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PubMed1999

[Lanthanide immunofluorescent analysis: virus detection and the serodiagnosis of viral infections].

This communication summarizes 10-year experience gained by the author in developing and using the lanthanide immunofluorescence assay (LIFA) for the laboratory diagnosis of viral infections. The bulk of studies has been conducted on natural focal viruses, including Venezuelan equine encephalitis, tick-borne encephalitis, Crimean Congo hemorrhagic fever, California serogroup, and other viruses. Moreover, test systems have been developed for diagnosis of infections caused by herpes simplex and cytomegaloviruses. The studies performed have demonstrated the sensitivity of LIFA in the indication of viruses in the laboratory materials and the samples from natural foci is 10-100 times higher than that of enzyme immunoassay and it is close to that of the biological isolation assay; the specificity of LIFA is comparable to that of the neutralization reaction, but it is more accessible in practice due to the fact it does not require the use of living viruses and biological models. The results of detection of herpes viruses in the clinical samples by LIFA are shown to rather well correlate with the data of virus isolation in the cultured cells, with other diagnostic methods and with the clinical manifestations of diseases. LIFA is recommended for use in large-scale studies involving the monitoring of infection foci and the screening of risk population groups for social infectious diseases.

Chelating Agents↗
PubMed2001

Comparison of systems performance for TT virus detection using PCR primer sets located in non-coding and coding regions of the viral genome.

BACKGROUND: the heterogeneity of the TT virus (TTV) DNA prevalence values reported from comparable human cohorts suggests that diagnostic PCR protocols still require to be optimized. OBJECTIVES: to design TTV PCR primer sets with low genotype restriction and to compare their performances with commonly used amplification systems. STUDY DESIGN: we compared full length TTV genomic sequences and identified conserved nucleotide patterns in the 5' and 3' non-coding regions of the viral genome. This permitted to design two new primer sets usable for the PCR amplification of the most divergent human isolates of TTV described to date. The performances of these amplification systems were compared with those of three other PCR systems earlier used for prevalence studies. RESULTS: the primer systems P5Bx and P3Bx exhibited higher PCR scores than the other systems tested; 14 to 34% improvement values were obtained, and divergent positive results of earlier described PCR systems were confirmed systematically by our new detection assays. CONCLUSIONS: an optimized detection of TT virus DNA is a pre-requisite for the accurate epidemiological survey of viral infection and for the realization of phylogenetic studies. Such PCR systems with low genotype restriction will be helpful in the future for a better knowledge of natural history of TT virus infection.

3' Untranslated Regions↗
PubMed1997

Bluetongue virus detection: a safer reverse-transcriptase polymerase chain reaction for prediction of viremia in sheep.

A reversible target capture viral RNA extraction procedure was combined with a reverse-transcriptase nested polymerase chain reaction (PCR) to develop a capture PCR assay providing a rapid and safe prediction method for circulating bluetongue virus in infected ruminants. This new assay was compared with virus isolation and a recently developed antigen-capture enzyme-linked immunosorbent assay (ELISA) for the detection of bluetongue virus. Eight Warhill crossbred sheep were inoculated subcutaneously with bluetongue virus serotype 10, and blood samples were taken sequentially over a period of 28 days. The capture PCR detected the peak of viremia, as determined by virus isolation and antigen-capture ELISA, from day 5 to day 14 after challenge. The results indicate that the rapid-capture bluetongue virus PCR provides a rapid indicator of samples in which virus can be isolated. In addition, this capture bluetongue virus PCR procedure does not require a lengthy phenol extraction or the use of the highly toxic methyl mercury hydroxide denaturant.

Animals↗
PubMed2003

Expression of VP2 gene protein of infectious bursal disease virus detected in Korea.

The VP2 gene DNA (1.4 kb in approximate) of a very virulent infectious bursal disease virus (vvIBDV) Chinju strain detected in Chinju, Korea was cloned into the bacmid, a baculovirus shuttle vector, through transposition of the gene from initially cloned pFastBacHTa plasmid, a baculovirus expression vector, and was subsequently expressed in Spodoptera frugiperda (Sf) cells. Biological properties of the expressed VP2 subunit protein were characterized to aid in the development of genetically engineered diagnostic reagents and vaccines against the vvIVDV. When the VP2 DNA-recombinant bacmid was transfected and propagated in the Sf cells, the cells showed no occlusion formation, which is a positive evidence for the insertion of the VP2 DNA into the polyhedrin gene of the bacmid, whereas the occlusions were observed in the cells infected by the Autographa californica nuclear polyhedrosis virus, a wild baculovirus. The expression of VP2 DNA was identified by strong positive reaction in fluorescent antibody test using chicken anti-IBDV serum. The VP2 protein was determined as a polypeptide band with Mr of 48 kDa by the sodium dodecyl-polyacrylamide gel electrophoresis for the lysate of the Sf cells infected with the recombinant bacmid. The VP2 protein was successfully purified from the cell lysate by Ni-NTA affinity chromatography. The expressed VP2 subunit protein reacted specifically with chicken anti-IBDV serum in Western blotting.

Animals↗
PubMed1995

Characterization of a CD4-expressing macaque cell line that can detect virus after a single replication cycle and can be infected by diverse simian immunodeficiency virus isolates.

Primate lentiviruses such as human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) are phenotypically diverse, and virus isolates vary in cytopathicity, replication rate, and cell tropism. While all virus isolates infect primary peripheral blood lymphocytes, only a subset of strains infect established CD4-expressing T-cell lines. Here, we describe the development and characterization of a macaque cell line that can be infected by all of the strains of SIV that we have tested, including macrophage- and T-cell-tropic strains, primary and cell-line adapted strains, and SIVmac, SIVMne, and SIVsm isolates. The cells can be infected by strains of HIV type 2 (HIV-2) to varying degrees, but not by either cloned or primary isolates of HIV type 1 (HIV-1). This cell line is a derivative of a rhesus macaque mammary tumor cell line (CMMT) engineered to express human CD4. For these studies, a CMMT-CD4 clone expressing an integrated copy of a truncated HIV-1 long terminal repeat fused to the beta-galactosidase gene (LTR-beta-gal) was established to allow detection of infectious SIV after a single round of replication. Here, we demonstrate the ability of the CMMT-CD4-LTR-beta-gal cell line to rapidly and quantitatively detect infectious SIV. Using these cells to assay virus, we could readily measure neutralizing antibody activity in animals infected with different SIV isolates. Neutralizing activity was detected against the homologous virus and lower, but detectable, activity was measured against heterologous virus. Thus, this system, which is highly sensitive and can detect infection by all of the SIV isolates we tested, is a rapid method for detecting infectious virus and quantitating neutralizing antibody activity.

Animals↗
PubMed1991

JC virus detection by in situ hybridization in brain tissue from elderly patients.

Brains from 10 patients aged 68 to 96 years at time of death were studied for JC viral DNA and common papovaviral capsid protein. In situ hybridization of JC viral DNA was performed by affinity cytochemistry using a biotinylated fragment of JC viral DNA. Immunohistochemistry was performed on brain tissue by the avidin DH-biotinylated horseradish peroxidase technique using polyclonal antibody raised against the papovaviral capsid protein. Viral protein and DNA were detected in 4 of 10 patients. JC virus may be present in the brains of aged patients more frequently than previously suspected.

Adult↗
PubMed1977

Virus diseases of berry crops in the Soviet Far East I. Identification of some mechanically transmitted viruses, detected in Primorye Territory.

In Primorye Territory, USSR, cucumber mosaic virus (CMV-I), arabis mosaic virus (AMV), raspberry, ringspot virus (RRSV), and tomato ringspot virus (TRSV) were identified on berry crops (currant, raspberry, honeysuckel). With respect to indicator plants and physico-chemical and serological properties, the isolates obtained do not differ from other isolates of these viruses, reported on berry crops in Europe and North America.

Fruit↗
PubMed1995

Partial nucleotide sequencing of six subtype 2c hepatitis C viruses detected in Italy.

The great majority of 121 hepatitis C virus (HCV) isolates obtained from 117 Italian patients with community-acquired infection could readily be typed by genotype-specific PCR. Subtype 1b was dominant (74 isolates); subtypes 2b, 2a, and 1a followed, with 19, 14, and 8 isolates, respectively. The six isolates that remained untyped by this method were classified as subtype 2c on the basis of sequence analysis of PCR amplicors obtained from the core and NS5 genes. These findings indicate that HCV subtype 2c has a relatively high prevalence in Italy. Sequencing the core region from positions 160 to 259 is sufficient to distinguish subtype 2c from other known HCV genotypes.

Adolescent↗
PubMed1979

Reticuloendotheliosis virus: detection of immunological relationship to mammalian type C retroviruses.

Reticuloendotheliosis virus (REV) p30 shares cross-reactive determinants and a common NH2-terminal tripeptide with mammalian type C viral p30's. An interspecies competition radioimmunoassay was developed, using iodinated REV p30 and a broadly reactive antiserum to mammalian virus p30's. The avian leukosis-sarcoma viruses and mammalian non-type C retroviruses did not compete in this assay. Previous data indicating that the REV group is not represented completely in normal avian cell DNA lead us to speculate that this may be the first example of interclass transmission, albeit in the remote past, among the Retroviridae.

Animals↗
PubMed2003

Epstein-Barr virus detected in a head and neck squamous cell carcinoma cell line derived from an immunocompromised patient.

BACKGROUND: DNA tumor viruses potentially play a role in the development of squamous cell carcinoma of the head and neck (SCCHN). Human papillomavirus is found in up to 50% of SCCHN specimens, and Epstein-Barr virus (EBV) has been detected in nasopharyngeal carcinoma, Burkitt lymphoma, and other lymphomas. However, the role of EBV in nonnasopharyngeal SCCHN has not been thoroughly investigated. METHODS: Twenty-one SCCHN cell lines derived from tumors of various subsites were used to screen for EBV DNA as well as latent viral protein expression. The method of EBV DNA detection was polymerase chain reaction with 3 independent primer sets from distinct regions of the genome. Expression of the viral protein EBNA-1, critical for the maintenance of the viral episome, was monitored by immunofluorescence using an antibody specific for EBNA-1. In addition, 12 paraffin-embedded tumor specimens and adjacent normal tissues were analyzed by polymerase chain reaction. These tumor specimens were further characterized by immunohistochemistry with the use of a mouse monoclonal antibody that recognizes EBNA-1. RESULTS: Little or no EBV DNA was detected in 20 of 21 cell lines or in any of the tumor specimens, while detecting approximately 40 genome copies in a control reaction. Accordingly, these cell lines and specimens were negative for EBNA-1 expression. In 1 cell line derived from an immunosuppressed patient, EBV DNA was detected, and on further examination a small percentage of cells expressed EBNA-1 as shown by immunofluorescence. CONCLUSIONS: Although EBV may not be a major cofactor contributing to the proliferation of SCCHN, the limited initial evidence suggests that EBV may be involved in development of SCCHN in immunosuppressed patients.

Carcinoma, Squamous Cell↗
PubMed2001

TT virus detection in oral lichen planus lesions.

Epidemiological studies have demonstrated a correlation between oral lichen planus and different liver diseases. The new virus termed TT virus (TTV) is highly prevalent in patients with chronic hepatitis of different etiology and it may be speculated that TT virus may be involved in the pathogenesis of oral lichen planus. This study examined the presence of TT virus DNA in serum by PCR and in oral mucosa biopsies by in situ hybridization from 20 patients with oral lichen planus (13 with chronic hepatitis and seven without liver disease). Serum and oral mucosa biopsies from six patients all with chronic hepatitis with leukoplakia were also studied as controls. TT virus DNA was positive in the serum of 17/20 (85%) of the patients with oral lichen planus and in all the controls. TT virus DNA hybridization signals were detected in mucosa biopsies from all the patients with TT virus DNA in serum but in none of the three cases without this marker. The percentage of positive cells ranged from 1.6-80%. No differences were found in the percentage of positive cells between TT virus positive patients with and without oral lichen planus and there was no relationship between the number of positive cells and the intensity of the inflammatory infiltrate. In conclusion, TT virus infects oral epithelial cells but the results do not support a role for TT virus in causing oral lichen planus.

Aged↗
PubMed2003

Hepatitis C virus detection in follicular fluid and culture media from HCV+ women, and viral risk during IVF procedures.

BACKGROUND: Hepatitis C virus (HCV) has been detected in sperm, but no data are available on follicular fluid (FF) collected on IVF procedures. The aim of this study was to: (i) search for HCV RNA in FF and in culture media at each step of IVF undergone by HCV(+) women; (ii) investigate the impact of blood contamination of FF induced by vascular injury associated with transvaginal ovarian puncture; (iii) assess risk for the embryo and the impact on the contamination rate of the newborn; and (iv) determine the viral risk presented by these fluids in order to define guidelines for the laboratory. METHODS: FF from 22 IVF procedures performed in 17 HCV(+) women were classified as either clear, lightly bloody or bloody FF. Oocytes from each FF were washed and incubated in separated fertilization media. At 20 h after puncture (day 1), the fertilized oocytes were washed and transferred to fresh media until embryo transfer. HCV RNA was detected and quantified in FF and media using Cobas Amplicor and Cobas Monitor HCV RNA kits. RESULTS: HCV RNA was positive in 39 of 44 FF samples, and viral loads increased with blood contamination. At day 1, after rinsing of oocyte-cumulus complexes, only 8 of 44 media were still positive. Viral loads were quantified in 5 of 8 positive media, were below the test sensitivity threshold in 4 of 5 HCV RNA-positive media, and just above it in the fifth medium. The day of transfer HCV RNA was undetectable in all media. CONCLUSIONS: HCV RNA was detected in 89% of FF irrespective of the degree of blood contamination, and in 25% of the media at day 1. FF must be considered as potentially infected. Blood contamination increases HCV load in the FF. Rinsing oocytes seems significantly to discard the HCV RNA. It is too early to assess the impact of IVF on the contamination rate of HCV mothers' offspring. After counselling, attempting IVF in HCV(+) women is justified. Universal guidelines prevent nosocomial infection, and IVF does not specifically increase the professional risk.

Adult↗
PubMed1992

Vertical transmission of HIV-1: lack of detectable virus in peripheral blood cells of infected children at birth.

OBJECTIVE: To evaluate the time-course of HIV-1 detection in peripheral blood mononuclear cells (PBMC) from newborns at risk of vertically acquired infection. DESIGN AND METHOD: Forty-six infants born to HIV-1-infected mothers were enrolled at birth and examined virologically and clinically in the perinatal period and every 30 days for the first 3 months of life. Follow-up was conducted at intervals of 2-3 months. HIV-1 detection in PBMC was performed using virus culture and polymerase chain reaction (PCR) assay. RESULTS: Only one out of 24 newborns tested within 48 h of delivery and two out of 22 infants tested between 3 and 15 days of age were found to be HIV-1-positive by both PCR and virus culture. Further testing performed between 30 and 60 days of life identified an additional eight HIV-1-positive children. Subsequent viral, immunological and clinical follow-up confirmed PCR and virus culture results obtained in 30-60-day-old children. CONCLUSIONS: Infected infants had detectable levels of HIV-1 in their PBMC at 1 month of age. The negative PCR and virus culture findings in PBMC of newborns indicate strongly that HIV-1 cannot be diagnosed at birth in the majority of cases, and suggests that viral transmission could occur during late pregnancy and/or delivery.

Antibodies, Viral↗
PubMed1986

Diagnosis of bovine respiratory syncytial virus infections improved by virus detection in lung lavage samples.

The potential of lung lavage to diagnose and study infections with bovine respiratory syncytial virus (BRSV) was investigated. The technique was simple to perform under field conditions, and even in calves with respiratory tract disease, serious drawbacks were not observed. In 9 epizootics of respiratory tract disease, BRSV infection was diagnosed in 1 or more calves. The BRSV was detected from a total of 21 of 32 calves by immunofluorescence in cells recovered from lavage fluids, and the virus was recovered by cell culture from 17 of these. In the 4 remaining calves, infectious bovine rhinotracheitis virus or parainfluenza type 3 virus was isolated. In calves with high levels of maternally derived antibodies, direct demonstration of the virus was found to be essential for making a diagnosis of BRSV. In 3 of the 9 epizootics, the majority of 4- to 8-month-old calves had a significant increase in antibody concentration against BRSV, whereas only a few 1- to 2-month-old calves present during the other 6 epizootics did so. However, at least in 4 of the 6 herds with younger calves, direct detection of the virus in 2 or more calves indicated that BRSV was involved in the epizootic. Cytologic examination of wash fluids from BRSV-infected animals identified an increased neutrophil/large mononuclear cell ratio. Occasionally, bronchiolar casts and syncytial giant cells with inclusion bodies were seen. In lung lavage fluids of 4 experimentally infected specific-pathogen-free calves, BRSV-containing cells were detected by immunofluorescence for 12 to 18 days after inoculation.

Animals↗
PubMed2026

Molecular characteristics, phylodynamics, and evolutionary changes of avian infectious bronchitis virus detected from chickens in Yunnan Province, 2021-2024.

Avian infectious bronchitis virus (IBV) is endemic in poultry flocks worldwide, posing a significant threat to the global poultry industry. Frequent mixing of free-range local chickens with introduced chickens in Yunnan Province, China, facilitates the transmission, recombination, and mutation of avian IBV, thereby complicating disease prevention and control. In this study, we aimed to investigate the presence of IBV in poultry populations in Yunnan Province. Samples were collected from live poultry markets (LPMs) and breeding farms, comprising 725 randomly sampled cloacal/fecal swabs and 55 tissue samples. IBV-positive samples were confirmed via polymerase chain reaction (PCR), with an overall positivity rate of 0.89% (7/780) for all tested samples. The positivity rate was 0.35% (2/564) in Kunming, 3.7% (2/54) in Zhaotong, 20% (1/5) in Yuxi, and 12.5% (2/16) in Baoshan, while no IBV was detected in samples from Lanping, Xichou, or Ninglang. Six IBV strains, including five GI-19 strains and one GVI-1 strain, were successfully isolated. Phylogenetic analysis further showed that the Yunnan GI-19 strains predominantly clustered with strains originating from Sichuan Province. Sequencing of the S1 gene revealed several amino acids substitutions per isolate in hypervariable regions HVR1-HVR3. Notably, a valine (V) and glycine (G) insertion between amino acid positions 88 and 89 was identified exclusively in isolate F210, a feature rarely reported in IBV. Protein-protein docking analysis indicated that the unique 88-89 insertion in isolate F210 S1 may alter its binding interactions with the host receptor ANPEP. Whole-genome comparison revealed that isolate YX3 shared 97.05% nucleotide identity with strain CK/CH/GX/YL17/2017 from Guangxi, whereas isolates Q47, F13, and F210 shared 96.40%-97.27% identity with strain CK/Henan/H1036/2021 from Henan. Recombination analysis detected obvious recombination events in isolates F13, F210, Q47, and YX3, with GI-22 strains serving as the major parental donors. These genetic characteristics, recombination patterns, and structural insights demonstrate the complex evolutionary dynamics of circulating IBV strains in Yunnan. Continuous molecular epidemiological surveillance combined with functional protein analysis is essential to monitor emerging variants and formulating targeted, effective disease control strategies.

Avian infectious bronchitis virus↗
PubMed1986

Herpes simplex virus detection by ELISA: effect of enzyme amplification, nature of lesion sampled and specimen treatment.

The relative sensitivity of two enzyme detection procedures was investigated in a simultaneous "monoclonal" ELISA for herpes simplex virus (HSV). A cyclical enzyme amplified detection system with alkaline phosphatase, rather than horse-radish peroxidase and a conventional chromogenic substrate, gave an increase in absolute sensitivity and a 20 to 30% increase in the detection of HSV in routine isolation-positive genital specimens collected in transport medium. The HSV detection rate, with both procedures, was shown to vary with the site and clinical stage of lesion sampled; it was highest with penile vesicular lesions. Direct extraction of the swab specimen in a small volume of diluent further increased the sensitivity of antigen detection giving positive and negative predictive values of 100 and 96% respectively. The overall sensitivity of HSV detection was equivalent to that obtained by isolation in cell culture. The amplified ELISA offers an alternative, rapid, simple, non-culture technique for routine HSV diagnosis that does not rely upon retention of virus viability.

Alkaline Phosphatase↗
PubMed2000

[Production of monoclonal antibodies to broad bean wilt virus and application in virus detection].

Six hybridoma cell lines secretion monoclonal antibodies(MAbs) against broad bean wilt virus(BBWV) were produced by fusing mouse myeloma cells(SP 2/0) with spleen cells from BAL B/c immunized by the BBWV particles. The hybridoma cell lines secreted MAbs stably after cultured in vitro for 3 months or stored in liquid nitrogen and then revived for several times. The titres of ascitic fluids of six MAbs ranged from 1:256,000 to 1:640,000 when measured by indirect ELISA. In agarose gel immunodiffusion test, it showed that the six MAbs represented the same isotype of murine antibodies, IgG1. Six MAbs could detect 4 tested BBWV isolates, but didn't crossreact with other 5 plant viruses. The result of Western blot showed that all the six MAbs can react with the 44.7 kD large coat protein subunit of BBWV. This is the first report of production of MAbs against BBWV.

Animals↗
PubMed1998

Comparison of different methods of RNA isolation for plum pox virus detection by reverse transcription-polymerase chain reaction.

The diagnosis of plum pox virus (PPV) is still considered one of the most important aspects of the "sharka" problem. In fact, different studies demonstrated an uneven distribution of the virus in infected trees due to a high variability in virus concentration. These aspects complicate the PPV diagnosis. To date, biological, serological and molecular assays have been successively developed in order to obtain sensitive and efficient PPV detection techniques. In particular, the polymerase chain reaction (PCR) technique seems to be promising and can be considered the most sensitive and reliable one. Preparation of viral RNA is still a fundamental step in reverse transcription-PCR (RT-PCR) technique, especially when applied to large scale testing, i.e., for certification purposes. In order to find the most rapid and efficient procedure, we have compared three different procedures of extraction of viral RNA to be processed RT-PCR. Their common characteristics is their capacity to extract the RNA from a small amount of plant tissue without organic solvents in the extraction fluid. The procedures were as follows: an immuno-capture (IC) method using a specific antiserum, a silica-capture (SC) method using a non-specific matrix, and a simple and rapid RNA extraction (RE) method. They all were followed by one-tube RT-PCR. The obtained results show that all the three techniques allowed a successful amplification and detection of PPV in tested samples except the SC-PCR method which proved less effective. In fact, the IC-PCR and RE-PCR methods amplified and detected PPV in all isolates tested, while the SC-PCR method was able to reveal the presence of the virus in apricot and infected control samples only.

Fruit↗