting B cells and reflects nonresponsiveness to interferon‐α
Authors: Lohr Hanns F. , Goergen Bernd , zum Biischenfelde Karl‐Hermann Meyer , Gerken Guido
Published: March 2007

Recently, it was demonstrated in chronic hepatitis C that the release of IgG and IgM anti‐HCV antibodies by mononuclear cells (PBMCs) correlated with inflammatory activity, HCV persistence in serum, and negative outcome from antiviral therapy. Thus, persistent antigenic stimulation of the antibody‐secreting B cells has been suggested. In this study, PBMCs were derived from 13 patients with chronic hepatitis C. Nucleic acids were extracted by the guanidine‐thiocy‐anate‐method, and plus‐ and minus‐stranded HCV‐RNAs were determined using primers from the 5′‐untranslated region of HCV. Simultaneously, unstimulated PBMCs were cultured for 8 days and anti‐HCV antibodies were detected in the supernatants by EIA and RIBA.

Seven patients (53.8%) had both plus‐ and minus‐stranded HCV‐RNA in PBMCs, while anti‐HCV antibodies were secreted in vitro. One of 2 patients with plus‐ but not minus‐stranded HCV‐ RNA in PBMCs was anti‐HCV positive in vitro, whereas 4 patients without HCV‐infected PBMCs were anti‐HCV negative in vitro.

Eight patients received antiviral therapy with interferon‐α2b. Four nonresponders and 1 partial responder had plus‐ and minus‐stranded HCV‐ RNA in PBMCs and anti‐HCV secretion in vitro. On the other hand, 2 complete responders and another partial responder showed neither HCV infection of PBMCs nor anti‐HCV secretion in vitro.

In conclusion, infection of PBMCs by HCV is observed frequently in patients with chronic hepatitis C, and may be related to the high rate of nonresponders to antiviral treatment. The close correlation of anti‐HCV secretion in vitro and viral replication suggests that the humoral response to HCV is triggered by viral antigens that are expressed on infected mononuclear cells. © 1995 Wiley‐Liss, Inc.
High proportion of false positive reactions among donors with anti‐HCV antibodies in a low prevalence area
Authors: Sakugawa Hiroshi , Nakasone Hiroki , Nakayoshi Tomofumi , Kinjo Fukunori , Saito Atsushi , Yakabi Shizuko , Zukeran Hiroki , Miyagi Yasuhiro , Taira Reiko , Koja Keishun , Uezu Tomio , Kina Morio , Omine Keisho
Published: March 2007

Among 39, 656 voluntary blood donors in Okinawa Prefecture, Japan, 115 (0.29%) were repeatedly reactive for antibody to hepatitis C virus (anti‐HCV) by second generation (2nd‐gen) passive hemagglutination assay (PHA). Positive serum samples were tested for anti‐HCV using three different enzyme immunosorbent assays (ELISAs; Abbott 2nd EIA, UBI‐HCV‐EIA, JCC‐2) and for HCV‐RNA by the polymerase chain reaction (PCR).

The 115 2nd‐gen PHA‐positive sera were divided into three groups according to the agglutination titers; >210 (high titer group), 27−29 (median), 25−26 (low). All but one serum (44/45) in the high PHA titer group reacted in each of the three second screening ELISAs. Furthermore, 43 (97.7%) of the 44 sera contained HCV‐RNA by PCR. In the median titer group, 11 of the 13 samples tested were positive by each of the three ELSIAs, and 4 (36.4%) of the 11 showed reaction by PCR. On the other hand, all of the 38 sera tested in the low titer group were negative for HCV‐RNA by PCR, and 24 of the 38 were also negative by each of the three ELISAs.

Most of the low titer positive reactions in the 2nd‐gen agglutination assay seemed to be false positive. In Okinawa Prefecture, the prevalence of anti‐HCV among blood donors is much lower than in the rest of Japan (0.29% vs. 1.11%). Moreover, a significant proportion of these sera were low titer by the PHA assay. The difference in the genuine anti‐HCV‐positive rate, or the prevalence of HCV carriage between Okinawa Prefecture and the rest of Japan may therefore be even greater than is presently assumed. © 1995 Wiley‐Liss, Inc.
Amplification and sequence analysis of the precore and core region of the HBV genome from sera of Spanish patients with HBV2‐like infections
Authors: Valliammai T. , Echevarría J. M. , León P. , Tusets C. , Harrison Tim J.
Published: March 2007

Hepatitis B virus (HBV) infections are occasionally associated with unusual serological profiles which may be attributed to the lack of immune responsiveness of the patients or to variants of the virus. The term HBV2 was introduced to describe potential variants of HBV which do not elicit a detectable immune response to the nucleocapsid (anti‐HBc) in apparently irnrnune‐cornpetent patients. The entire nucleotide sequences of the nucleocapsid genes of viruses from two Spanish patients with HBV2‐like infections are reported here. In‐frame deletions and other mutations in these sequences may account, at least in part, for the unusual serological profiles of the patients. Antibody responses to viral proteins containing deletions may not be detected by commercially available assays. © 1995 Wiley‐Liss, Inc.
Prevalence of precore‐defective mutant of hepatitis B virus in HBV carriers
Authors: Niitsuma Hirofumi , Ishii Motoyasu , Saito Yukie , Miura Masahito , Kobayashi Koju , Ohori Hitoshi , Toyota Takayoshi
Published: March 2007

Two hundred and seventy‐three serum specimens from hepatitis B virus (HBV) carriers were examined for the presence of a characteristic one point mutation at nucleotide (nt) 1896 from the EcoRI site of the HBV genome in the precore region (the preC mutant) using restriction fragment length polymorphism (RFLP) analysis. This assay approach could detect preC mutants or wild‐type sequences when either form constituted more than 10% of the total sample. Overall, 65.5% (76/116) of HBeAg‐positive carriers had only the preC wild‐type. All HBeAg‐positive asymptomatic carriers (n = 14) had only the preC wild‐type. In patients with chronic hepatitis B and in anti‐HBe‐positive asymptomatic carriers, increased prevalence of the preC mutant was associated with the development of anti‐HBe antibodies and normalization of the serum alanine aminotransferase concentration. Furthermore, 27 (29.0%) of 93 HBeAg‐negative carriers had unexpectedly preC wild‐type sequences only. Direct sequencing of the HBV precore region of HBV specimens from 24 patients revealed no mutation at nt 1896, supporting the specificity of the RFLP analysis. These results suggest that RFLP analysis was accurate for the detection of the preC mutation and that the absence of serum HBeAg cannot be explained solely by the dominance of the preC mutant. © 1995 Wiley‐Liss, Inc.
Comparison of two immunoassay procedures for detecting enterovirus IgM
Authors: Hodgson Julian , Bendig Justin , Keeling Philip , Booth James C.
Published: March 2007

Enteroviruses cause significant illness in man but viral diagnosis is problematic. Enterovirus specific IgM tests have been developed but due to the difficulties of obtaining reliable control sera the interpretation of assay data remains mainly arbitrary and empirical. The present study was undertaken to assess the reliability of such assays by comparing two tests performed independently in two different laboratories: a μ‐capture radioimmunoassay (MACRIA) which utilizes 35S‐labelled Coxsackie virus antigens and an enzyme immunoassay (EIA). A feature of the MACRIA was that sera were tested in one large batch whereas the EIA was in routine use in a reference laboratory. The MACRIA was easy to perform but more suitable for research investigations than routine diagnostic use. Similar results were detected in the majority of sera tested in the two assays with 85% concordance achieved on testing 120 sera. Of the 18 discrepant results, 11 were positive by EIA only and 7 by MACRIA only. 89‐95% concordance was obtained on testing sera against individual Coxsackie B1‐5 serotypes, moreover 52% of the serapositive in MACRIA were reactive against only one viral antigen and the results on certain of the more strongly reactive sera suggested the existence of a measure of type specificity in the MACRIA test. Qualitative differences between the two tests highlighted problems of interpretation in the absence of a gold standard and cautioned against sole reliance on serology for diagnosis of enteroviral infections. © 1995 Wiley‐Liss, Inc.
Rapid and sensitive streptavidin‐biotin amplified fluorogenic enzyme‐linked immunosorbent‐assay for direct detection and identification of dengue viral antigens in serum
Authors: Malergue Fabrice , Chungue Eliane
Published: March 2007

Each of the four serotypes of dengue viruses is responsible for a spectrum of illnesses that range from nonspecific febrile syndrome with good prognosis to dengue haemorrhagic fever or dengue shock syndrome. Definite diagnosis of dengue is provided by the detection of virus in acute‐phase sera of patients. Virus isolation can be accomplished with mosquito cell lines or mosquito inoculations. However, these methods are time consuming and labour intensive. The reverse‐transcriptase polymerase chain reaction (RT‐PCR) provides a potential means of rapid diagnosis but requires specialised facilities and equipment and is expensive. Therefore a rapid, simple, sensitive, and economical method for direct detection of viral antigens in viraemic sera is needed for clinical and epidemiological investigations. An amplified fluorogenic enzyme‐linked immunosorbent assay (F‐ELISA) is described for the detection and identification of dengue‐3 viruses in serum specimens. This assay utilizes biotinylated mouse IgG antibody directed against dengue antigens captured by anti‐dengue monoclonal antibody coated onto polystyrene microplate wells. lt takes advantage of the high affinity of biotin for the multivalent binding sites of streptavidin‐labelled β‐galactosidase, and combines the amplification effect of biotin‐streptavidin interaction with the high sensitivity of fluorogenic detection methods. Following optimisation of the procedure by reducing nonspecific binding of proteins and enhancing the specific binding of antigens, F‐ELISA was tested on 259 sera submitted routinely to our laboratory for confirmation of dengue diagnosis. The sensitivity of the F‐ELISA was 90%, the specificity was 99% and the agreement rate was 98% between F‐ELISA and virus isolation results. © 1995 Wiley‐Liss, Inc.
Antibody prevalence and immunoglobulin IgG subclass pattern to norwalk virus in Sweden
Authors: Hinkula Jorma , Ball Judy M. , Löfgren Sture , Estes Mary K. , Svensson Lennart
Published: March 2007

The antibody prevalence in Sweden to Norwalk virus (NV) was investigated using a baculovirus expressed capsid antigen. One hundred thirty‐two serum samples were examined for IgA, total IgG and IgG subclass antibodies to Norwalk virus. In young children, NV IgG antibody prevalence was higher than the IgA prevalence, whereas no difference was found in individuals older than 21 years. The IgG antibody prevalence was 50% in children below 5 years of age and increased to >8O% in individuals older than 10 years of age. To examine the IgG antibody response in more detail, IgG subclass patterns were characterized. IgG 1 predominated in all age groups. IgG 4, usually detected after repeated exposure to antigen, was the second most prevalent subclass, but was only found in individuals olderthan 21 years of age. IgG 3 subclass antibodies were found in 13% and IgG 2 in 3% of the sera examined. IgG 3 subclass antibodies have been recognized as a marker for recent or ongoing viral infections. © 1995 Wiley‐Liss, Inc.
Rapidly recurrent enteroviral meningitis in non‐immunocompromised infants caused by different viral strains
Authors: Aintablian Nectar , Pratt R. Douglas , Sawyer Mark H.
Published: March 2007

Repeated episodes for enteroviral meningitis occurring within a 1‐month priod in two non‐immunocompromised infant were investigated using molecular techniques to distinguish persistent or recrudescent infection from new infection. Viral RNA from cerebrospinal fluid was amplified by polymerase chanin reaction, cloned, and the nucleic acid sequences determined. This analysis demonstrated that both infants had recurrent episodes of meningitis caused by new infection with a distincy enterovirus strain. The moleular methods that were employed should prove useful in similar clinical settings as well as for the study of enteroviral outbreaks. These cases also illustrate the potential for rapid reinfection of infants with different viruses of the same genus. © 1995 WiIey‐Liss, Inc.
Enterovirus 71 isolated from China is serologically similar to the prototype E71 BrCr strain but differs in the 5′‐noncoding region
Authors: Zheng Zhi‐Ming , He Pei‐Jun , Caueffield Diane , Neumann Mark , Specter Steven , Baker Carl C. , Bankowski Matthew J.
Published: March 2007

Enterovirus71 H (E71 H), an isolate from an adult patient with hand‐foot‐mouth disease (HFMD) in China, was serologically similar to the prototype strain E71 BrCr, which was isolated from a patient with aseptic meningitis. The study further analyzed the similarity of E71 H to E71 BrCr at the 5′‐noncoding region (NCR), a location in genomic RNA that recently was found to be related to neurovirulence in poliovirus and Venezuelan equine encephalitis virus. Using a reverse transcription‐polymerase chain reaction (RT‐PCR) technique and a unique primer pair I, a 397 bp product was detected from E7 I BrCr, Cox A9 (Griggs), Cox A16 (NIH), Cox B1 (HA antigen 201–4681, Cox 65 (wild type), and ECHO 11 (Gregory), but not from E71 H, Cox A24 (Joseph), and ECHO 5 (Noyce). However, all of the viruses generated a 154 bp product using a universal enterovirus primer pair II. Further comparative analysis using primer‐directed sequencing of both the E71 H and E71 BrCr 154 bp products revealed that they differed by 12 bases. The variations between the two viruses were clustered in two loci, one in the region of nucleotides 43–61 with eight variations, and the other in the region of nucleotides 120–133 with three variations. The differences within the 5′‐NCR between the E71 H (HFMD) and the E71 BrCr (aseptic meningitis) viruses might provide a clue to explain why E71 was associated with two different clinical patterns: polio‐like disease in the United States, Australia, and Eastern Europe, HFMD in China, Japan, and Singapore. © 1995 Wiiey‐Liss, Inc.
Herpesvirus‐like DNA sequences in classic Kaposi's Sarcomas
Authors: Sarcomas Kaposi'S , Rady Peter L. , Yen Angela , Martin Robert W. , Nedelcu Ioan , Hughes Thomas K. , Tyring Stephen K.
Published: March 2007

Kaposi's sarcoma (KS) accounts for more than 15% of AIDS‐related malignancies. The etiology of KS is unresolved but is postulated to be multi‐factorial, involving viruses and overexpression of cellular growth factors and/or oncogenes. Recently, herpesvirus‐like sequences (KSHV) were identified with high prevalence in AIDS‐KS (AKS), endemic KS, and in classic KS biopsies (CKS). To confirm the presence and the prevalence of the KSHV sequences, 18 CKS and 13 AKS samples were tested using polymerase chain reaction (PCR) analysis. To our knowledge this is the highest number of CKS samples that has ever been included in a single study, and it is also important that the biopsies were obtained from different institutions and geographical locations. KSHV sequences were detected in 100% of the AKS samples and 72% of the CKS biopsies using PCR analysis. The presence of the unique KSHV sequences was confirmed by direct sequencing of representative PCR products obtained from AKS and CKS samples. Reverse transcriptase (RTI‐PCR experiments showed that the KSHV sequences were transcribed to mRNA in both AKS and CKS samples. Our results confirm that the putative new herpesvirus‐like agent is associated with both AKS and CKS and may have an etiological role in the pathogenesis of this malignancy. © 1995 WiIey‐Liss, Inc.
Analysis of wild‐type and e antigen‐defective hepatitis B viruses during the course of a short‐term corticosteroid therapy in chronic hepatitis B
Authors: Daikoku Manabu , Nakata Keisuke , Hamasaki Keisuke , Ido Akio , Nakao Kazuhiko , Kato Yuji , Koga Michiaki , Yano Michitami , Nagataki Shigenobu
Published: March 2007

Hepatitis B virus (HBV), with a G‐to‐A point mutation at nucleotide 83 in the precore region (mutant HBV83), accounts for most cases of hepatitis B e antigen (HBeAg)‐defective HBV. However, it is still not clear how mutant HBV83 is associated with HBe seroconversion. Twenty‐six HBeAgpositive patients with chronic hepatitis B who received oral prednisolone (30 mg/day) for 3 weeks were studied to clarify the prevalence of mutant HBV83 during the treatment using polymerase chain reaction with a restriction fragment length polymorphism assay. Twelve (46%) patients seroconverted to anti‐HBe 1 year after treatment, whereas 14 (54%) did not. The proportion of mutant HBV83 to whole HBV remained unchanged in both groups during an acute exacerbation induced by withdrawal of corticosteroids. Among 12 anti‐HBe‐0seroconverted patients, five (56%) of nine patients with only wild‐type HBV at baseline developed detectable levels of mutant HBV83 while all three patients with a mixed viral population of wild‐type HBV and mu tant HBV83 at baseline developed a higher pro portion of mutant HBV83 one year after treat ment. In contrast, these changes were observed in only one (14%) of seven who failed to seroconvert. The results indicate that a flare‐up of hepa titis precedes emergence or selection of mutant HBV83, followed by HBe seroconversion in patients with chronic hepatitis B. © 1995 WiIey‐Liss, Inc.
Perinatal infection and persistence of human papillomavirus types 16 and 18 in infants
Authors: Cason John , Kaye Jeremy N. , Jewers Richard J. , Kambo Parminder K. , Bible Jon M. , Kell Barbara , Shergill Bhavneet , Pakarian Farzin , Raju K. Shanti , Best Jennifer M.
Published: March 2007

Perinatal transmission of genital human papillomaviruses (HPVs), including HPV‐16 and ‐18 which are associated with anogenital carcinomas have been described previously [Pakarian et al. (1994): British Journal of Obstetrics and Gynaecology 101:514‐517; Kaye et al. (1994) Journal of Medical Virology 44:415‐421]. A study was undertaken to investigate whether HPV‐16 and ‐18 DNA in infants contaminated at delivery persists until they are 6 months of age. Of 61 pregnant women recruited, 42 (68.8%) were HPV‐16 and 13 (21.3%) were HPV‐18 DNA positive. At 24 hr there were transmission rates from HPV DNA positive mothers to their infants of about 73% (HPV‐16: 69%; HPV‐18: 76.9%). Ten mothers who were both HPV‐16 and ‐18 DNA positive produced six (60%) infants who were also doubly positive at 24 hr. HPV DNA persisted to 6 weeks in 79.5% (HPV‐16: 84%; HPV‐18: 75%) of those infants who were positive at birth. At 6 months of age, persistent HPV‐16 DNA was detected in 83.3% of cases, but HPV‐18 DNA persistence at this time was 20%. To extend these observations over a greater age range of children HPV‐16 L1 and L2 proteins were expressed in insect cells via recombinant baculoviruses and sera from 229 children were examined to determine at what age IgM antibodies to HPV were acquired. There was a bimodal distribution of IgM seropositivity which peaked between 2 and 5 and 13 and 16 years of age, suggesting that two distinct modes of transmission may occur. The observation that infection with high cancer risk genital HPVs may occur in early life and persist is of considerable importance for HPV vaccine strategies. © Wiley‐Liss, Inc.
PCR detection of JC virus DNA in brain tissue from patients with and without progressive multifocal leukoencephalopathy
Authors: Ferrante Pasquale , Caldarelli‐Stefano Rita , Omodeo‐Zorini Elisabetta , Vago Luca , Boldorini Renzo , Costanzi Giulio
Published: March 2007

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system, which is thought to be a result of the reactivation of JC virus (JCV), a human polyomavirus. The disease occurs in individuals with immunosuppression and in recent years there has been an increase in PML cases due to AIDS.

A nested polymerase chain reaction (n‐PCR) was employed to detect JCV and BK virus (BKV) DNA in brain tissue collected postmortem from 28 AIDS patients with PML and from 13 patients without PML, but with other diagnoses, including solid tumors, Alzheimer's disease, thromboembolism, myocardial infarction and acute cerebrovascular diseases.

All 28 brain specimens from the patients with PML were positive for JCV DNA when tested by n‐PCR and three of the latter were also positive for BKV DNA. These results were confirmed by an enzyme restriction analysis and a DNA hybridization assay.

Interestingly, in this study, JCV DNA was also found in 6 brain tissue specimens from 4 subjects with diseases unrelated to PML or AIDS. All the brain specimens from the control group were negative for BKV DNA.

The results confirm that the n‐PCR is a useful tool for PML diagnosis. The presence of JCV DNA in the brain tissue of patients without PML is particularly important since it indicates that JCV could be latent in the brains of immunocompetent individuals. Moreover, detection of simultaneous presence of JCV and BKV in the brain tissue of the patients with PML demonstrates that BKV may also infect the human brain without causing any apparent neurological disease. © Wiley‐Liss, Inc.
Pathogenesis of hepatitis A in orally inoculated owl monkeys (Aotus trivirgatus)
Authors: Asher Ludmila V. Shavrina , Binn Leonard N. , Mensing Timothy L. , Marchwicki Ruth H. , Vassell Russell A. , Young G. David
Published: March 2007

The pathogenesis of hepatitis A virus (HAV) infection was studied in owl monkeys following oral administration of the wild‐type HM‐175 strain of HAV. Stools were collected daily and blood and pharyngeal swabs twice weekly for viral isolation, and animals were necropsied at various intervals after inoculation. Organs were examined for the presence of virus by isolation in cell culture and for viral antigens by immunofluorescence. Monkeys excreted HAV in the stools for 1–4 days after inoculation, presumably due to the residual unabsorbed inoculum. No virus was found in stools for the next 2–3 days. HAV re‐appeared on days 4–7 and then persisted through day 39. Viremia occurred on the 10th day and continued until day 35. Virus was isolated occasionally from throat swabs 1 or 2 weeks after it was detected in stools and blood, and there was no evidence that HAV replicated in the pharyngeal tissues. Animals acquired anti HAV antibody by the 4th week, and alanine aminotransferase (ALT) was elevated 5–5.5 weeks after inoculation. HAV was isolated from liver 5 days after inoculation; however, viral antigens were first detected in Kupffer cells of the liver at 14 days and in hepatocytes at 21 days. HAV antigen was detected in epithelial cells of the intestinal crypts and in the cells of the lamina propria of the small intestine 3 days postinoculation and thereafter until the 5th week, suggesting that these cells might represent an additional site of HAV replication. © Wiley‐Liss, Inc.

This article is a US Government work and, as such, is in the public domain in the United States of America.
Comparison of monoclonal antibodies for immunostaining in the cytomegalovirus shell vial assay on 4, 388 specimens
Authors: George Kirsten St. , Rinaldo Charles R.
Published: March 2007

The shell vial assay is a sensitive, rapid test for the detection of cytomegalovirus (CMV) in a variety of specimens. The sensitivity of this assay is dependent on a number of factors including the antibodies used for immunostaining. Monoclonal antibodies to the CMV major immediate early antigen (p72) from Chemicon (MAB810) and Dupont (NEA‐9221) were assessed side by side in duplicate vials on 4, 388 specimens from a patient population consisting of > 90% organ transplant recipients. A total of 240 specimens (5.5%) were CMV positive in either one or both vials. Positivity rates were variable across different specimen types but highest (12.9%) in urine Specimens. Of the positive specimens, 175 (72.9%) tested positive in both vials, 43 (17.9%) tested positive in the Chemicon‐stained vial only, and 22 (9.2%) tested positive in the Dupontstained vial only (P < 0.01, McNemar's chisquare test). This gave an overall positivity rate of 5.0% for Chemicon antibodies and 4.5% for Dupont. There was no difference in the fluorescent focus counts produced by the two antibody sets. It is concluded that use of the Chemicon antibodies provides increased sensitivity of detection of CMV in the shell vial assay above that afforded by the Dupont antibody. © Wiley‐Liss, Inc.
Tumor necrosis factor‐α and interferon‐γ inhibit synergistically viral replication in hepatitis B virus‐replicating cells
Authors: Kawanishi Yuko , Hayashi Norio , Katayama Kazuhiro , Ueda Keiji , Takehara Tetsuo , Miyoshi Eiji , Mita Eiji , Kasahara Akinori , Fusamoto Hideyuki , Kamada Takenobu
Published: March 2007

The effects of tumor necrosis factor‐a and/or interferon‐γ on the replication of hepatitis B virus were examined using HB611 cells. These cells were derived from human hepatoblastoma cells, Huh6, by integrating hepatitis B virus DNA, and produce hepatitis B virus continuously. Each of the cytokines inhibited hepatitis B virus replication in the cells assessed as the amount of episomal hepatitis B virus DNA, without a decrease in cell viability. When the two cytokines were administered together, the inhibitory effect became greater. Incubation of the cells with 1, 000 U/ml tumor necrosis factor‐α decreased HBV DNA replicative intermediates by 55%, and that with 1, 000 U/ml interferon‐γ decreased these by 51%. Furthermore, incubation with 1, 000 U/ml tumor necrosis factor‐α and 1, 000 U/ml interferon‐γ in combination decreased HBV DNA replicative intermediates by 71%. In contrast, the amount of hepatitis B virus RNA and secretion of hepatitis B e antigen were not apparently reduced by the cytokines, and 2′, 5′‐oligoadenylate synthetase activity was not detected in the supernatant. These results suggest that tumor necrosis factor‐α and interferon‐γ inhibit hepatitis B virus replication by blocking some step in reverse transcription and that the 2′, 5′‐oligoadenylate synthetase is not involved in the mechanism underlying the inhibition by these two cytokines. © Wiley‐Liss, Inc.
Role of tight junctions of polarized epithelial MDCK cells in the replication of herpes simplex virus type 1
Authors: Hayashi Kyoko
Published: March 2007

Before completion of polarization, Madin‐Darby canine kidney (MDCK) cells showed high infectivity and progeny production of herpes simplex virus type 1 infection. After polarization or formation of tight junctions, the infectivity and virus replication in MDCK cells was restricted significantly. The disruption of tight junctions by depletion of Ca2+ resulted in increasing virus infectivity and productivity. Mechanical disruption of tight junctions by scratching the cell monolayers with injection needle allowed markedly the replication of HSV‐I in the cells aligned along the injured area. In polarized MDCK cells the progeny were released preferentially from the apical surface of the cells. These data suggest that because polarized MDCK cells mimic the epithelial cell layers, this cell line is helpful for determining the factors which regulate viral transmission in the human body. © Wiley‐Liss, Inc.
Use of polymerase chain reaction and quantitative antibody tests in children born to human immunodeficiency virus‐1‐infected mothers
Authors: Newell Marie‐Louise , Loveday Clive , Dunn David , Kaye Steve , Tedder Richard , Peckham Catherine , De Maria Andrea , Giaquinto Carlo , Omenaca Felix , Canosa Cipriano , Mǔr Antonio
Published: March 2007

The diagnosis of human immunodeficiency virus (HIV) infection in children born to HIV‐infected mothers is complicated by the presence of passively acquired maternal antibodies, and exclusion of infection in these infants remains problematic. The use of genome detection by polymerase chain reaction (PCR) amplification and the quantification of anti‐HIV‐I antibodies were examined as methods for early diagnosis. Blood samples were taken from 84 non‐breastfed infants of HIV‐infected mothers in five Italian and Spanish centres, a subgroup of children enrolled in the European Collaborative Study (ECS) for whom clinical and immunological information has been documented from birth. Whole blood was added to glycigel cryopreservative, stored, and tested in the United Kingdom by a nested PCR method. Antibody to HIV‐1 was detected and quantified by titration using a gelatin particle agglutination test. PCR sensitivity and specificity were assessed. Twenty‐one of the 84 children tested were infected. The estimated PCR sensitivity ranged from 0% (95% CI 0–26%) on day 1, 57% (19–85) on day 7, to 63% (33–92) on day 30. The negative predictive value of PCR ranged from 85% (83–88) on day 0 to 98% (94–100) at 3 months of age. On average, the level of maternal antibody halved every 33 days (31–36.5) in uninfected children. Between 6 and 9 months of age, increases in antibody titres in infected children were not more informative than absolute levels. These findings suggest that antibody measurement may supplement genomic diagnosis and that this collection method provides an alternative to the use of dried blood spots. © Wiley‐Liss, Inc.
Productive HIV‐1 infection of human vascular endothelial cells requires cell proliferation and is stimulated by combined treatment with interleukin‐1β plus tumor necrosis factor‐α
Authors: Conaldi P. G. , Serra C. , Dolei A. , Basolo F. , Falcone V. , Mariani G. , Speziale P. , Toniolo A.
Published: March 2007

Vascular endothelial cells (EC) play a key role in viral tropism in vivo. Since conflicting reports have been published on the capability of HIV to infect EC in vitro, we analyzed some factors potentially capable of influencing the susceptibility of human umbilical vein endothelial cells (HU‐VEC) to HIV‐1. Both primary cultures and differentiated immortalized HUVEC lines were used. HUVEC were negative for the expression of CD4, but weakly CD26‐ and galactosylceramide‐positive. Although binding of HIV to EC was substantial, the virus was apparently incapable of replicating in nonproliferating cultures. In resting cultures, the content of cell‐associated HIV disappeared 4–6 days after infection without production of p24 and infectious progency. In contrast, infection of proliferating EC cultures led to the transient release of p24 and infectious virus (102.5‐103.5 SFU/ml) peaking 2–6 days postinfection. Antibody neutralization of cytokines that may be produced by EC (IL1, IL6, IL8, TNF, IFN‐β) failed to modify virus adsorption and replication, whereas treatment with IL1‐β plus TNF‐α stimulated both virus binding and virus release. As seen by gag polymerase chain reaction (PCR) the viral genome persisted up to 15 days in untreated EC cultures, but over 20 days in cultures exposed to IL1‐β plus TNF‐α. This study shows that: (a) CD4‐negative HUVEC are capable of binding substantial amounts of HIV‐1; (b) binding is enhanced by proinflammatory cytokines; (c) the establishement of productive infection is favored by cell proliferation; and (d) exposure to IL1‐β plus TNF‐α enhances virus replication. © Wiley‐Liss, Inc.
Pathogenesis of ocular cytomegalovirus infection in the immunocompromised host
Authors: Hayashi Kozaburo , Suwa Yuzo , Shimomura Yoshikazu , Ohashi Yuichi
Published: March 2007

Balb/C nude and C.B‐17 SCID mice were inoculated with salivary gland passaged cytomegalovirus (SG‐MCMV) intraperitoneally. Dissemination of the virus in the systemic and ocular tissues was studied by the direct immunofluorescence test, and the virus growth in each tissue was titrated in mouse embryonic fibroblasts. The mode of viral spread was assessed by inhibiting macrophage function by silica and administering polyclonal murine anti‐MCMV antibody in the circulation. The virus first reached the eyelid, conjunctiva, and cornea. Subsequently, it spread in the outer ocular muscles and chorioretinal layer. Ocular tissues were involved as part of a generalized infection. Abrogation of macrophage function by silica did not affect the outcome of the viral distribution. Administration of antibody prior to and 3 days after the viral infection prevented virus dissemination. Ocular CMV infection occurred initially at the anterior segment of the eye in an immunocompromised host. Free virus, not macrophage‐bound virus, disseminated via the bloodstream. © Wiley‐Liss, Inc.
Two different PCR assays to detect enteroviral RNA in CSF samples from patients with acute aseptic meningitis
Authors: Casas Inmaculada , Klapper Paul E. , Cleator Graham M. , Echevarria Juan E. , Tenorio Antonio , Echevarria Jose M.
Published: March 2007

Two polymerase chain reaction (RT‐PCR) assays were developed to allow rapid detection of enteroviral RNA in cerebrospinal fluid samples (CSF). Primers homologous to the conserved 5′ noncoding region of the enterovirus genome were designed. The RT‐PCR product size was‐500 bp (479 bp for Poliovirus, 500 bp for Cox‐sackievirus) and was visualized using ethidium bromide‐stained gels. Assay 1 utilized Moloney Murine Leukaemia Virus Reverse Transcriptase (MMLV‐RTase) for reverse transcription and Taq polymerase for subsequent PCR. Assay 2 utilized a thermoactive DNA polymerase of Thermus thermophilus (rTth enzyme) for both reverse transcription and DNA amplification. In addition, in Assay 2 reverse transcription and PCR wer eaccomplished within the same reaction tube. Both assays detected between 1 and 0.02 TCID50 of prototype strains of Polio and Coxsackie type B viruses propagated in VERO cell and spiked in a pooled preparation of CSF samples from patients with noninfective neurological disorders. However, Assay 1 was 10‐fold more sensitive than Assay 2 when applied to the detection of enteroviral RNA in CSF samples from patients with etiologically well characterized acute aseptic meningitis. © Wiley‐Liss, Inc.
Broadly reactive reverse transcriptase polymerase chain reaction for the diagnosis of SRSV‐associated gastroenteritis
Authors: Green J. , Gallimore C. I. , Norcott J. P. , Lewis D. , Brown D. W. G.
Published: March 2007

A limitation to date of reverse transcriptase polymerase chain reactions (RT‐PCRs) for the detection of small, round structured viruses (SRSVs) has been that they have detected only a narrow range of SRSVs due to the marked genomic diversity among strains. A total of 331 faecal samples collected from 136 separate incidents of gastroenteritis occurring in the UK between 1992 and 1994 were examined by RT‐PCR employing a single primer pair (NI/E3). SRSV RNA was detected in samples from 93 of 101 (91%) incidents shown to be SRSV‐associated by electron microscopy (EM) and in 5 of 35 (14%) SRSV‐negative incidents. Amplification products were tested by Southern blot hybridisation with a pool of four digoxigenin (DIG)‐labelled oligonucle‐otides derived from genomic sequence data of SRSV SPIEM types UK 1 to 4. Products from −5% of amplified strains did not hybridise. The NI/E3 primer pair were shown to be SRSV‐specific by their failure to amplify other faecal viruses including other human caliciviruses with typical calicivirus morphology.

Hybridisation of PCR products with the individual oligonucleotides relating to SRSV SPIEM types UK 1–4 was investigated: 1 of 60 (1.7%) reacted with the UK1 probe, 2/60 (3.4%) reacted with the UK2 probe, 51/60 (85%) with the UK3 probe, and 27/60 (45%) reacted with the UK4 probe. All PCR products that hybridised with the UK4 probe hybridised with the UK3 probe; 6 (10%) failed to hybridise. Identification of this primer pair facilitates routine diagnosis of SRSV infection by RT‐PCR and offers the potential for direct detection in food and environmental samples. © Wiley‐Liss, Inc.
Persistent rubella infection after erroneous vaccination in an immunocompromised patient with acute lymphoblastic leukemia in remission
Authors: Geiger R. , Fink F. M. , Sölder B. , Sailer M. , Enders G.
Published: March 2007

A 16‐year‐old male patient with acute lymphoblastic leukemia in complete remission and on maintenance treatment with weekly oral rnethotrexate and daily oral 6‐mercaptopurine for 3 months was immunized in error with the WI‐RA 27/3‐HDC live attenuated rubella vaccine. Increasing rubella HAI antibodies were noted from 3 to 7 months post‐vaccination as well as high levels of IgM antibody up to 8 months in three different tests. High HAI antibody titers persisted for 12–18 months after vaccination. Persisting rubella virus was indicated by PCR detection of rubella‐specific nucleic acid in whole blood, non‐stimulated and stimulated mononuclear cells 8 months following vaccination. Further attempts to detect rubella virus RNA in two subsequent blood samples were negative. Since acute arthritis and arthralgia occurred in the second month (days 51–63) after vaccination, antileukemic chemotherapy had to be interrupted. Evidence of higher risk for chronic or relapsing rubella‐associated arthropathy in immunologically compromised patients and the need to interrupt antileukemic chemotherapy should warrant imrnunoprophylaxis with polyvalent immune globulin in rubella‐susceptible patients who are immunocompromised. © Wiley‐Liss, Inc.
Polymerase chain reaction for prenatal diagnosis of congenital human cytomegalovirus infection
Authors: Revello Maria Grazia , Baldanti Fausto , Furione Milena , Sarasini Antonella , Percivalle Elena , Zavattoni Maurizio , Gerna Giuseppe
Published: March 2007

The reliability of the polymerase chain reaction (PCR) for prenatal diagnosis of human cytomegalovirus (HCMV) infection was determined by retrospective testing of 35 amniotic fluids identified previously as positive or negative for HCMV by virus isolation. Amniocentesis was performed in 26 pregnant women with primary HCMV infection at 14–36 weeks gestation, 3–21 weeks after maternal infection. Blood samples were obtained from 20 fetuses for IgM determination and/or virus isolation. Amniotic fluid culture led to antenatal diagnosis of HCMV in 9 of the 13 infected fetuses (sensitivity 69.2%) with one case diagnosed at a second sampling. PCR was able to detect one additional infected fetus (10/13, sensitivity 76.9%). Nested PCR did not increase sensitivity of prenatal diagnosis. Three cases were not diagnosed by all the techniques employed. The specificity of virus isolation from and DNA detection by PCR in amniotic fluid was 100%. The negative predictive value for virus isolation from amniotic fluid was 76.5% and for DNA detection by PCR 81.2%, whereas the positive predictive value was 100% for both techniques. The results showed that neither approach can detect all cases of congenital HCMV infection prenatally, and that the time interval between maternal infection and sampling seems to be a major factor affecting the reliability of prenatal diagnosis. © Wiley‐Liss, Inc.