bluuebunny/crossref_metadata_2025 huggingface dataset:
Replication of varicella zoster virus in primary human keratinocytes
Authors: Sexton Connie J. , Navsaria Harshad A. , Leigh Irene M. , Powell Kenneth
Published: March 2007

The ability of varicella zoster virus (VZV) to infect and replicate in human keratinocytes in culture was examined. Primary human keratinocytes derived from the abdomen, breast, and foreskin were plated as monolayers and infected by co‐cultivation with VZV infected fibroblasts (MRC‐5 cells). Replication and spread of the virus was assayed by plaque assay and immunofluorescence of infected cells using a VZV specific monoclonal antibody. Although all three types of keratinocytes tested were capable of supporting productive VZV infection, the keratinocytes showed a 1.5 to 2 log reduction in virus yield as compared to infection of monolayer cultures of MRC‐5 cells. Results from immunofluorescence studies and plaque assays indicate a slower rate of cell‐to‐cell spread of the virus. Testing of an anti‐VZV compound in this novel assay system demonstrated an interesting sensitivity compared to that observed in conventional assay Systems. © 1992 Wiley‐Liss, Inc.
Detection of respiratory syncytial virus in acute bronchiolitis in infants
Authors: Cubie Heather A. , Inglis J. M. , Leslie Eleanor E. , Edmunds A. T. , Totapally B.
Published: March 2007

Direct detection of respiratory syncytial (RS) virus antigen i n nasopharyngeal secretions (NPS) provides the most rapid diagnostic test for RS infection, but more sensitive methods might be more beneficial in the study of virus shedding. RS virus RNA was extracted from cells stored at −70°C either in suspension with added RNAse inhibitor or as a pellet without inhibitor. The RNA was reverse transcribed, the resultant cDNA amplified by the polymerase chain reaction and detected by ethidium bromide staining after electrophoresis through agarose gel (RT‐PCR). Of 217 specimens tested, 106 were positive by antigen detection, 99 by RT‐PCR, and 92 by virus isolation. In a series of 97 sequential NPS specimens from 15 infants in whom RS virus induced bronchiolitis was confirmed, antigen detection proved most sensitive in the first week after on‐set and RT‐PCR detected most positive specimens in the second week. Storage of the cells as a pellet proved more satisfactory than storage as a suspension. A further round of amplification using nested primers increased the number of positive results obtained by RT‐PCR. The sensitivity of antigen detection using directly labelled monoclonal antibody to RS virus was slightly higher than that of RT‐PCR, but the specificity was slightly lower. © 1992 Wiley‐Liss, Inc.
Venezuelan equine encephalitis‐specific immunoglobulin responses: Live attenuated TC‐83 versus inactivated C‐84 vaccine
Authors: Engler R. J. M. , Mangiafico J. A. , Jahrling P. , Ksiazek T. G. , Pedrotti‐Krueger M. , Peters C. J.
Published: March 2007

Venezuelan equine encephalitis (VEEI‐specific immunoglobulin responses to the two vaccines, TC‐83 (a live attenuated vaccine) and C‐84 (a formalin inactivated vaccine derived from the TC‐83 strain of virus) were evaluated using an antigen and isotype‐specific enzyme‐linked immunoadsorbent assay (ELISA). The VEE‐specific ELISA for IgG, IgG subclasses, IgA and IgM were developed and standardized using sera from vaccine‐ exposed and unexposed human subjects. Paired human sera (before and 28 days after immunization) were tested from laboratory workers vaccinated with either TC‐83 (Group A: 20 paired sera from subjects receiving a single TC‐83 vaccine and with no prior history of vaccination) or C‐84 in varying schedules (Group B: 19 paired sera from subjects who had a distant vaccination history to TC‐83 but no evidence of neutralizing antibody; Group C: 19 paired sera from subjects receiving their first C‐84 vaccination and no prior documented history of vaccination; Group D: 15 paired sera from subjects receiving a C‐84 booster vaccination with prior history of C‐84 but no TC‐83 exposure). Sera were all tested for viral neutralization in vitro using a Vero cell monolayer for culturing virus and establishing 80% plaque reduction for each serum tested.

All pre‐sera tested demonstrated no plaque reduction neutralization at a level of 80% for a dilution of 1 :10. ELSA antibody titers for all pre‐sera with no prior VEE exposure through vaccination or possible environmental factors were negative at a titer of 1 :160 for IgM, 1 :80 for IgG, IgA, and G subclasses. All vaccine types and strategies generated a significant IgG response postvaccination (P < 0.0001) and this response correlated with the 80% plaque reduction neutralization titer (PRN‐80) for VEE‐specific IgG, GI, G3 and IgA at a P value of < 0.001 for both Group A and B. No such correlation was observed for G2 and no G4 responses to immunization were noted in any of the groups tested. There was a significant difference between geometric mean (GM) titers postvaccination for Group A or Group B versus Group C (P< 0.001) and for Group C versus Group D (P < 0.001) for IgG. Neither group C or D (1 or 2 doses of C‐84 alone) demonstrated an IgA response in contrast to the TC‐83 exposed groups (Groups A and B). C‐84 was an effective booster vaccine in subjects previously exposed to the live attenuated vaccine and generated a significant neutralization antibody response mirrored in the IgG, G1, G3 and IgA titer increases by ELISA. © 1992 Wiley‐Liss, Inc.
Treatment with recombinant α‐interferon of chronic hepatitis C in anti‐HIV‐positive patients
Authors: Marriott Eduardo , Navas Sonia , Romero Jorge Del , Garcia Soledad , Castillo Inmaculada , Quiroga Juan Antonio , Carreño Vicente
Published: March 2007

A pilot study of chronic hepatitis C treatment was conducted in 14 patients (13 had chronic active hepatitis and 1 had liver cirrhosis). All patients were asymptomatic for the human immunodeficiency virus (HIV) type 1 (mean CD4 count of 584 ± 283 cells/mm3). Patients received 9 MU rlFN‐α2A per day for three months. After this, patients received 9 MU three times weekly for three months, 6 MU for another three months on the same protocol, and finally 3 MU again three times weekly for the last three months. After the first month of ALT treatment in 9 patients (64%) returned to normal; a significant decrease in ALT levels was observed with respect to the pretreatment values (mean of 42 lU/l, range 15–75 vs 152 IUI, range 69–355; P < 0.01). Of the 9 patients who completed the treatment period, 5 had a complete response, and 4 of these 5 continued with normal ALT values during follow‐up (sustained response) while the other patient relapsed within one month after cessation of therapy. The remaining 4 patients were non‐responders (including one case with a break‐through of the response). HCV‐RNA was not detectable in 3 of the 5 responders at the end of therapy while during follow‐up viral RNA became undetectable in the other 2 patients. 2/4 non‐responder patients had detectable HCV‐RNA during follow‐up. Liver histology improved in all the patients. No changes were observed in the immunological status or HIV infection. © 1993 Wiley‐Liss, Inc.
Hypervariable 5′‐terminus of hepatitis C virus E2/NS1 encodes antigenically distinct variants
Authors: Lesniewski Richard R. , Boardway Keith M. , Casey James M. , Desai Suresh M. , Devare Sushil G. , Leung Tat K. , Mushahwar Isa K.
Published: March 2007

Synthetic peptides representing sequences encoded at the 5′‐terminus of E2/NS1 in hepatitis C virus (HCV) were constructed. Peptides synthesized based on the sequences of four distinct HCV isolates were used to develop enzyme immunoassays (EIAs) for detection of antibodies in chronic HCV patients and in HCV‐infected plasma donors. HCV sequence‐specific antibodies were detected among patients with chronic HCV from the United States and Italy at frequencies of 22.2% and 55.8%, respectively. Similarly, sequence‐specific antibodies were detected in 54.6% of U.S. and 55.6% of Japanese commercial plasma donors who had previous evidence of HCV exposure. Our data support earlier findings of geographic variability among HCV variants. The region encoded by amino acids (aa) 380–436 was shown to contain at least one variant‐specific and one conserved epitope. The data further indicate that a majority of patients chronically infected with HCV (58.1% U.S., 68.8% Italy) have antibodies directed to the 5′‐terminus of the E2/NS1 gene product. We conclude that genotypic variability within the E2/NS1 gene of HCV results in antigenically distinct variants. © 1993 Wiley‐Liss, Inc.
Detection of low level of human papilloma virus type 16 DNA sequences in cancer cell lines derived from two well‐differentiated nasopharyngeal cancers
Authors: Huang Eng‐Shang , Gutsch David , Tzung Keh‐Weei , Lin Chin‐Tarng
Published: March 2007

Human papilloma virus type 16 (HPV16) related sequences were detected in two EBV‐negative nasopharyngeal carcinoma (NPC) cell lines derived from two well‐differentiated NPC by polymerase chain reaction. E2 and E6 related sequences of HPV16 were demonstrated using two pairs of primers derived from these two regions. DNA sequence analysis of amplified products excluded the possibility of laboratory viral DNA contamination. © 1993 Wiley‐Liss, Inc.
The E2 region of HPV 16 in relation to different types of cervical lesions
Authors: Krajinovic Maja , Lazic Jelena , Stanimirovic Branko , Diklic Vukosava , Savic Ana
Published: March 2007

The presence of the E2 region of HPV 16 was determined by the polymerase chain reaction in biopsy samples with different histopathological diagnoses. The absence of the E2 gene was observed in 17 of 30 cases examined. The E2 deletions were not confined exclusively to invasive carcinomas or to integrated viral forms. In the majority of cases, the course of cervical lesions was consistent with the state of the E2 gene.
Two successive outbreaks of SRSV‐associated gastroenteritis in South Africa
Authors: Taylor Maureen B. , Schildhauer Carmen I. , Parker Simon , Grabow Wilhelm O. K. , Jiang Xi , Estes Mary K. , Cubitt W. David
Published: March 2007

Two successive outbreaks of gastroenteritis in South Africa were investigated to identify the aetiological agents. Some patients were involved in both outbreaks. Enteropathogenic bacteria or parasites were not evident in either outbreak. Small round structured viruses (SRSVs) were demonstrated in both outbreaks by direct electron microscopy. SRSV UK3/Hawaii virus was identified by immune electron microscopy as the causative agent in the first outbreak. Using new recombinant Norwalk virus (rNV) immunoassays for antibodies and antigen, Norwalk virus was implicated in the second outbreak. Preexisting antibodies to Norwalk virus were not protective and there was no cross protection between Hawaii and Norwalk viruses. There was no anamnestic response to Norwalk virus following the SRSV UK3/Hawaii outbreak although those affected had preexisting antibodies to Norwalk virus. To our knowledge, this is the first definitive diagnosis of SRSV‐associated gastroenteritis in South Africa.
Immunoglobulin M antibody response to measles virus following primary and secondary vaccination and natural virus infection
Authors: Erdman Dean D. , Heath Janet L. , Watson John C. , Markowitz Lauri E. , Bellini William J.
Published: March 2007

The use of IgM antibody detection for the classification of the primary and secondary measles antibody response in persons following primary and secondary vaccination and natural measles virus infection was examined. Of 32 nonimmune children receiving primary measles vaccination, 31 (97%) developed IgM antibodies, consistent with a primary antibody response. Of 21 previously vaccinated children with low levels of preexisting IgG antibodies who responded to revaccination, none developed detectable IgM antibodies, whereas 33 of 35 (94%) with no detectable preexisting IgG antibodies developed an IgM response. Of a sample of 57 measles cases with a prior history of vaccination, 55 (96%) had detectable IgM antibodies. Of these, 30 (55%) were classified as having a primary antibody response and 25 (45%) a secondary antibody response based on differences in their ratios of IgM to IgG antibodies. Differences in the severity of clinical symptoms between these 2 groups were consistent with this classification scheme. These findings suggest that 1) an IgM response follows primary measles vaccination in the immunologically naive, 2) an IgM response is absent on revaccination of those previously immunized, and 3) an IgM response may follow clinical measles virus infection independent of prior immunization status.
Long‐term carriage of hepatitis C virus with normal aminotransferase after interferon treatment in patients with chronic hepatitis C
Authors: Kakumu Shinichi , Yoshika Kentaro , Tanaka Kazuma , Higashi Yasuyuki , Kurokawa Susumu , Hirofuji Hideo , Kusakabe Atsuhiko
Published: March 2007

Studies were undertaken to investigate whether interferon therapy could induce hepatitis C virus (HCV) carriage with normal serum alanine aminotransferase (ALT) values using an assay that combined reverse transcription and polymerase chain reaction. The subjects studied were 53 patients with chronic active hepatitis C who received interferon (α, 33 cases; β, 20 cases) therapy. All were seropositive for HCV RNA prior to therapy. In all 22 complete responders, whose ALT levels fell to normal during therapy and for at least 24 weeks after therapy, HCV RNA became persistently negative except in two cases. The two had sustained viremia on treatment and for 1.0‐1.5 years of follow‐up, although their biochemical tests were normal. In 15 patients with a transient response in whom the disease recurred when interferon was stopped, HCV RNA was undetectable in 80% of the cases at the end of therapy, but the virus reappeared with subsequent elevation of ALT in all patients. However, 3 patients in this group had normal enzyme levels with viremia for 2.1‐2.8 years of follow‐up after acute deterioration of illness. In 16 patients who did not respond to interferon, HCV RNA was persistently positive during and after therapy. These findings suggest that interferon therapy induces a long‐term carrier state of HCV infection with normal ALT levels in some patients.
Attenuation of a reactivated cardiovirulent coxsackievirus B3: The 5′‐nontranslated region does not contain major attenuation determinants
Authors: Zhang Hong Yi , Yousef Galal E. , Cunningham Louise , Blake Neil W. , Yang Xiaomei Ou , Bayston Trevor A. , Kandolf Reinhard , Archard Leonard C.
Published: March 2007

To investigate the molecular basis of pathogenicity of Coxsackieviruses, a virus was reactivated by transfection from a full‐length cDNA clone derived from cardiovirulent Coxsackievirus B3 (CVB3). The reactivated virus, rCVB3, was passaged serially in human dermatofibroblasts (HDF). No cytopathic effect was observed up to 12 days after inoculation with rCVB3 or earlypassage virus, although disintegration of the monolayers was observed with late‐passage virus (10th to 14th passages). Approximately 10% of HDF inoculated with rCVB3 were positive for viral antigens by immunofluorescence using enterovirus‐ or CVB3‐specific monoclonal antibodies. These observations, together with the low infectivity titre of rCVB3 in HDF, suggest that HDF initially support only carrier state infection. After the 14th passage, the cardiovirulence of passaged virus (p14V) in mice was attenuated by a factor of >104. Phenotypic changes of plaque size were also noticed in p14V: An attenuated variant (p14V‐1) that produced larger plaques than rCVB3 in Vero cells has been plaque purified. The 5′‐terminus of the genome of attenuant p14V‐1 was amplified by polymerase chain reaction (PCR) and its sequence determined. Only one point mutation was found within the 5′‐nontranslated region (5′NTR) at position 690 (A to U) compared to the viral RNA sequence obtained for rCVB3. An intertypic chimeric virus was reactivated from a cDNA clone after replacing the 5′‐terminal 891 nucleotides of the wild‐type genome with the corresponding region of the attenuant p14V‐1. This chimeric virus, CB3/p14V1/1, produced wild‐type plaques in Vero cells and showed cardiovirulence similar to that of rCVB3 in mice. It is concluded that, unlike polioviruses, the 5′NTR of p14V‐1 does not contain major determinants of attenuation.
Prevalence of antibody to hepatitis C virus (HCV) in HIV‐1‐infected patients (nice seroco cohort)
Authors: Quaranta J. F. , Delaney S. R. , Alleman S. , Cassuto J. P. , Dellamonica P. , Allain J. P.
Published: March 2007

The prevalence of antibody to hepatitis C virus (HCV) in a cohort of 272 HIV‐infected patients was assessed by means of 4 anti‐HCV assays: a 1st generation and a neutralization test, a 2nd generation test, and a confirmatory test, the dot‐blot Matrix HCV™ immunoassay. The cohort included, as a single risk factor, 35.7% intravenous drug users (IVDUs), 25% homosexual men, 30.1% heterosexual individuals, 5.9% transfused patients, 0.7% occupational infections, and 2.6% patients with unknown infection source, and was studied on entry and in samples collected for up to 36 months. Results showed that on entry (i) sera of 83 out of 272 members of the cohort were positive by the HCV 1st generation EIA (30.5%); 70 were confirmed by the neutralization test (84.3%); (ii) 115 of the cohort were reactive with the 2nd generation HCV EIA (41.3%); (iii) with the dot‐blot immunoassay 99 (86.1%) of the cohort were confirmed and 16 remained indeterminate. The overall confirmed HCV antibody‐positive rate in these 272 patients was 36.4%. Antibody to HCV was detected in 78.3% of IVDUs, 18.3% of heterosexual individuals, 31.2% of transfused patients, and only 2.9% of homosexual men. The 36‐month follow‐up of this cohort revealed that 4/145 patients became anti‐HCV positive by second generation assay. Hepatitis B markers were frequently associated with HCV in IVDUs (71.1%) but infrequently in heterosexual (8.5%) or homosexual (1.5%) individuals. Our results suggest that HCV 2nd generation EIA used in combination with the semiautomated dot‐blot assay as a confirmatory test improves the specificity and sensitivity for HCV antibody detection. Homosexual males are at low risk of HCV infection among HIV risk groups. © 1994 Wiiey‐Liss, Inc.
Antibody response to a synthetic peptide derived from the human papillomavirus type 6/11 L2 protein in recurrent respiratory papillomatosis: Correlation between southern blot hybridization, polymerase chain reaction, and serology
Authors: Tachezy Ruth , Hamsikova Eva , Valvoda Jaroslav , Van Ranst Marc , Betka Jan , Burk Robert D. , Vonka Vladimir
Published: March 2007

Recurrent respiratory papillomatosis (RRP) is the most common benign tumour of the larynx, affecting both children and adults. We present a series of 25 patients, including 10 cases of juvenile multiple, 8 cases of adult solitary, and 7 cases of adult multiple RRP. Biopsy tissue from each patient was screened by Southern blot hybridization and polymerase chain reaction for the presence of human papillomavirus (HPV) DNA. Sera from patients and age‐ and sex‐matched controls were tested for the presence of HPV‐specific antibodies using a synthetic pep‐tide derived from the minor capsid protein (L2) of HPV 6/11. By Southern blot hybridization and/or polymerase chain reaction, biopsies from all patients were positive for HPV 6/11 DNA. There was no difference in antibody response between cases and controls. Female cases and controls had significantly higher antibody titers than male subjects. A correlation was observed between the HPV‐specific antibody level and the number of surgery‐necessitating recurrences. © 1994 Wiiey‐Liss, Inc.
Hepatitis C virus‐RNAs in plasma and in peripheral blood mononuclear cells of hemophiliacs with chronic hepatitis C: Evidence for viral replication in peripheral blood mononuclear cells
Authors: Willems M. , Peerlinck K. , Moshage H. , Deleu I. , Van Den Eynde C. , Vermylen J. , Yap S. H.
Published: March 2007

Hemophiliacs who have been exposed to unbeaten and/or dry heated pooled clotting factor concentrates are at high risk of chronic hepatitis C. Although the mechanism and site of hepatitis C virus (HCV) replication are not yet known, HCV is thought to replicate through a complementary negative RNA strand, as has been shown for fla‐viviruses. The detection of negative RNA strands has therefore been regarded as a marker of replication. We investigated the prevalence of HCV‐RNA and of negative HCV‐RNA strands in peripheral blood mononuclear cells (PBMC) and plasma of hemophiliacs.

Forty‐three of 47 patients studied (91%) had anti‐HCV antibodies and in 36 patients HCV‐RNA was detectable in PBMC. In one group of 20 patients negative HCV‐RNA strands were present in PBMC and 10 of these patients also had negative HCV‐RNA strands in plasma. In another group of nine patients HCV‐RNA was detected in PBMC, although cDNA synthesis was carried out in the absence of primers. Only in two of these nine patients negative and positive HCV‐RNA strands were demonstrated specifically in PBMC using a modified reverse transcription step.

If the presence of negative HCV‐RNA strands can be considered as marker of viral replication, the findings indicate that HCV can replicate in PBMC. Furthermore, in certain patients it is impossible to use the currently available technique to detect selectively positive or negative HCV‐RNA Strands. © 1994 Wiley‐Liss, Inc.
Complete nucleotide sequence of a beta‐cell tropic variant of coxsackievirus B4
Authors: Titchener P. A. , Jenkins O. , Szopa T. M. , Taylor K. W. , Almond J. W.
Published: March 2007

A mouse pancreas‐adapted variant of coxsackievirus B4 (P‐CB4) has been shown to replicate in, and cause an excessive release of insulin from, pancreatic beta cells cultured in vitro. The prototype CB4 strain (JVB Benschoten), from which the adapted variant was derived, although able to replicate in cultured islets does not cause a similar release of insulin from the beta cells. The pancreas‐adapted virus has also been shown to cause host cell protein synthesis shut‐off in beta cells and to inhibit (pro)insulin biosynthesis. These metabolic changes occur in the absence of cytolytic damage [Szopa et al.: Bioscience Reports 5:63‐69, 1985 and Cell Biochemistry and Function 4:181‐187, 1986].

To investigate the genetic basis for this beta cell tropism, the complete nucleotide sequence of P‐CB4 has been determined and compared to that of the previously published sequence of the prototype CB4 strain (JVB Benschoten) [Jenkins et al.: Journal of General Virology 68:1835‐1848, 1987]. Twenty‐five nucleotide sequence differences were observed. Of these, six occur in the 5′ noncoding region of the genome and 19 in the coding region (resulting in seven amino acid changes). The possible significance of these changes in relation to the beta cell tropism of the pancreas‐adapted virus is discussed. © 1994 Wiley‐Liss, Inc.
Genomic characterization of rotavirus strains obtained from hospitalized children with diarrhoea in Bangladesh
Authors: Tabassum Shahina , Shears P. , Hart C. A.
Published: March 2007

Faecal samples were obtained from 111 children hospitalized with acute watery gastroenteritis in a children's hospital in Bangladesh from January to December, 1989. Rotavirus was detected as the aetiologic agent in 35 (32%) of these patients. The electrophoretic pattern of ds‐RNA extracted from the rotaviruses showed 11 different migration patterns (six short and five long), as well as some short and long mixed profiles. All four major G serotypes were identified by amplification of a segment of the gene for VP7 using the polymerase chain reaction. A specific serotype could be assigned in 32 (91%) cases. Serotypes G1‐G4 accounted for 11%, 37%, 11%, and 23% of isolates respectively. Three (9%) mixed serotypes were identified by this method and were found to be mixtures of serotypes G1 and G4, G2 and G4, and G3 and G4. © 1994 Wiley‐Liss, Inc.
Tumor necrosis factor, interleukin‐2, and interferon‐gamma in adult varicella
Authors: Wallace Mark R. , Woelfl Ingrid , Bowler William A. , Olson Patrick E. , Murray Nancy B. , Brodine Stephanie K. , Oldfield Edward C. , Arvin Ann M.
Published: March 2007

Adult varicella can be a severe illness complicated by pneumonia, encephalitis, or prolonged fever. This study measured levels of tumor necrosis factor (TNF)‐alpha, interleukin‐2 (IL‐2), and interferon gamma (IFN‐G) in a consecutive group of 31 adult varicella patients presenting within 24 hours of rash onset. All cytokines were assayed using an ELISA technique. TNF‐alpha was detectable in 71% of patients with a mean level of 52 pg/ml. IL‐2 was detectable in 29% with a mean level of 1040 pg/ml. IFN‐gamma was detectable in only 9%. There was no correlation between TNF, IL‐2, or IFN‐G level and clinical severity as determined by duration and severity of cutaneous findings, duration of fever, frequency of hepatitis, or thrombocytopenia. © 1994 Wiley‐Liss, Inc.
Mutation specific PCR and direct solid phase sequencing assay for the detection of hepatitis B virus Pre‐C/C mutants in anti‐HBe‐positive, chronic hepatitis B
Authors: Goergen Bernd , zum Büschenfelde Karl‐Herrmann Meyer , Gerken Guido
Published: March 2007

Sequence analysis of the HBV DNA from patients with anti‐HBe+, chronic hepatitis B revealed that the lack of HBeAg is mostly due to a single G→A transition at nucleotide position 1896, resulting in a translational stop codon. A point mutation‐specific polymerase chain reaction (msPCR) for the detection of this genetic variant was established. Two serologically defined groups of patients with symptomatic chronic hepatitis B (HBeAg+ n = 14, anti‐HBe+ n = 11) were included in this study. Viral DNA from 43 sera (26 eAg+/17 anti‐HBe+) was amplified twice, using two different sets of PCR primers. Each set contained the same — strand primer, but the + strand primers differed at their 3′‐end, thus being complementary only to the wild‐type or to the mutant DNA. Unspecific amplification was ruled out by choosing high annealing temperatures (66°C) and cloned HBV‐DNA as specificity controls. Furthermore, the results were compared to our findings obtained with direct solid‐phase sequencing of the amplified viral DNA. Using msPCR, we found the mutation in four of 26 of the eAg+ sera and in 17 of 17 of our anti‐HBe+ samples. Mixed virus populations were identified in 13 of 21 cases. Compared to the sequencing results, msPCR is more sensitive and less time consuming for the detection of the stop codon and thus is suitable as a rapid and specific screening method. © 1994 Wiley‐Liss, Inc.
Identification of the major late human cytomegalovirus matrix protein pp65 as a target antigen for CD8+ virus‐specific cytotoxic T lymphocytes
Authors: McLaughlin‐Taylor Elizabeth , Pande Hema , Forman Stephen J. , Tanamachi Becky , Li Cheng‐Rong , Zaia John A. , Greenberg Philip D. , Riddell Stanley R.
Published: March 2007

Primary Cytomegalovirus (CMV) infection and reactivation of persistent CMV are associated with significant morbidity and mortality in immuno‐compromised individuals. Although recovery from CMV disease is correlated with the development of CMV‐specific cytotoxic T lymphocytes (CTL), the major viral target antigens to which the response is directed are ill‐defined, though they may comprise viral structural elements. We now identify the CMV matrix protein pp65 as a significant target antigen for CD8+ class I major histocompatibility complex (MHC)‐restricted CMV‐specific CTL derived from the peripheral blood of four of five latently infected individuals. CMV‐specific CTL recognition of pp65 on target cells occurs prior to the onset of viral gene expression and persists throughout the duration of the replicative cycle. Recognition in the absence of viral gene expression suggests that abundant viral protein enters the normal trafficking pathway upon viral penetration and is readily made available to MHC molecules for presentation at the cell surface. Thus pp65 specific CTL may represent an important effector population for early control and limitation of CMV infection and disease. The observation that CMV‐specific CTL can be induced in vitro using peptide fragments derived from pp65 supports the future use and manipulation of this and similar effector populations in a clinical setting. © 1994 Wiley‐Liss, Inc.
Use of confirmatory assays for diagnosis of hepatitis C viral infection in patients with hepatocellular carcinoma
Authors: Mangia Alessandra , Vallari David S. , Di Bisceglie Adrian M.
Published: March 2007

Serum samples from 87 patients with hepatocellular carcinoma (HCC) in the United States were tested for evidence of hepatitis C viral (HCV) infection using an immunoblot assay for antibodies to the hepatitis C virus and the polymerase chain reaction to detect HCV RNA. The findings with these assays were compared to those with a first generation enzyme‐linked immunoassay (EIA). Antibody to HCV (anti‐HCV) was detected in 14 patients (16%) by EIA; only eight of these were also positive by immunoblot and four had HCV RNA by reverse transcription polymerase chain reaction (RT‐PCR). An additional four cases, negative by EIA, were found to be positive by immunoblot; two of these had HCV RNA in serum. Evidence of previous hepatitis B viral infection was noted in 15 patients (17%). Only two patients with antibody to hepatitis B core antigen also had anti‐HCV by the immunoblot assay, suggesting that concomitant infection with the hepatitis B and C viruses was not common. Thus, HCV infection appears to play a less important role in the pathogenesis of HCC in the United States than in southern Europe and Japan and other etiologic factors should be sought in this population. © 1994 Wiley‐Liss, Inc.

This article is a US Government work and, as such, is in the public domain in the United States of America.
Infectivity titration of a prototype strain of hepatitis E virus in cynomolgus monkeys
Authors: Tsarev Sergei A. , Tsareva Tatiana S. , Emerson Suzanne U. , Yarbough Patrice O. , Legters Llewellyn J. , Moskal Thomas , Purcell Robert H.
Published: March 2007

The infectivity titer of a standard stock of the SAR‐55 strain of hepatitis E virus (HEV) was determined in cynomolgus macaques (Macaca fas cicularis) and the effect of dose on the course of the infection was examined by weekly monitoring of alanine aminotransferase (ALT) and anti‐HEV levels. Antibody to HEV (anti‐HEV) was measured with ELISAs based on ORF‐2 recombinant antigens consisting of either a 55 kDa region expressed in insect cells or shorter regions expressed as fusion proteins in bacteria. The ELISA based on the 55 kDa antigen was generally more sensitive. The infectivity titer of SAR‐55 was 106 cynomolgus 50% infectious doses per gram of feces. The infectivity titer corresponded to the HEV genome titer of the inoculum as determined by reverse transcriptase‐polymerase chain reaction (RT‐PCR). Anti‐HEV IgM was detected in only a portion of the animals that had an anti‐HEV IgG response. Biochemical evidence of hepatitis was most prominent in animals that were inoculated with the higher concentrations of virus and the incubation period to seroconversion was prolonged in animals that received the lower doses. © 1994 Wiley‐Liss, Inc.

This article is a US Government work and, as such, is in the public domain in the United States of America.
Low correlation of human cytomegalovirus DNA amplification by polymerase chain reaction with cytomegalovirus disease in organ transplant recipients
Authors: Weber B. , Nestler U. , Ernst W. , Rabenau H. , Braner J. , Birkenbach A. , Scheuermann E.‐H. , Schoeppe W. , Doerr H. W.
Published: March 2007

Seventy‐five organ transplant recipients underwent prolonged virological and serological follow‐up for early detection of human Cytomegalovirus (HCMV) infection after transplantation. HCMV DNA detection by nested polymerase chain reaction (PCR) and HCMV early structural antigen (pp65) detection were carried out in 576 peripheral blood leucocyte (PBL) samples. Furthermore, 563 blood specimens were investigated by a commercially available enzyme‐linked immunosorbent assay (ELISA) for the detection of specific immunoglobulins G, M, and A against HCMV structural antigens.

In eight of nine symptomatic organ transplant recipients, HCMV DNA was detected in one or more consecutive blood samples. HCMV DNA PCR was also positive in one or more samples from eight patients who never developed HCMV‐related symptoms. HCMV pp65 antigen was detected almost exclusively in PBL samples from organ transplant recipients suffering from HCMV disease. However, antigenaemia was not detected in four PCR positive patients presenting clinical signs attributable to HCMV infection. Two of the initially HCMV DNA positive samples were not confirmed by retesting and hybridisation.

The results of the present study demonstrate that despite the high specificity of nested PCR, HCMV DNA may be detected in the absence of clinical symptoms attributable to HCMV infection. In asymptomatic reactivation, limited replication of viral DNA may be responsible for positive results of PCR without any clinical relevance. In this context, pp65‐antigen detection from PBL seems to have a better prognostic value, but is not always detected when clinical symptoms are present. © 1994 Wiley‐Liss, Inc.
Human papillomavirus DNA and anti‐HPV secretory IgA antibodies in cytologically normal cervical specimens
Authors: Veress György , Konya József , Csiky‐Mészáros Tibor , Czeglédy Judit , Gergely Lajos
Published: March 2007

Cervical specimens collected from 163 cytologically healthy women were screened for the presence of human papillomavirus (HPV) DNA and anti‐HPV secretory IgA antibodies. HPV DNA was detected by a general primer mediated polymerase chain reaction (PCR), which amplifies a conserved region from the L1 ORF of genital HPVs. The PCR products were typed by restriction enzyme digestion. A total of 35 samples (21.5%) were positive for HPV DNA (13 samples for HPV 6, 6 for HPV 16, 3 for HPV 18, and 13 for untypeable HPV X). HPV DNA positivity was significantly higher among women under 25 years of age (34.8%) than among the older patients (12.4%) (P< 0.001).

An enzyme‐linked immunosorbent assay (ELISA) using synthetic peptide antigens was carried out to detect local secretory IgA antibodies against the following HPV specific antigens: HPV 16 E2, HPV 16 E7, HPV 16 L1, HPV 16 L2, and HPV 11 L2. Thirty‐four secretions (20.9%) were found to react with at least one of the oligopeptides. Anti‐HPV IgA positivity was the highest among women aged 25–32 years, and it was significantly lower in both the younger and the older age groups (P < 0.05). Correlation between HPV DNA and anti‐HPV IgA detection was rather weak: anti‐peptide IgA positivity was 34.3% (12 of 35) among HPV DNA positive patients compared to 17.2% (22 of 128) among HPV DNA negative women (P < 0.05). The fluctuating course of latent HPV infections should be considered in evaluating the low level of correlation between HPV DNA and anti‐HPV IgA positivity. © 1994 Wiley‐Liss, Inc.
Typing of human papillomaviruses in cervical carcinoma biopsies from Cape Town
Authors: Williamson Anna‐Lise , Brink Nicola S. , Dehaeck Catherine M. C. , Ovens Samantha , Soeters Robbert , Rybicki Edward P.
Published: March 2007

Sixty‐eight cervical carcinoma biopsies from women at Groote Schuur hospital in Cape Town were screened for human papillomavirus (HPV) DNA by polymerase chain reaction (PCR) amplification with subsequent typing by hybridisation with specific oligonucleotides. Amplified DNAs which hybridised with the HPV group‐specific oligonucleotide probes, but with none of the type‐specific probes, were categorized as unclassified HPV types. Eighty‐one percent of samples were HPV positive and the following distribution and types were detected: 46% HPV 16, 1.5% HPV 18, 6% HPV 31, 6% HPV 33, 1.5% HPV 45, and 25% unclassified HPVs. Four of the tumours had detectable dual infections with the following combinations of HPVs: HPV31/HPV33; HPV16/ HPV33; HPV18/HPV16; HPV45/HPV33. Tumour DNA from the 17 PCR unclassified HPVs was analysed by Southern blotting and gave the following results: 11 were negative, 4 were positive on hybridisation but could not be typed (unclassified HPV), 1 was HPV 16, and 1 resembled the HPV 16 “variant” described previously [Williamson et al. (1989): Journal of Medical Virology 28:146‐149]. Only 2 of 13 samples that were negative for HPV by PCR were positive for unclassified HPVs by Southern blot analysis. Sequencing of a PCR product revealed that one of the unclassified HPVs was HPV 52. This is the first record of HPV 52 and HPV 45 in Africa. © 1994 Wiley‐Liss, Inc.
Enhanced detection of antibodies to hepatitis C virus by use of a third‐generation recombinant immunoblot assay
Authors: Buffet C. , Charnaux N. , Laurent‐Puig P. , Chopineau S. , Quichon J. P. , Briantais M. J. , Dussaix E.
Published: March 2007

Fifty‐seven sera with indeterminate results by the second generation RIBA (RIBA 2) for confirmation of hepatitis C virus (HCV) enzyme linked immunoassay (ELISA) reactivity were tested by the new third generation RIBA (RIBA 3). Thirty three (57.9%) displayed reactivity for at least one other band and were therefore classified as positive; two became negative and 22 (38%) remained indeterminate. The incidence of HCV viremia, as determined by the RNA polymerase chain reaction (PCR), was 75% for the latter sera. The data show that it is important to subject RIBA 3 indeterminate samples to PCR. © 1994 Wiley‐Liss, Inc.
Comparison of quantitative cDNA‐PCR with the branched DNA hybridization assay for monitoring plasma hepatitis C virus RNA levels in haemophilia patients participating in a controlled interferon trial
Authors: Bresters D. , Cuypers H. T. M. , Reesink H. W. , Mauser‐Bunschoten E. P. , van den Berg H. M. , Schaasberg W. P. , Wilber J. C. , Urdea M. S. , Neuwald P. , Lelie P. N.
Published: March 2007

The branched DNA (bDNA) assay was compared with a semi‐quantitative cDNA‐polymerase chain reaction (cDNA‐PCR) assay for monitoring HCV RNA levels in plasma in 17 haemophilia patients participating in a controlled a‐interferon trial. Good correlation between the HCV RNA levels as detected by the two assays was observed, with a correlation co‐efficient of 0.83 (P < 0.0001) and 0.90 (P < 0.0001) at week 0 and 24, respectively. Hepatitis C virus RNA (HCV RNA) levels could be assessed with the bDNA assay in 14/17 (82 percent) HCV cDNA‐PCR positive pre‐treatment samples. The bDNA assay apparently failed to detect low viral titres. (riterferon treated patients (n = 11) showed either a complete response, being a large reduction in HCV RNA level to below the detection limit of the HCV cDNA‐PCR assay (6/11) or no significant reduction in HCV RNA level (5/11). A “partial” virological response was not observed. The changes in HCV RNA plasma levels in non‐responders during interferon (IFN) treatment were similar to the (small) natural fluctuations in viral load observed in controls (untreated patients). Although the bDNA assay was not as sensitive as cDNA‐PCR, given its user friendliness and quantitative results, it is concluded that it is a useful test for monitoring HCV RNA levels in patients treated with interferon. However, patients who are non‐reactive in the bDNA assay have to be retested by cDNA‐PCR because low viral titres are not detected by the bDNA assay. © 1994 Wiley‐Liss, Inc.
Detection of respiratory syncytial virus by RNA‐polymerase chain reaction and differentiation of subgroups with oligonucleotide probes
Authors: van Milaan A. J. , Sprenger M. J. W. , Rothbarth Ph. H. , Brandenburg A. H. , Masurel N. , Claas E. C. J.
Published: March 2007

The polymerase chain reaction (RNA‐PCR) was used for specific detection of respiratory syncytial virus (RSV) genomes in clinical specimens. A set of primers was selected from conserved regions of the 1Band N genes for detection of both subgroups. The primers were found to be RSV specific, all RSV strains generated a 218 bp product, and no RSV specific amplified product was obtained when nucleic acids from a variety of micro‐organisms from the respiratory tract were subjected to the RNA‐PCR. We took advantage of the sequence heterogeneity of the amplified products to discriminate between the A and B strains by hybridisation with subgroup specific Oligonucleotide probes. This additional hybridisation assay increased the sensitivity of the RNA‐PCR tenfold. The RNA‐PCR was tested on clinical specimens from children with symptoms of an infection of the respiratory tract. The results were compared with isolation of RSV in cell culture and direct immunofluorescence. From 93 specimens tested, 31 were found positive by all three techniques. Six additional positive results were detected using RNA‐PCR. From these 37 RSV positive specimens 33 (92%), including all 6 additional positives, were subgroup A and only 4 were subgroup B strains.

Thus, the RNA‐PCR is a specific and sensitive technique for the detection and subgroup classification of RSV genomes. © 1994 Wiley‐Liss, Inc.
Sequence analysis of proviral HIV RT amplified directly by a semi‐quantitative technique from AZT treated patients
Authors: Stein Christian A. , Levantis P. , Oxford J. S.
Published: March 2007

To minimise possible arbitrary selective effects of culturing HIV, proviral RT DNA was isolated directly from PBMCs of four patients treated for 6–14 months with AZT, RT DNA was amplified by PCR and sequenced directly without further in vitro manipulation. Eighteen changes additional to those 4 or 5 changes previously shown by genetic reconstruction experiments [Kellam et al.: Proceedings of the Nationa Academy of Sciences of the United States of America 89:1934–1938, 1992] were found in the 14 different sequences analysed. Substitutions clustered in two defined areas of the RT, from amino acids 60 to 70 and from 180 to 220. Mutations were observed at each of the two areas independently or at both sites simultaneously. Amino acid changes in RT from patients harbouring resistant strains of HIV‐1 were found in positions 60 (Val), 62 (Ala), 93 (Gly), 100 (Phe), 161 (Pro), 173 (Asn), 177 (Glu), 180 (lle), 181 (Tyr), 182 (Leu), 186 (Asp), 194 (Gln), 196 (Glu), 200 (lle), 209 (Val), 210 (Trp), 211 (Lys), and 214 (Phe) in addition to those described previously. It was anticipated that multiple proviral DNAs would be present in a single clinical sample. Therefore end point dilution PCR methodology was used, which allowed sequence analysis of separate proviral DNA molecules from the patients' proviral DNA. Even in patients who had received AZT for more than 10 months wild‐type “AZT‐sensitive” RTs co‐existed with mutated “AZT‐resistant” RTs in the same patient sample. © 1994 Wiley‐Liss, Inc.
Homologous and heterologous neutralization antibody responses after immunization with Japanese encephalitis vaccine among Taiwan children
Authors: Ku Chia‐Chi , King Chwan‐Chuen , Lin Ching‐Yuang , Hsu Huei‐Chu , Chen Li‐Yen , Yueh Yi‐Yuen , Chang Gwong‐Jen J.
Published: March 2007

Because 21 immunized children (13%) among the 162 confirmed Japanese encephalitis (JE) cases during 1986–1991 occurred in Taiwan, we collected 320 serum samples from Taiwan children aged 15–31 and 27–44 months immediately before the 1st dose (n = 41) and 1–3 months after the 2nd dose (n = 78, 27 pairs), and immediately before (n = 58) and 1–3 months after the 3rd dose (n = 143,44 pairs) to determine neutralization antibody (Nt Ab) against the Nakayama (N) and Beijing‐1 (B) strains and two Taiwan wild type JE viruses (JEV): CC‐27 and CH‐1392. Our Nt results showed that (1) B vaccine stimulated a better homologous Ab response than N vaccine for Nt Ab seropositivity rate (NASR), produced a higher level of Nt titer after the primary immunization [2 doses = 100% vs. 91%, geometric mean titer (GMT) = 115 vs. 22], had a greater booster effect (3 doses: 100% vs. 95%; GMT = 320 vs. 33), and showed a better capability to neutralize two local Taiwan JEV strains, particularly only after 3 doses (ave. NASR for B vs. N = 90% vs. 10%; and GMT for B vs. N = 154 vs. 1); (2) the two wild type JEV strains had different plaque morphology and antigenic variation and the CC‐27 strain was not neutralized as well as the CH‐1392 strain after 3 doses of vaccine (BBB or NNN or NNB); and (3) 30% of the children had lost JEV Nt Ab one year after the 2nd dose of N vaccine and natural infection with JE virus did occur among those children after immunization. In conclusion, (1) three doses of mouse‐brain vaccine are the minimum requirement to protect children against the local Taiwan JEV; (2) the best strain for a JE vaccine depends on level of Nt Ab it induced, the molecular epidemiology and antigenic variation of the JEV in each local area; and (3) future vaccine must produce better B‐ and T‐cell memory. © 1994 Wiley‐Liss, Inc.
Biological and genetic characterization of a hemagglutinating coronavirus isolated from a diarrhoeic child
Authors: Zhang X. M. , Herbst W. , Kousoulas K. G. , Storz J.
Published: March 2007

The coronavirus strain HECV‐4408 was isolated from diarrhea fluid of a 6‐year‐old child with acute diarrhea and propagated in human rectal tumor (HRT‐18) cells. Electron microscopy revealed coronavirus particles in the diarrhea fluid sample and the infected HRT‐18 cell cultures. This virus possessed hemagglutinating and acetylesterase activities and caused cytopathic effects in HRT‐18 cells but not in MDBK, GBK and FE cells. One of four S‐specific monoclonal antibodies reacted in Western blots with HECV‐4408, BCV‐L9 and BCV‐LY138 but not with HCV‐OC43, and two reacted with BCV‐L9 but not with HECV‐4408, BCV‐LY138 and HCV‐OC43. One S‐specific and two N‐specific monoclonal antibodies reacted with all of these strains. cDNA encompassing the 3′ 8.5 kb of the viral RNA genome was isolated by reverse transcription followed by polymerase chain reaction amplification had size and restriction endonuclease patterns similar to those of BCV‐L9 and BCV‐LY138. In contrast, the M gene of HCV‐OC43 differed in restriction patterns from HECV‐4408 and BCV. A genomic deletion located between the S and M within the nonstructural genes of HCV‐OC43 was not detected in HECV‐4408. DNA sequence analyses of the S and HE genes revealed more than 99% nucleotide and deduced amino acid homologies between HECV‐4408 and the virulent wild‐type BCV. Forty‐nine nucleotide and 22 amino acid differences were found between the HE genes of HECV‐4408 and HCV‐OC43, while only 16 nucleotide and 3 amino acid differences occurred between the HE genes of HECV‐4408 and BCVLY138. We thus conclude that the strain HECV‐4408 is a hemagglutinating enteric coronavirus that is biologically, antigenically and genomically more closely related to the virulent BCVLY138 than to HCV‐OC43. © 1994 Wiley‐Liss, Inc.
Serotyping of human rotaviruses in the Tokyo area (1990–1993) by enzyme immununoassay with monoclonal antibodies and by reverse transcription and polymerase chain reaction amplification
Authors: Ushijima Hiroshi , Mukoyama Atsushi , Hasegawa Ayako , Nishimura Shuichi , Konishi Kyoko , Bosu Kim
Published: March 2007

Serotyping of human rotavirus in the Tokyo area was conducted from 1990 to 1993 by enzyme immunoassay with monoclonal antibodies (EIAMAbs) against VP7 and by reverse transcription and polymerase chain reaction (RT‐PCR) amplification of the VP4 and VP7 genes.

The results by EIA‐MAbs were very similar to those obtained by RT‐PCR. Evidence of intraserotypic variations was suggested because strains of undetermined serotypes were detected by either EIA‐MAb or RT‐PCR. This kind of study is required for vaccine development. © 1994 Wiley‐Liss, Inc.