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This paper is aimed at the development of a biosensor for direct detection of Hepatitis C virus (HCV) surface antigen: envelope protein (E2). A recombinant LEL fragment of biological cell receptor CD81 and two short synthetic peptides imitating the fragment of LEL sequence of CD81 (linear and loop-like peptides) capable of specific binding to E2 were tested as molecular recognition elements of the biosensor. For this purpose the selected ligands were immobilized to the surface of a screen-printed electrode utilized as an electrochemical sensor platform. The immobilization parameters such as the ligand concentration and the immobilization time were carefully optimized for each ligand. Differential pulse voltammetry used to evaluate quantitatively binding of E2 to the ligands revealed their similar binding affinity towards E2. Thus, the linear peptide was selected as a less expensive and easily prepared ligand for the HCV biosensor preparation. The resulting HCV biosensor demonstrated selectivity towards E2 in the presence of interfering protein, conalbumin. Moreover, it was found that the prepared biosensor effectively detected E2 bound to hepatitis C virus-mimetic particles (HC VMPs) at LOD value of 2.1∙10 -5  mg/mL both in 0.01 M PBS solution (pH 7.4) and in simulated blood plasma., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Although travel-related tick-borne encephalitis (TBE) cases have been increasingly registered worldwide, very few published case studies are available to date. The present report describes a travel-related TBE case and provides genotypic characterization of two viral isolates. Laboratory diagnostics were based on complement fixation test and virus isolation. This report is unique because the TBE case was first confirmed by virus isolation from the engorged tick and only later from the patient's blood. Moreover, this case demonstrated a successful prophylaxis performed on day 8 post tick exposure although it is generally recommended that anti-TBEV immunoglobulins should be administered not later than on day 4 after tick bite. Sequences of E protein gene fragments were used to phylogenetically characterize the two isolates. The results demonstrated that both viral isolates belonged to clusteron 3A (Zausaev group) of the Asian lineage of the TBEV Siberian subtype. The synonymous nucleotide substitution, C351 T, was identified in E protein gene fragments of TBEV 88 and TBEV 89, which could have been induced by virus transmission. A few important take-home messages can be gleaned from the reported case. First, travelers should be aware of TBE endemic areas that they plan to visit and be proactive when exposed to Ixodes ticks. Second, medical practitioners should always consider travel history and potential tick exposure of patients. Lastly, engorged Ixodes spp. ticks removed from the patients, who have arrived from endemic areas, should be tested for TBEV even in the absence of TBE clinical signs., (Copyright © 2021 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The transporter associated with antigen processing (TAP) directly participates in the immune response as a key component of the cytosolic peptide to major histocompatibility complex (MHC) class I protein loading machinery. This makes TAP an important target for viruses avoiding recognition by CD8+ T lymphocytes. Its activity can be suppressed by the UL49.5 protein produced by bovine herpesvirus 1, although the mechanism of this inhibition has not been understood so far. Therefore, the main goal of our study was to investigate the 3D structure of bovine herpesvirus 1 - encoded UL49.5 protein. The final structure of the inhibitor was established using circular dichroism (CD), 2D nuclear magnetic resonance (NMR), and molecular dynamics (MD) in membrane mimetic environments. In NMR studies, UL49.5 was represented by two fragments: the extracellular region (residues 1-35) and the transmembrane-intracellular fragment (residues 36-75), displaying various functions during viral invasion. After the empirical structure determination, a molecular docking procedure was used to predict the complex of UL49.5 with the TAP heterodimer. Our results revealed that UL49.5 adopted a highly flexible membrane-proximal helical structure in the extracellular part. In the transmembrane region, we observed two short α-helices. Furthermore, the cytoplasmic part had an unordered structure. Finally, we propose three different orientations of UL49.5 in the complex with TAP. Our studies provide, for the first time, the experimental structural information on UL49.5 and structure-based insight in its mechanism of action which might be helpful in designing new drugs against viral infections., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objectives: Rapid diagnostic tests targeting virus-specific antigen could significantly enhance the diagnostic capacity for chikungunya virus infections. We evaluated the accuracy of an immunochromatographic antigen test for diagnosis of chikungunya in a reference laboratory for arboviruses., Methods: An immunochromatographic rapid test that uses mouse monoclonal antibodies as a tracer against the E1-envelope protein of chikungunya (ARKRAY, Inc. Kyoto, Japan) was evaluated. Sensitivity was tested in sera from travellers with RT-PCR confirmed chikungunya virus infection (Eastern/Central/Southern African (ECSA) genotype) (n=9) and from patients diagnosed during the 2014-2015 chikungunya outbreak on Aruba (Asian genotype, n=30). Samples from patients with other febrile and non-febrile illnesses (n=26), sera spiked with Flavivirus and Alphavirus reference strains (n=13, including non-spiked serum), and samples containing other selected pathogens (n=20) were used to test specificity of the E1-antigen test., Results: Sensitivity of the E1-antigen test was 8/9 (88.9%, 95% CI 56.5-98.0) for the ECSA genotype, but only 10/30 (33.3%, 95% CI 19.2-51.2) for the Asian genotype. Overall diagnostic specificity was 49/59 (83.1%, 95% CI 71.5-90.5)., Conclusions: The E1-antigen test we evaluated had fair diagnostic sensitivity for ECSA genotype chikungunya, but low sensitivity for Asian genotype, and poor overall specificity. Antibodies that react across genotypes will be required for further development of a rapid test for chikungunya. Performance of new tests should be evaluated against different chikungunya genotypes., (Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

The aim of this study was to establish the effect of a 70% ethanol extract of Elaeocarpus sylvestris (ESE) on varicella-zoster virus (VZV) replication and identify the specific bioactive component(s) underlying its activity. ESE induced a significant reduction in replication of the clinical strain of VZV. Activity-guided fractionation indicated that the ethyl acetate (EtOAc) fraction of ESE contains the active compound(s) inhibiting VZV replication. High-Performance Liquid Chromatography coupled to Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry (HPLC-Q-TOF-MS/MS) analysis of the EtOAc fraction of ESE facilitated the identification of 13 chemical components. Among these, 1,2,3,4,6-penta-O-galloyl-ß-D-glucose (PGG) markedly suppressed VZV-induced c-Jun N-terminal kinase (JNK) activation, expression of viral immediate-early 62 (IE62) protein and VZV replication. Our results collectively support the utility of PGG as a potential candidate anti-viral drug to treat VZV-associated diseases., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A chitosan-modified carbon fiber electrode (CFE) for dengue virus envelope protein (DENV) was developed. Antibodies against DENV were covalently immobilized on the chitosan (CHIT) matrix after activation with sodium periodate. Cyclic voltammetries and scanning electron microscopies analysis were performed to monitor steps involved in the CFE surface modification. Amperometric response of the competitive immunoassays was generated by hydrogen peroxide reaction with the peroxidase conjugated to DENV and 2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as mediator. The immunosensor showed a lower limit of detection for DENV (0.94 ng mL(-1)) than previously described and a linear range from 1.0 to 175 ng mL(-1), in concentration levels clinically relevant for dengue virus diagnosis. The intra- and inter-assay were respectively 5.8% and 3.6%. The unique and simple design of this immunoassay format provides an economical alternative for the manufacture of other sensitive sensors., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Each year, up to 10,000 cases of infections with the flavivirus tick-borne encephalitis (TBE) virus that affect the central nervous system are reported in Europe and Asia. Due to the potentially severe adverse effects of post-exposure prophylaxis with TBE virus hyperimmunoglobulin, TBE can currently only be treated symptomatically. An RNA interference (RNAi) approach to inhibit TBE virus replication was therefore developed. In this study we demonstrate for the first time that small interfering RNAs (siRNAs) targeted at the TBE virus genome reduce the quantity of infectious TBE virus particles, TBE virus genome, and TBE virus protein in vitro by up to 85%. The 50% inhibitory dose (DI(50)) of the shRNA plasmid was only 0.05μg/ml. As RNAi-based therapeutics for other diseases are already being evaluated in phases II and III clinical trials, it is possible that RNAi could become valuable tool for controlling TBE virus infection., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Infectious bronchitis virus (IBV) is a highly contagious respiratory coronavirus of domestic chickens. Although mortality is low, infection with IBV results in substantial losses for the egg and meat chicken industries. Despite the economic importance of IBV and decades of research into the pathogenesis of infection, significant gaps in our knowledge exist. The aim of this study was to compare the early progression of air sac lesions in birds receiving a vaccine strain of the virus or a more virulent field strain. The air sacs are lined by different types of epithelia and are relatively isolated from the environment, so they represent a unique tissue in which to study virus-induced lesions. Both the pathogenic and vaccine strains of the virus produced significant lesions; however, the lesions progressed more rapidly in the birds receiving the pathogenic strain. Immunohistochemistry demonstrated that in birds infected with the pathogenic strain of virus, IBV spike protein is detected first in the ciliated cells lining the air sac. These preliminary data provide important clues regarding potential mechanisms for IBV tissue tropism and spread and show that the nature of the virus isolate influences the early progression of IBV infection., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

SIV infection of macaques is the most widely employed model for preclinical AIDS vaccine and pathogenesis research. In macaques, high-titer virus-specific antibodies are induced by infection, and antibody responses can drive evolution of viral escape variants. However, neutralizing antibodies (Nabs) induced in response to SIVmac239 and SIVmac251 infection or immunization are generally undetectable or of low titer, and the identification and cloning of potent Nabs from SIVmac-infected macaques remains elusive. Based on recent advances in labeling HIV-specific B lymphocytes [1-3], we have generated recombinant, secreted, soluble SIVmac envelope (Env) proteins (gp120 and gp140) for detection and quantification of SIVmac Env-specific B lymphocytes. In contrast to HIV-1, we found that soluble SIVmac239 gp140 retains the ability to form stable oligomers without the necessity for introducing additional, stabilizing modifications. Soluble oligomeric gp140 reacted with rhesus anti-SIV Env-specific monoclonal antibodies (MAbs), and was used to deplete Env-specific antibodies with SIV neutralization capability from plasma taken from a rhesus macaque immunized with live attenuated SIVmac239∆nef. Soluble gp120 and gp140 bound to SIV-specific immortalized B cells, and to SIV Env-specific B lymphocytes in peripheral blood of immunized animals. These reagents will be useful for analyzing development of Env-specific B cell responses in preclinical studies using SIV-infected or vaccinated rhesus macaques., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Development of an electrochemical DNA biosensor, using a gold electrode modified with a self-assembled monolayer composed of a peptide nucleic acid (PNA) probe and 6-mercapto-1-hexanol, is described. The sensor relies on covalent attachment of the 14-mer PNA probe related to the hepatitis C virus genotype 3a (pHCV3a) core/E1 region on the electrode. Covalently self-assembled PNA could selectively hybridize with a complementary sequence in solution to form double-stranded PNA-DNA on the surface. The increase of peak current of methylene blue (MB), upon hybridization of the self-assembled probe with the target DNA in the solution, was observed and used to detect the target DNA sequence. Some hybridization experiments with noncomplementary oligonucleotides were carried out to assess whether the suggested DNA sensor responds selectively to the target. Diagnostic performance of the biosensor is described and the detection limit was found to be 5.7 x 10(-11)M with a relative standard deviation of 1.4% in phosphate buffer solution, pH 7.0. This sensor exhibits high reproducibility and could be used for detection of the target DNA for seven times after the regeneration process., (Copyright 2009 Elsevier Inc. All rights reserved.)

Objectives: The frequency of hepatitis G virus exposure in blood donors varies between 2.5% in Japan to 24.2% in Poland. Therefore there is a geographic difference in distribution of hepatitis G virus (HGV) in the world. We aimed to determine the frequency of HGV exposure in Iranian blood donors., Methods: Blood samples from 478 Iranian volunteer blood donors were tested. Positive anti-E2 samples were tested for HGV RNA by reverse transcriptase polymerase chain reaction (RT PCR) using primers derived from the NS5A region of the viral genome., Results: Of the 478 donors enrolled in our study, five (1%) were positive for anti-E2. Only one donor out of a total of three HBsAg-positive donors was co-infected with HGV, but we did not find HGV and HCV co-infection in our subjects. HGV RNA was not observed in the five anti-E2-positive subjects. We did not find HGV viremia and antibody at the same time., Conclusion: A low frequency of HGV exposure in blood donors was found in this study. We did not observe co-infection of HGV with HCV in our subjects, supporting the theory that although the parenteral route is the most effective means of transmission, other routes such as sexual contact and intra-familial contact may also play a role in HGV transmission.

Little information is available on persistent infection of severe acute respiratory syndrome (SARS) coronavirus (CoV). In this study, we established persistent infection of SARS-CoV in the Vero E6 cell line. Acute infection of Vero E6 with SARS-CoV produced a lytic infection with characteristic rounding cytopathic effects (CPE) and the production of a large number of infectious particles in the culture fluid within 3 days post-infection. Upon subsequent culturing of the remaining adherent cells, the cells gradually proliferated and recovered normal morphology similar to that of the parental cells, and continued to produce large numbers of infectious viral particles during the observation period of 5 months. Among a total of 87 cell clones obtained from the persistently infected Vero E6, only four cell clones (named #13, #18, #21, and #34) were positive for viral RNA. Clones #13, #18, and #34 shifted to viral RNA-negative during subsequent cultures, while #21 continuously produced infectious particles at a high rate. The SARS-CoV receptor, angiotensin-converting enzyme 2, was almost completely down regulated from the cell surface of persistently infected cells. Western blot analysis as well as electron microscopy indicated that the ratios of spike to nucleocapsid protein in clone #21 as well as its parental persistently infected cells were lower than that in the cells in the acute phase of infection. These Vero E6 cells persistently infected with SARS-CoV may be useful for clarifying the mechanism of the persistent infection and also for elucidating the possible pathophysiologic significance of such long-term maintenance of this virus.

Purpose: To standardize and apply a polymerase chain reaction (PCR) on the glycoprotein D gene to differentiate Herpes simplex virus (HSV) 1 & 2 serotypes in culture negative intraocular specimens., Methods: Twenty-one intraocular fluids collected from 19 patients were subjected to cultures for HSV and uniplex PCR (uPCR) for DNA polymerase gene. To differentiate HSV serotypes, as 1 & 2, a seminested PCR (snPCR) targeting the glycoprotein D gene was standardised and applied onto 21 intraocular fluids. The specificity of the snPCR was verified by application onto ATCC strains of HSV 1 and 2, clinical isolates and DNA sequencing of the amplified products. All specimens were also tested for the presence of cytomegalovirus (CMV) and varicella zoster virus (VZV) by nucleic acid amplification methods., Results: Four of the 21 intraocular fluids were positive for HSV by uPCR. snPCR detected HSV in three additional specimens (total of seven specimens), and identified three as HSV 1 and four as HSV 2. DNA sequencing of PCR products showed 100% homology with the standard strains of HSV 1 and 2 respectively. None of the samples were positive in culture. Among the other patients, CMV DNA was detected in two and VZV DNA in five others., Conclusions: The standardized snPCR can be applied directly onto the culture negative specimens for rapid differentiation of HSV serotypes.

The tertiary structures of the S1 and S2 domains of the spike protein of the coronavirus which is responsible of the severe acute respiratory syndrome (SARS) have been recently predicted. Here a molecular assembly of SARS coronavirus peplomer which accounts for the available functional data is suggested. The interaction between S1 and S2 appears to be stabilised by a large hydrophobic network of aromatic side chains present in both domains. This feature results to be common to all coronaviruses, suggesting potential targeting for drugs preventing coronavirus-related infections.

Background: Although the genome of severe acute respiratory syndrome coronavirus (SARS-CoV) has been sequenced and a possible animal reservoir identified, seroprevalence studies and mass screening for detection of subclinical and non-pneumonic infections are still lacking., Methods: We cloned and purified the nucleocapsid protein and spike polypeptide of SARS-CoV and examined their immunogenicity with serum from patients with SARS-CoV pneumonia. An ELISA based on recombinant nucleocapsid protein for IgG detection was tested with serum from 149 healthy blood donors who donated 3 years previously and with serum positive for antibodies against SARS-CoV (by indirect immunofluorescence assay) from 106 patients with SARS-CoV pneumonia. The seroprevalence of SARS-CoV was studied with the ELISA in healthy blood donors who donated during the SARS outbreak in Hong Kong, non-pneumonic hospital inpatients, and symptom-free health-care workers. All positive samples were confirmed by two separate western-blot assays (with recombinant nucleocapsid protein and recombinant spike polypeptide)., Findings: Western-blot analysis showed that the nucleocapsid protein and spike polypeptide of SARS-CoV are highly immunogenic. The specificity of the IgG antibody test (ELISA with positive samples confirmed by the two western-blot assays) was 100%, and the sensitivity was 94.3%. Three of 400 healthy blood donors who donated during the SARS outbreak and one of 131 non-pneumonic paediatric inpatients were positive for IgG antibodies, confirmed by the two western-blot assays (total, 0.48% of our study population)., Interpretation: Our findings support the existence of subclinical or non-pneumonic SARS-CoV infections. Such infections are more common than SARS-CoV pneumonia in our locality.

In this study, 13 samples of liver biopsies from patients with chronic hepatitis C were studied by transmission electron microscopy (EM) and immunoelectron microscopy (IEM). The 13 biopsies showed ultrastructural cell damage typical of acute viral hepatitis. In four of the 13 liver biopsies enveloped virus-like particles (VLPs) inside cytoplasmic vesicles and in the cytoplasm of hepatocytes were observed. We also detected the presence of unenveloped VLPs mainly in the cytoplasm and in the endoplasmic reticulum. IEM using anti-core, E1 and E2 monoclonal antibodies (mAbs) confirmed the specific localization of these proteins, in vivo, inside cytoplasm and endoplasmic reticulum. Thus, this work provided evidence for hepatocellular injury related to HCV infection. It also suggested the presence of HCV-related replicating structures in the cytoplasm of hepatocytes and raised the possibility of hepatitis C virion morphogenesis in intracellular vesicles.

Background/aims: A simple and reproducible hepatic immunohistochemical staining (IHS) for hepatitis C virus (HCV) is not available. We aimed to validate hepatic IHS with monoclonal antibody (Mab) IG222, directed against the HCV-envelope 2 (E2) protein., Methods: A three-step indirect immunoperoxidase method was used for frozen sections and a two-step indirect EnVision technique was used for paraffin-embedded sections., Results: Naturally or in vitro HCV infected primary human hepatocytes were immunoreactive to HCV-E2. In the patient study (n=253), IHS had a sensitivity of 96% and a specificity of 91%. Six patients who showed positivity in the liver with Mab IG222, but remained anti-HCV and HCV-RNA negative, had hepatitis C-like changes in their liver biopsy. In one patient HCV-RNA could be detected in the liver biopsy. We confirmed early graft reinfection in patients transplanted for HCV-related disease (34 patients with serial biopsies). Treatment for acute cellular rejection with steroids was associated with an increase in staining intensity. In nine patients with clearance of HCV-RNA during antiviral therapy, seven achieved negativation of immunoreactivity and two a marked reduction., Conclusions: The IHS with Mab IG222 is an accurate tool for diagnosis and clinical management of chronic hepatitis C.

Guinea-pigs and non-human primates have traditionally been used as animal models for studying Ebola Zaire virus (EBO-Z) infections. The virus was also recently adapted to the stage of lethal virulence in BALB/c mice. This murine model is now in use for testing antiviral medications and vaccines. However, the pathological features of EBO-Z infection in mice have not yet been fully described. To identify sites of viral replication and characterize sequential morphological changes in BALB/c mice, adult female mice were infected with mouse-adapted EBO-Z and killed in groups each day for 5 days post-infection. Tissues were examined by light microscopy, immunohistochemistry, electron microscopy and in-situ hybridization. As in guinea-pigs and non-human primates, cells of the mononuclear phagocytic system were the earliest targets of infection. Viral replication was observed by day 2 in macrophages in lymph nodes and spleen. By the time of onset of illness and weight loss (day 3), the infection had spread to hepatocytes and adrenal cortical cells, and to macrophages and fibroblast-like cells in many organs. Severe lymphocytolysis was observed in the spleen, lymph nodes and thymus. There was minimal infection of endothelial cells. All of these changes resembled those observed in EBO-Z-infected guinea-pigs and non-human primates. In contrast to the other animal models, however, there was little fibrin deposition in the late stage of disease. The availability of immunodeficient, "gene-knockout" and transgenic mice will make the mouse model particularly useful for studying the early steps of Ebola pathogenesis., (Copyright Harcourt Publishers Ltd.)

To gain further insight into the pathogenesis of classical swine fever (CSF), the changes induced by hog cholera (HC) virus in pulmonary intravascular macrophages (PIMs) were examined. Twelve pigs were inoculated by the intramuscular route with a virulent strain of HC virus (Quillota strain) and killed in groups of three at 4, 7, 10 and 14 days post-inoculation. Immunohistochemical and ultrastructural examination revealed HC virus infection in endothelial cells, PIMs, and interstitial and alveolar macrophages. In addition to viral replication, a predominant feature was the secretory activation of PIMs, characterized by expanded rough endoplasmic reticulum and hyperplastic Golgi complexes. The results obtained suggest that macrophage activation and the subsequent release of pro-inflammatory mediators play an important role in the pathogenesis of CSF., (Copyright Harcourt Publishers Ltd.)

To study the effect of hog cholera virus on the epithelial cells of the bronchiolar mucosa, 12 pigs were inoculated with a highly virulent strain. Immunohistochemical and ultrastructural examination of the ciliated epithelial cells demonstrated an increase in the number of atypical cilia. The latter showed alterations in the microtubular pattern, possibly resulting from viral interference with the normal metabolism of the epithelial cells.

To investigate whether the recently discovered hepatitis G virus (HGV) influences the clinical outcome of heart transplant recipients under immunosuppression, we determined the prevalence of HGV infections correlated with liver function and survival in 51 patients. Presence of HGV RNA and anti-E2, a marker for resolved HGV infection, were serially tested in sera from patients before and after heart transplantation (HTX) by nested RT-PCR and ELISA. Four of 51 (7.8%) patients before transplantation, and 22 of 50 patients (44%) after transplantation showed signs of persistent or resolved HGV infection. HGV infection was not associated with impairment of liver function or with patient survival. In summary, presence of HGV infection does not influence the clinical outcome in heart transplant patients.

A suite of 3D NMR experiments for measuring 15N-¿1H¿ NOE, 15N T1, and 15N T1rho values in large proteins, uniformly labeled with 15N and 13C, is presented. These experiments are designed for proteins that exhibit extensive spectral overlap in the 2D 1H-15N HSQC spectrum. The pulse sequences are readily applicable to perdeuterated samples, which increases the spectral resolution and signal-to-noise ratio, thereby permitting the characterization of protein dynamics to be extended to larger protein systems. Application of the pulse sequences is demonstrated on a perdeuterated 13C/15N-labeled sample of the 44 kDa ectodomain of SIV gp41.

Japanese encephalitis still occurs in endemic and epidemic forms over a wide area of Asia. Although the vaccine against Japanese encephalitis virus (JEV) is widely used, no antiviral drug has been reported. We used several different kinds of furanonaphthoquinone derivatives and found antiviral activity against JEV. Especially, 2-methylnaphtho[2,3-b]furan-4,9-dione (FNQ3) indicated the highest antiviral activity, followed by 2-(1-hydroxyethyl)-, 5(or 8)-hydroxy-, and 2-methyl-5(or 8)-hydroxy-analogs of naphtho[2,3-b]furan-4,9-dione. In the presence of 3 microg/ml FNQ3, the virus yields in Vero cells were 2 x 10(5) PFU/ml at 24 h after infecting with the virus and 10% of the control level. Western blot analysis using anti-E rabbit sera or anti-NS3 showed that the expression of viral proteins was inhibited by treatment with FNQ3. In addition, Northern blot analysis indicated that the appearance of JEV-RNA was also inhibited by FNQ3. These results suggest that FNQ3 inhibits JEV replication through viral RNA and protein synthesis.

The parallel measurement of serum antibodies to the hepatitis G virus (anti-HGV) and of viremia (HGV-RNA) should improve our understanding of HGV transmission by coagulation factor concentrates. The aim of this study was to assess the relationship between HGV, the type of concentrate infused, and liver disease in multitransfused hemophiliacs. To this end, anti-HGV and HGV-RNA were evaluated by an enzyme-linked immunosorbent assay and a nested-polymerase chain reaction assay in patients treated lifelong with nonvirus-inactivated plasma-derived concentrates (n = 128), virus-inactivated concentrates (n = 33), or recombinant factors (n = 7), and in 200 regular blood donors. The prevalence of serum HGV-RNA and anti-HGV was higher in the recipients of nonvirus-inactivated factors than in blood donors (HGV-RNA: 9% v 1.5%, P = .002; anti-HGV: 32% v 5%, P < .0001). In the recipients of virus-inactivated concentrates the prevalences of these markers were similar to those in blood donors (HGV-RNA: 3% v 1.5%; anti-HGV: 15% v 5%). The prevalence of either marker in the recipients of nonvirus-inactivated concentrates was higher than in the recipients of virus-inactivated factors (39% v 18%, P = .04). The former group had serum hepatitis C virus (HCV) RNA or anti-HCV more frequently than the latter group (HCV-RNA: 86% v 15%, P < .0001; anti-HCV: 96% v 18%, P < .0001). Serum alanine aminotransferase was persistently high in 83 (81%) patients with HCV-RNA alone, in 8 (89%) with HCV/HGV coinfection, and in none of the three patients with HGV-RNA only. Thus, HGV infection in hemophiliacs is more common than previous studies of HGV-RNA prevalence have suggested, but it resolved in most cases and caused chronic viremia only in a small number of patients, without biochemical evidence of persistent liver damage.

A comprehensive index of IV1(Tipula iridescent virus, or TIV)-associated polypeptides has been established using enrichments of "empty" and "filled" virions that are considered to be maturation-phase-related. The mapping strategy which involved one- and two-dimensional polyacrylamide gel electrophoresis, silver staining, disulphide bond reduction and 125I iodination of putative surface proteins revealed 103 and 116 polypeptides for empty and filled capsids, respectively. These estimates could be reduced to < or = 70 by reclassing multicharged polypeptides of approximately identical masses as single entitles. At least 10 polypeptides from empty and filled virions were involved in intermolecular sulphhydryl linkages and another 11 species were identified as putative outer shell (surface) polypeptides. These data provide useful criteria for iridovirus classification and identification of candidate polypeptides involved in capsid formation and maturation.

In this study, we designed a capture ELISA using a monoclonal antibody specific for the N protein, the major protein of the rabies virus nucleocapsid, to measure the N protein content in rabies vaccines. Free N protein content was compared in the two types of rabies vaccine currently used in humans: suckling mouse brain (SMB) vaccine prepared from rabies virus-infected brain tissue, and tissue culture (TC) vaccine prepared from supernatants of rabies virus-infected cells. It was found that SMB vaccines contained considerably higher amounts of N protein than most of the TC vaccines. Possible implications concerning the efficacy of rabies vaccines are discussed.

To obtain a hepatitis C virus (HCV) proliferation system, we examined the susceptibility of various cultured cell lines to HCV infection. We found that a human T-lymphotropic virus type I infected cell line MT-2 was fairly sensitive to HCV infection. Using the polymerase chain reaction, intracellular positive-stranded HCV RNA was detected until at least 15 days postinoculation (p.i.). Intracellular negative-stranded HCV RNA was also detected at 10 days p.i., although not at 7 days p.i., suggesting that HCV is replicating in MT-2 cells 10 days p.i. Sequence analysis of hypervariable region 1 (HVR1) revealed that HVR1 sequences from cells 10 days p.i. had become homogeneous, although HVR1 sequences from the inoculum showed the typical quasi-species. We also found a lack of anti-HVR1 antibody against the HVR1 species which became homogeneous at 10 days p.i., although we easily detected antibody against the other HVR1 species obtained from the inoculum. These findings suggest that MT-2 cells are susceptible to HCV infection and are capable of supporting HCV replication.

By performing immunofluorescence staining assays in confluent COS-7 cells, hepatitis B virus core, small surface and large surface proteins were found to localize largely in the nucleus, diffusely in the cytoplasm, and accumulatively in a perinuclear area, respectively. When two proteins were co-expressed, the core protein was found co-localized with the large but not small surface protein in the cytoplasm. By performing subcellular fractionation experiments, the large surface protein was found to reduce the amount of the core protein in the nuclear fraction to an undetectable level. These results indicate that the large but not small surface protein can alter the subcellular localization of the core protein.

Two groups of synthetic oligopeptides (namino acid = 7 and 17) were prepared by solid-phase peptide synthesis using the Boc-polystyrene strategy. After deprotection, cleavage and gel permeation, the crude products were analysed by conventional RP-HPLC methods. Separation and isolation of major components were performed on a semi-preparative RP-HPLC column. In order to clarify the primary structure of these products, amino acid analysis, Edman degradation sequence determination and analytical RP-HPLC characterization were applied. The isolated fractions were further assessed by direct molar mass investigation utilizing the fast atom bombardment and 252Cf plasma desorption mass spectrometry. The results with an interleukin-6 oligopeptide corresponding to the 179LRALRQM185 sequence indicate that the single peak product obtained by RP-HPLC separation contains only one component, as verified by amino acid analysis and mass spectrometry. In contrast, the analysis of 268LAPEDPEDSALLEDPVG284-NH2 from HSV-1 gD protein suggests that this large peptide amide showing a single peak after repeated purification by RP-HPLC contains microheterogeneities as revealed by mass spectrometry and sequencing, but not by amino acid analysis.

[35S]Methionine-labelled envelope polypeptides of herpes simplex virus type 1, strain F, propagated in mammalian cell culture of various origins, were separated by ion-exchange high-performance liquid chromatography on a TSK DEAE-3SW column. Analysis of the fractions by radioimmunoprecipitation followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis of the immunoprecipitates showed similarities as well as distinct differences in the number, migration patterns and molecular mass of the synthesized polypeptides, depending on the host cell. The results show that this method can be used to demonstrate species-specific or organ-specific differences in the processing of virus-specified polypeptides synthesized in host cells.

A relatively simple method for immobilizing synthetic peptides as a receptor onto a gold surface using the self-assembling monolayer (SAM) technique has been investigated. A synthetic peptide with an amino acid sequence similar to the 9-21 gD sequence of herpes simplex virus type 1 was modified with an alkylthiol chain and adsorbed in combination with an alkylthiol chain without peptide according to the SAM procedures. The resulting self-assembled receptor layers (SARs) were able to specifically interact with a monoclonal antibody directed against the 9-21 gD peptide. Binding studies monitored with a surface plasmon resonance setup showed that the response to the antibody depended on the composition of the SAR; a maximum response was reached at approximately 3 mol% peptide coverage. The response itself exceeded the value obtained from passive adsorption of the antibodies under similar conditions, indicating that the formed antibody layer is highly oriented. The equilibrium binding properties of the immobilized receptor molecules were found to be identical to those found when the immobilized antibodies interacted with the receptor molecule. The presence of a hydrophobic moiety in the alkylthiol derivates contributes to the ordering of the monolayer: a less specific interaction occurred when SARs without hydrophobic moiety were used.

The Epstein-Barr virus (EBV) has been implicated in the pathogenesis of Hodgkin's disease (HD). This study was undertaken to determine whether the association of EBV with HD showed geographical variation, as in Burkitt's lymphoma. We studied 32 formalin-fixed, paraffin-embedded cases of HD occurring in Peru. EBV DNA-RNA in situ hybridization was performed using a 30-base biotinylated antisense oligonucleotide complementary to the EBER1 gene of EBV. EBV immunohistochemistry was also performed, using a monoclonal antibody (MoAb) to the latent membrane protein (LMP1) of EBV. Identification of the precise cellular subset staining with EBV was accomplished via double-labeling with MoAbs directed against Reed-Sternberg cells (LeuM1/CD15) and B cells (L26/CD20). EBV RNA was identified in all or virtually all of the Reed-Sternberg cells and variants in 30 of the 32 (94%) cases of HD by in situ hybridization. LMP1 expression was identified in 83% of the EBER1-positive cases. Double-labeling studies confirmed the localization of EBV RNA to CD15-expressing Hodgkin's cells. This study found an extremely high prevalence of EBV in Peruvian HD, in contrast to the much lower percentage of EBV-associated cases of HD occurring in "Western" patients.

A simple, reliable ELISA for the quantitative detection of the envelope glycoproteins of both HIV and SIV is described. It incorporates the snowdrop lectin GNA to capture the glycoprotein antigens and combines the high selectivity of GNA binding with its broad reactivity with the glycoproteins of HIV-1, HIV-2 and SIV.

An immunodominant HTLV-I-specific epitope in the HTLV-I envelope glycoprotein (GP) 46 has been described. To determine if the analogous region of HTLV-II contains a similarly immunogenic and specific epitope, the polymerase chain reaction (PCR) was used to amplify HTLV-II DNA fragments encoding various portions of the putative epitope. The synthesized DNAs were cloned into lambda-phage gt11 and screened for production of immunoreactive fusion protein using sera from HTLV-II- or HTLV-I-infected individuals. Antisera from HTLV-II-infected individuals identified three of four recombinant clones when tested in a plaque immunoassay. Fusion protein from one of the clones, GH2-K15, was purified and analyzed by Western blot against a panel of HTLV-I and HTLV-II antisera. Twenty-one of 22 HTLV-II-infected sera were reactive with the GH2-K15 epitope. Sera from HTLV-I-infected and HTLV-I-uninfected individuals did not cross-react with GH2-K15. Western blot analysis of recombinant proteins encoding portions of the HTLV-II sequences in the Gh2-K15 antigen localized the HTLV-II-specific epitope to a 17-amino acid sequence. Recombinant antigens containing this epitope should be useful for type-specific serologic diagnosis of HTLV-II infection.

The detection of Epstein-Barr virus (EBV) nucleic acids by in situ hybridization (ISH) with biotinylated BamHI-W probes was correlated with the expressions of EBV latent membrane protein (LMP) and EB nuclear antigen 2 (EBNA2), in 107 cases of Hodgkin's disease (HD) of different immunomorphologic subtypes. Epstein-Barr virus nucleic acids were present and restricted to the pathogenic cells in 4 of 40 (10%) cases of nodular sclerosis (NS) and 33 of 55 (60%) cases of mixed cellularity (MC), but were undetectable in other subtypes. Of the 37 cases positive for EBV nucleic acids, 35 (95%) showed the expression of LMP. Epstein-Barr virus nucleic acids and LMP were restricted to Reed-Sternberg cells and variants. Only 1 case (MC) showed LMP expression in the absence of EBV detection. The correlation was strengthened by the finding of LMP expression at first diagnosis in 6/7 EBV positive cases at relapse (14-126 months) (5/5 EBV negative cases at relapse were LMP negative at first diagnosis). EBNA2 was absent in all 13 (NS, 2; MC, 11) EBV+ and LMP+ cases tested. Both LMP and EBNA2 were expressed in control EBV-positive tissues and cell lines. EBV serology in MC HD was indicative of latent EBV infection, but neither serology nor clinical parameters correlated with the presence or the absence of EBV, over a short-term follow-up (median, 20 months). The findings, although not proving EBV as the etiologic agent of HD, suggest that: 1) LMP expression alone may be adequate for identifying EBV-associated HD, 2) the MC subtype has a stronger relation with EBV presence, and 3) the regulation of EBV genes in HD is different from other EBV-associated disorders. The clinical implications still remain to be discovered.

Serial sera were collected prospectively and from early on in the clinical course of ten patients with fulminant hepatitis B. These were analysed for HBV DNA (dot-blot technique), HBsAg, HBeAg, pre-S2-Ag and their respective antibodies. Two patterns emerged in nine of the patients. The first and well-recognised pattern of rapid clearance of antigens and appearance of antibodies was seen in four patients, all of whom survived. The second pattern seen in five patients was one of persistence of HBsAg and pre-S2 antigen and failure to detect antibodies but only one patient survived. The first pattern may reflect a more rapid cessation of virus replication and this may favour liver cell regeneration and recovery. In contrast, the second pattern may indicate continuing virus replication and liver cell damage which could contribute to the high mortality in some patients with fulminant hepatitis B.

Anti-carbohydrate antibodies were isolated from Human immunodeficiency virus (HIV) negative human serum by affinity chromatography using yeast mannan followed by protein A. The purified mannan-binding IgG (MBIgG) bound to HIV glycoproteins gp 160, gp 120 and gp 41 in Western blot. Immunofluorescence revealed that MBIgG bound to HIV/IIIB-infected H9 cells but not to uninfected H9 cells, suggesting that carbohydrate structures recognized by MBIgG are specifically expressed on HIV-infected cells. MBIgG did not neutralize infectivity of HIV. These results show that normal human serum contains natural antibodies reactive to carbohydrate structures of HIV glycoproteins propagated in human cells.

ELISA methodology was adapted to measure antigen-specific immune complexes (ASIC) containing retroviral antigens. Using ICs artificially generated from MuLV gp70 and monoclonal antibodies against gp70 we showed that ICs can be quantified by measuring the levels of antibody and antigen and comparing these to the total content of ICs. Furthermore, this method permits determination of the composition of ICs containing an excess of antigen or antibody (i.e., small or large complexes). The levels of the ASIC were correlated with the levels of ICs containing undefined antigen components as determined by the C1q ELISA. Using this method it was possible to determine levels of MuLV gp70 specific ICs in various mouse sera.

Sendai virus envelopes have been a useful tool in studying the mechanism of membrane-membrane fusion and have served as a vehicle for introducing foreign molecules (e.g., membrane proteins) into recipient cells. Reconstituted Sendai virus envelopes are routinely obtained following solubilization of virus particles with Triton X-100. This detergent has a low critical micellar concentration which precludes it from being the best detergent of choice in reconstitution studies. Nevertheless, it has remained in use since other detergents such as sodium deoxycholate and sodium cholate rendered the resultant vesicles inactive. Triton X-100 may be suboptimal for studies of some proteins that need be coreconstituted with the viral envelopes. Thus, alternative advantageous detergents, which retain the envelope fusogenic activity, have been sought. In this study we show that the synthetic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) effectively solubilizes the Sendai virions, and that the vesicles formed by simple reconstitution protocols appear structurally and biochemically similar to those obtained with Triton X-100. The resultant vesicles retain functional integrity as assessed in both fusion and hemolysis assays. This protocol seems to be useful in sendai envelope-mediated reimplantation of Fc epsilon receptors into the plasma membranes of rat basophilic leukemia cells.

Approximately half of patients with advanced acquired immune deficiency syndrome (AIDS) develop a subcortical dementia. The brains of all autopsies on AIDS patients performed at UCSD between 1982 and 1986 (N = 93) were studied. Neuropathologic changes consistent with a viral encephalitis were present in 54 brains (58%). Human immunodeficiency virus (HIV) antigens were detected in 37 of the brains (40%), most frequently in macrophages, multinucleated giant cells, and endothelial cells. Cytomegalovirus (CMV) was detected in 31 of the brains (33%), 22 of which also contained HIV. Cellular localization of CMV antigens suggests that CMV disseminates to the central nervous system hematogenously where the virus can infect endothelial cells, glia, and neurons. While the temporal course of the appearance of these two viruses within the CNS is not clear, the common simultaneous occurrence of both viruses within the brains of AIDS patients suggests that in vivo interaction between them may play a role in the pathogenesis of AIDS-associated encephalitis. Given the significant neurologic symptoms described in AIDS patients, the paucity of viral antigens suggests a pathogenic mechanism of indirect CNS damage rather than direct viral infection.

By electron microscopy and immunocytochemistry we have examined the retroviruses endogenous to AtT20 D16V cells, a cloned line of murine pituitary tumour cells. In addition to the C-type retrovirus particles related to Rauscher murine leukemia virus (MuLV) previously reported to bud from these cells we observed cytoplasmic A-type particles and intracisternal A-type particles (IAP). In the cytoplasm the A-type particles occur in large clusters often associated with sheets of material with a fine structure resembling the shells of the particles. At the plasma membrane individual A-type particles bud to give rise to extracellular virions. The IAP are restricted to the rough endoplasmic reticulum (RER) into which they bud: they are not transported out of the RER to the Golgi apparatus and beyond. We describe a new monoclonal antibody (designated 83E7) which is specific for an epitope of the major core protein (MTVp27) of mouse mammary tumour virus (MMTV). Using immunogold labelling procedures we have specifically labelled both the A-type particles and the associated sheets of material with this antibody. We conclude that the A-type particles and the virions they give rise to are MMTV. The sheets of material must also at least in part be made up of the major core protein of MMTV or its precursor polypeptide. AtT20 cells, therefore, contain endogenous MuLV and MMTV as well as IAP.

The role of protein glycosylation in the tumor lysis mediated by effector cells derived from Moloney-sarcoma-virus(MSV)-immune mice was studied. Treatment of the Moloney-virus-induced H-2b lymphoma target cells, MBL-2, with tunicamycin (TM), an inhibitor of the protein-N-linked glycosilation, was found to cause a loss of susceptibility to lysis by MSV-immune syngeneic effectors cells, while the same target cells remained fully sensitive to the lytic action of anti-H-2b-immune lymphocytes. Examination of MBL-2 cell surface by lactoperoxidase, 125I iodination, and immunoprecipitation by antiviral protein sera revealed that env but not gag viral gene-encoded products were expressed on the surface of this lymphoma. The TM-induced alteration of cell surface expression of H-2Db, H-2Kb, and gp70 antigens was examined by a combined approach of serological and biochemical techniques. The results were concordant in indicating that (1) after 16 h of TM treatment the cells showed a decreased expression of the three glycoproteins, (2) H-2Db (the restriction element in this system) resulted more affected by the treatment than its counterpart H-2Kb (75% vs 50% reduction as compared to untreated cells), (3) an additional lighter form of H-2Kb was found on the surface of TM-treated cells. In the context of an "associative recognition' of Db and gp70 by MSV-immune effector cells, our results may explain the loss of susceptibility to antitumor effectors of TM-treated MBL-2 cells by a quantitative reduction in the expression of both molecules which interact to create the target structure of syngeneic effectors.

A computer program was developed for use on an Apple IIe that utilized the parameters developed by Hopp and Woods (T. P. Hopp and K. R. Woods, 1983, Mol. Immunol. 20, 483-489) for predicting the hydrophilic regions of a given protein. This program will produce a listing of the hydrophilic sequence averages and graphically illustrates the peak areas. The hydrophilic averages over a hexapeptide length can be used to predict protein structure. In conjunction with the Chou-Fasman predictive scheme for protein secondary structure determination, the possible antigenic determinants for the envelope glycoprotein of three viruses isolated from patients with acquired immunodeficiency syndrome (AIDS) were predicted. These predicted determinants could be used to generate synthetic peptides that represent a potential vaccine preparation or in developing a diagnostic assay that specifically detects the agent.

High resolution 15N NMR studies of proteins in solution can be performed efficiently by combining the use of isotopically enriched proteins and pulse sequences that generate polarization transfer from protons and result in two-dimensional heteronuclear chemical shift correlation spectra. The coat proteins of the filamentous bacteriophages fd and Pf1 solubilized in detergent micelles give one- and two- dimensional NMR spectra with resolved resonances for nearly all of the nitrogen sites in the proteins. The resonances from the amide sites with slowly exchanging protons can be obtained as a subset of the resonances of all amide sites by comparing the spectra of proteins in D2O and H2O solutions at pH = 4.0.

Glycoprotein B (gB) of Herpes simplex virus type 1 (HSV-1) plays an essential role in viral entry. A set of more than 100 HpaI (GTTAAC) linker insertion mutations and their derivatives were isolated in plasmids specifying the gB coding and flanking sequences. Mutations including addition, deletion and nonsense mutations at 34 independent sites were identified by DNA sequence analysis of 48 plasmids. A map was constructed for the ability of addition mutants to complement a gB-null virus. The expression of gB activity for some plasmids was temperature-dependent. Many complementation-negative plasmids inhibited the complementation activity of a plasmid specifying wild-type gB, suggesting an interaction between active and inactive molecules to form oligomers. The interaction was localized to 328 of the total of 904 amino acids comprising gB. Partial Endo H digestion of nonsense polypeptides revealed that five of the six potential N-linked oligosaccharide sites are glycosylated; the most C-terminal site appears not to be glycosylated. A number of mutations, including some on the cytoplasmic side, were identified that blocked processing, transport and secretion. Addition mutations that blocked processing of membrane polypeptides also blocked processing and secretion when combined into a nonsense mutant that by itself was processed and secreted. The previously predicted membrane spanning domain and the membrane orientation of the N-terminal portion of gB were confirmed.

Thymosin alpha 1 has many effects on immune function and its absence in primary immunodeficiency states produce a clinical presentation similar to the one encountered in acquired immune deficiency syndrome (AIDS). Human immunodeficiency virus (HIV), the etiologic agent of AIDS, binds to T4 helper/inducer lymphocytes through specific surface receptors which include the CD4 glycoprotein. Octapeptide T, a component of the HIV envelope, mediates the binding of HIV to its receptor. In this report, we draw attention to the similarity between the amino acid sequence of thymosin alpha 1 and peptide T and its analogues. This similarity can produce a cross-reactivity between thymosin alpha 1 and HIV and may be a factor in the pathophysiology of the acquired immuno-deficiency syndrome.

Fluorescent labeling of proteins was found to be a very sensitive and reliable alternative to conventional methods for monitoring proteins on Western blots. Proteins were labeled with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) before SDS-PAGE. After electrophoresis and subsequent electro-blotting the fluorescent-labeled proteins were visible upon ultraviolet illumination of the nitrocellulose membranes, and could be photographed to yield an accurate record of the blots before subsequent serological analysis. The sensitivity for detecting MDPF-labeled proteins on nitrocellulose was 100-200 ng, 50 to 100 fold less sensitive than on gels. Fluorescent-labeled TMV and MStpV capsid proteins that were blotted onto nitrocellulose still reacted in serological tests and were detected when present in quantities as low as 100 pg. Fluorescent labeling allows accurate photographic records of the SDS-gel, blot and probed blot using only one sample, and no subsequent staining steps are required.

Envelopment of herpes simplex virus type-1 (HSV-1) was investigated in relation to membrane differentiation in dissociated anterior pituitary cells. The number of cells stained positively with anti-HSV-1 serum was increased from 16 h to 31 h post infection. During this period, electron microscopy revealed that a number of nucleocapsids (unenveloped particles) were accumulated in the Golgi area, where they frequently became surrounded by a double membrane of short Golgi cisternae or by one with a Golgi associated endoplasmic reticulum lysosome (GERL)-like structure. The inner membrane of the cisterna surrounding the nucleocapsids showed regional specialization which was characterized by increased thickness and electron opacity. Acid phosphatase activity, a marker for GERL or trans Golgi cisternae, appeared in the cytoplasmic short cisternae surrounding the nucleocapsids, whereas glucose-6-phosphatase activity, a marker for the nuclear envelope or for endoplasmic reticulum, was not demonstrated in such cisternae. Monoclonal antibody against glycoprotein gD revealed that gD was localized in the trans Golgi membrane as well as in the envelope of the virion. The antibody-binding sites were highly concentrated in the area where Golgi membranes showed increased opacity. Furthermore, nucleocapsids were surrounded exclusively by gD-positive cisternal (Golgi or Golgi-derived) membranes. Thus, our results indicate that the envelope of HSV is derived from trans Golgi cisterna (GERL), and that some viral components, including gD, destined for the envelope may be assembled initially in the Golgi membrane, which is thereby transformed into the envelope of the virus.