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PubMed2003

Virus detection protocols for west nile virus in vertebrate and mosquito specimens.

The recent outbreaks of West Nile virus (WNV) infection in the northeastern United States and other regions of the world have made it essential to develop efficient, sensitive, and rapid protocols for virus surveillance. Laboratory testing is the backbone of any surveillance program. Protocols to detect the presence of WNV have been refined since 1999 for sensitivity, speed, efficiency, and specificity. This paper presents the protocols currently used by the New York State Department of Health to handle vertebrate and mosquito specimens that have been submitted for WNV testing to the Arbovirus Laboratories of the Wadsworth Center.

Algorithms↗
PubMed2006

Diagnostic value of real-time polymerase chain reaction to detect viruses in young children admitted to the paediatric intensive care unit with lower respiratory tract infection.

INTRODUCTION: The aetiology of lower respiratory tract infections in young children admitted to the paediatric intensive care unit (PICU) is often difficult to establish. However, most infections are believed to be caused by respiratory viruses. A diagnostic study was performed to compare conventional viral tests with the recently developed real-time PCR technique. METHOD: Samples from children aged under 5 years presenting to a tertiary PICU suspected of having a lower respiratory tract infection were tested using conventional methods (viral culture and immunofluorescence) and real-time PCR during the winter season from December 2004 to May 2005. Conventional methods were used to check for respiratory syncytial virus, influenzavirus, parainfluenzavirus 1-3, rhinoviruses and adenoviruses. Real-time PCR was used to test for respiratory syncytial virus, influenzavirus, parainfluenzavirus 1-4, rhinoviruses, adenoviruses, human coronaviruses OC43, NL63 and 229E, human metapneumovirus, Mycoplasma pneumoniae and Chlamydia pneumoniae. RESULTS: A total of 23 patients were included, of whom 11 (48%) were positive for a respiratory virus by conventional methods. Real-time PCR confirmed all of these positive results. In addition, real-time PCR identified 22 more viruses in 11 patients, yielding a total of 22 (96%) patients with a positive sample. More than one virus was detected in eight (35%) children. CONCLUSION: Real-time PCR for respiratory viruses was found to be a sensitive and reliable method in PICU patients with lower respiratory tract infection, increasing the diagnostic yield twofold compared to conventional methods.

Child, Preschool↗
PubMed1991

[Veterinary medicine and molecular biology: possibilities and limits of virus detection].

In the last years molecular biology has gained more and more influence in veterinary medicine, e.g. in animal breeding. In addition, methods of molecular biology may open new areas in the diagnosis of viral diseases, because they are fast and very sensitive. The following article displays an overview of molecular biological techniques that are currently in use for virus detection. In addition, the limits of these methods are discussed. In particular, the polymerase chain reaction (PCR) is described in some detail. Since this method has a considerable impact on the progress in molecular biology, it may as well be of importance for the detection of viruses. The potential of PCR in virus detection is illustrated using sheep and goat lentivirus and Bovine Viral Diarrhea/Mucosal Disease virus.

Animals↗
PubMed2002

Simple method for plant virus detection: effect of antibody immobilization technique.

The possibility has been demonstrated for applying a surface plasmon resonance for detecting plant viruses in real samples. An optimal mode for antiviral immunoglobulin immobilization on sensor surfaces is described. Out of three proposed techniques for sensor surface treatment, namely, unmodified gold surface, gold surface treated with (a) thiocyanate and (b) thiocyanate and protein A (Staphylococcus aureus), the latter was chosen as most suited for retention of the formed native immunoglobulin layer.

Antibodies, Viral↗
PubMed1985

Perinatal hepatitis B virus detection by hepatitis B virus-DNA analysis.

Maternal transmission of hepatitis B virus infection in relation to the hepatitis B e antigen/antibody system and serum hepatitis B virus-DNA were evaluated. Results indicate that hepatitis B virus-DNA analysis can identify hepatitis B serum antigen positive mothers who may transmit infection to their offspring.

Autoradiography↗
PubMed1990

Progressive multifocal leukoencephalopathy (PML) in AIDS and in the pre-AIDS era. A neuropathological comparison using immunocytochemistry and in situ DNA hybridization for virus detection.

Twenty-five brains with definite, and three brains with possible, progressive multifocal leukoencephalopathy (PML), including six brains of AIDS patients, were studied with special regard to the detection of papovaviruses. Formalin-fixed serial paraffin sections were immunostained with monospecific anti-JC virus (JCV) and genus-specific anti-simian virus (SV) 40 antisera, and hybridized in situ with DNA probes for JCV and SV 40, respectively. Immunocytochemistry (ICC) and in situ hybridization (ISH) were similarly sensitive in detecting virus in classical PML lesions. In all but one definite PML cases at least one method detected virus (96%). Possible PML tissue was never labeled. Labeling patterns were generally similar in ICC and ISH: mainly oligodendroglia and, less frequently, astroglia harbored virus, whereas labeling of neurons and endothelia was absent. Bizarre giant astrocytes were occasionally labeled by ICC and ISH. Burnt-out lesions harbored JCV DNA but not virus antigens. SV 40 DNA was never detectable. PML morphology in AIDS cases did not usually differ from the disease process seen in the pre-AIDS era. However, two AIDS brains presented extremely extended and, in one case, unusually necrotizing PML damage; in the latter case, PML lesions contained large amounts not only of JCV, but also of human immunodeficiency virus (HIV) antigens. We conclude that ICC and ISH are methods of comparable sensitivity for detection of papovavirus in fluorishing PML lesions. In burnt-out PML lesions only ISH may detect virus. The possibility of an exceptional non-JCV (e.g., SV 40) etiology of PML could be neither confirmed nor disproved. In AIDS, massive coinfection by HIV of PML lesions may increase damage to tissue, resulting in unusually extended and necrotizing PML.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Evaluation of sensitivity of different antigen and DNA-hybridization methods in African swine fever virus detection.

ELISA, immunodot and DNA hybridization methods have been adapted to detect African swine fever virus (ASFV), and their sensitivities were compared using virus obtained from cell cultures. About 2.3 x 10(2) 50% hemadsorbing doses (HAD50) of virus were detected with ELISA sandwich using an anti-ASFV IgG biotinylated followed by avidin-peroxidase. The immunodot technique showed similar sensitivity, detecting about 4.6 x 10(2) HAD50 of virus. ASFV-DNA was detected using radioactive DNA probes and molecular hybridization. The maximal viral detection capacity of this technique was about 1.8 x 10(3) HAD50. The antigenic and DNA detection of ASFV during the infection of animals with virulent and attenuated viruses, was also studied. For this purpose, sera and red blood cells from several infected pigs were obtained at different days post-inoculation. The virus was detected at the third day after infection by the three methods. However, ASFV-DNA detection was more efficient than antigenic detection at nine days post-inoculation, when antigen detection failed, because immunocomplexes with circulating viruses were formed in the subacute infection.

African Swine Fever↗
PubMed2000

Influence of environmental factors on virus detection by RT-PCR and cell culture.

The effects of clay, humic acid, u.v. light and shellfish tissue residues on the detection of poliovirus type 2 from environmental samples by culture and RT-PCR were investigated. RT-PCR showed 10-100 times greater sensitivity for PV2 detection in the absence of sample contaminants than did culture by plaque assay in BGM cell monolayers. Bentonite clay (100-1000 mg l-1) and shellfish tissue residues reduced virus detection by plaque assay, but the effect of bentonite was mitigated by simple elution procedures. Bentonite clay, humic acid (5-150 mg l-1) and mussel tissue reduced virus detection by RT-PCR by between 1 and 8 logs, although this was mitigated in part by elution and Sephadex filtration of extracts. Sephadex filtration of samples reduced culturable PV2 by 32-50%. Exposure of PV2 in water to u.v. light reduced culturability of PV2 but not detection by RT-PCR. This study demonstrates that virus detection in environmental samples is strongly influenced by naturally occurring substances and disinfection approaches. The accuracy of results of viral analyses of this nature should be carefully scrutinized with respect to sample constituents.

Animals↗
PubMed2005

[Quantitative fluorogenic real-time PCR assay for respiratory syncytial virus detection].

OBJECTIVE: To Establish a rapid and objective quantitative fluorogenic real-time PCR assay for early detection of human respiratory syncytial virus (hRSV). METHODS: Two pairs of primers and one TaqMan Fluorogenic probe that are specific for the recognition of the most conservative N gene of hRSV for virus detection with LighCycler PCR in 93 nasopharyngeal secretion specimens collected from infants and young children. The assay was compared with virus isolation, routine PCR, nested PCR, and enzyme-linked immunosorbent assay (ELISA). RESULTS: This TaqMan assay had a sensitivity of 1 x 10(2) cDNA copies/microl with a dynamic range between 1 x 10(2) and 1 x 10(7) cDNA copies/microl, which was the same as that of nested PCR, but 10 times more sensitive than routine PCR. The specificity of the assay was evaluated by comparing hRSV with polivirus type 1, coxsackie virus type 2, influenza A, influenza B and adenovirus type 7. A PCR product of the expected size (195 bp) was produced and fluorescence signal detected for hRSV, but not for any of the other viruses. The results in LightCycler and Rotor-Gene instrument were consistent. Forty-four specimens (43.9%) were hRSV-positive with this assay and 4 (4/93,4.3%) were hRSV-positive with ELISA, showing rather low correlation between the two methods. No visible relation was found between the concentration of hRSV RNA and severity of the disease. CONCLUSION: This assay is rapid, sensitive, specific and quantitative, and has the potential of wide application for early diagnosis of hRSV infection and evaluation of the therapeutic effect.

Child, Preschool↗
PubMed2002

Biosensor for dengue virus detection: sensitive, rapid, and serotype specific.

A serotype-specific RNA biosensor was developed for the rapid detection of Dengue virus (serotypes 1-4) in blood samples. After RNA amplification, the biosensor allows the rapid detection of Dengue virus RNA in only 15 min. In addition, the biosensor is portable, inexpensive, and very easy to use, making it an ideal detection system for point-of-care and field applications. The biosensor is coupled to the isothermal nucleic acid sequence-based amplification (NASBA) technique with which small amounts of virus RNA are amplified using a simple water bath. During the NASBA reaction, a generic sequence is attached to all RNA molecules as described earlier (Wu, S. J.; Lee, E. M.; Putvatana, R.; Shurtliff, R. N.; Porter, K R.; Suharyono, W.; Watt, D. M.; King, C. C.; Murphy, G. S.; Hayes, C. G.; Romano, J. W. J. Clin. Microbiol. 2001, 39, 2794-2798.). It has been shown earlier that Dengue virus can be detected specifically using two DNA probes: a first probe hybridized with the attached generic sequence and, therefore, bound to every amplified RNA molecule; and a second probe either bound to all four Dengue virus serotypes or chosen to be specific for only one serotype. These probes were utilized in the biosensor described in this publication. For a generic Dengue virus biosensor, the second probe is complementary to a conserved region found in all Dengue serotypes. For identification of the individual Dengue virus serotypes, four serotype-specific probes were developed (Wu, S. J.; Lee, E. M.; Putvatana, R.; Shurtiff, R. N.; Porter, K. R.; Suharyono, W.; Watt, D. M.; King, C. C.; Murphy, G. S.; Hayes, C. G.; Romano, J. W. J. Clin. Microbiol. 2001, 39, 2794-2798.). The biosensor is a membrane-based DNA/RNA hybridization system using liposome amplification. The generic DNA probe (reporter probe) is coupled to the outside of dye-encapsulating liposomes. The conserved or Dengue serotype specific probes (capture probes) are immobilized on a polyethersulfone membrane strip. Liposomes are mixed with amplified target sequence and are then applied to the membrane. The mixture is allowed to migrate along the test strip, and the liposome-target sequence complexes are immobilized in the capture zone via hybridization of the capture probe with target sequence. The amount of liposomes present in the immobilized complex is directly proportional to the amount of target sequence present in the sample and can be quantified using a portable reflectometer. The different biosensor components have been optimized with respect to sensitivity and, foremost, specificity toward the different serotypes. An excellent correlation to a laboratory-based detection system was demonstrated. Finally, the assay was tested using a limited number of clinical human serum samples. Although Dengue serotypes 1, 2 and 4 were identified correctly, serotype 3 displayed low cross-reactivity with biosensors designed for detection of serotypes 1 and 4.

Animals↗
PubMed2004

National disease burden of respiratory viruses detected in children by polymerase chain reaction.

BACKGROUND: The epidemiology of community-acquired respiratory viruses at the Children's Hospital of Wisconsin between 1996 and 1998 was examined with molecular [multiplex (M) PCR] and standard virologic methods. METHODS AND RESULTS: A total of 3325 patients with lower respiratory infection (LRI) [bronchiolitis (42%), pneumonia (38%) and croup (12%)] were identified. It is estimated that 545,000 LRI hospitalizations occur each year in the United States in children younger than 18 years old (viral, 428,000; pneumonia, 221,000; bronchiolitis, 222,000; croup, 65,000), including a continued increase in bronchiolitis hospitalizations (47.8/1000; <1 year). The most common viruses detected each year in hospitalized children included respiratory syncytial virus (A and B; 117,000), parainfluenza virus (PIV-1 and -2, 48 000; PIV-3, 18,000) and influenza (A and B, 39,000). With multiplex reverse transcription polymerase chain reaction (MPCR), different patient populations demonstrated different LRI epidemiology in relation to specific syndromes and viral causes. At least 21% of LRI hospitalizations were in children with significant medical problems.PIVs were detected much more frequently in immunocompromised children than in previously healthy children (33% vs. 16%). Similarly 15% of respiratory syncytial virus was detected between May and October, mostly in immunocompromised children. Influenza caused 19% of bronchiolitis in previously healthy children, and no virus could be reliably predicted based on clinical presentation, including wheezing. Of children hospitalized for LRI, 40% tested positive for the seven most common respiratory viruses by MPCR. CONCLUSIONS: Work is under way to expand the number of viruses detected by MPCR and to determine whether newly discovered viruses, such as human metapneumovirus, contribute to the burden of pediatric LRI hospitalizations.

Adolescent↗
PubMedDate not supplied

Comparative West Nile virus detection in organs of naturally infected American Crows (Corvus brachyrhynchos).

Widespread deaths of American Crows (Corvus brachyrhynchos)were associated with the 1999 outbreak of West Nile (WN) virus in the New York City region. We compared six organs from 20 crow carcasses as targets for WN virus detection. Half the carcasses had at least one positive test result for WN virus infection. The brain was the most sensitive test organ; it was the only positive organ for three of the positive crows. The sensitivity of crow organs as targets for WN virus detection makes crow death useful for WN virus surveillance.

Bird Diseases↗
PubMed1987

A rapid method for the detection of papillomavirus in warts: the frequency of virus detection in various types of warts.

A rapid method was devised for the detection of virus particles in wart specimens. The upper layer of warts was cut perpendicularly to the surface, and the freshly cut surface was lightly touched to an electron microscope grid. The grid was then stained with a small drop of phosphotungstate and observed electron microscopically. On the specimen grid thus prepared, papillomavirus particles were easily discriminated from tissue debris. Papillomavirus particles were detected in 71% of verrucae plantares, 78% of verrucae palmares, 50% of verrucae vulgares and 75% of condylomata acuminata by the present method.

Condylomata Acuminata↗
PubMed2005

West Nile virus detection in the organs of naturally infected blue jays (Cyanocitta cristata).

Blue jays (Cyanocitta cristata) are an effective indicator species for West Nile virus (WNV) and may be regionally important in surveillance efforts. The sites of WNV replication and sensitivity of virus detection techniques are undefined for blue jays. The objectives of this study were to describe the gross and microscopic pathology associated with natural WNV infection in blue jays, as well as determine the most appropriate tissues to be used for virus isolation, reverse transcription-nested polymerase chain reaction, and immunohistochemistry (IHC) techniques. Blue jays were collected in Georgia, USA, between May and September 2001. Initial screening by virus isolation indicated that 36 of 59 blue jays chosen for evaluation were WNV positive. From this group, 20 positive and five negative birds were chosen to compare virus detection techniques. Six positive and five negative birds were selected for histopathology examination. Splenomegaly and poor body condition were the most consistent gross findings among positive birds. The most consistent histopathologic findings in the tissues of WNV-positive blue jays were mononuclear leukocytosis and epicarditis/myocarditis. Brain, heart, and lung had the highest viral titers, and WNV antigen was most often detected by IHC in heart, kidney, liver, and lung. Reverse transcription-nested polymerase chain reaction proved to be the most sensitive diagnostic test applied in this study irrespective of the tissue type. Brain tissue could be used effectively for both virus isolation and RT-nPCR, and this tissue is simple to remove and process. The success of IHC is highly dependent on tissue selection, and the use of multiple tissues including heart, kidney, liver, or lung is recommended.

Animals↗
PubMed2004

DNA microarrays for virus detection in cases of central nervous system infection.

A low-density, high-resolution diagnostic DNA microarray comprising 38 gene targets for 13 viral causes of meningitis and encephalitis was constructed. The array has been used for the detection of multiplex PCR-amplified viruses in cerebrospinal fluid (CSF) and non-CSF specimens. A total of 41 clinical specimens were positive for echoviruses (23 samples), herpes simplex virus type 2 (4 samples), varicella-zoster virus (4 samples), human herpesvirus 7 (1 sample), human herpesvirus 6A (1 sample) and 6B (2 samples), Epstein-Barr virus (three samples), polyomavirus JC (1 sample), and cytomegalovirus (2 samples). Probes for herpes simplex virus type 1, polyomavirus BK, and mumps and measles viruses were also included on the array. Three samples were false negative by the microarray assay due to discordant results between the multiplex PCR for all 13 viruses simultaneously and the virus-specific PCR alone. Fifteen CSF specimens were true negative. The clinical sensitivity, specificity, and negative and positive predictive values of the assay were 93, 100, 100, and 83%, respectively, when the results were compared to those of the single-virus PCR, which was used as the "gold standard." The microarray-based virus detection assay is qualitative and provides a single-format diagnostic tool for the detection of panviral CNS infections.

Central Nervous System Viral Diseases↗
PubMed1986

Multisite monoclonal immunoassay for dengue viruses: detection of viraemic human sera and interference by heterologous antibody.

A monoclonal radioimmunoassay (RIA) was developed for detection of dengue virus in infected cell culture fluids and blood samples from dengue patients. Antibodies used to construct the RIA were selected on the basis of high binding avidity, the demonstration of synergism in competitive binding assays and empirical trials with different antibody combinations. Optimal binding of all four dengue virus serotypes was achieved by use of a flavivirus group-reactive and a dengue virus complex-reactive antibody as radiolabelled probe. A 'simultaneous sandwich' format and prolonged (18 h) incubation at 37 degrees C yielded optimal results. The limit of sensitivity of the RIA for detection of dengue type 2 virus was 2.7 log10 mosquito 50% infectious doses (MID50). The assay was tenfold more sensitive for dengue type 2 than for dengue types 1 and 3 viruses and 100-fold more sensitive than for dengue type 4 virus. Specificity, assessed using over 500 disease control human sera, was increased by addition of monoclonal anti-tetanus blocking antibodies, resulting in a false positive rate of only 0.2%. Heterologous dengue virus antibodies were shown to inhibit the RIA in assays performed with artificial immune complexes. Acute phase human sera containing 10(4.2) to 10(7.6) MID50 but no detectable antigen by RIA, were also shown to inhibit binding of the homologous dengue virus serotype; this effect was attributed to heterologous antibody from a prior infection. Among 116 viraemic sera from dengue patients, the RIA was positive in 43 to 47% of patients with dengue type 1, 2 or 3 infections but in only 10% of the dengue type 4 cases. Virus was more frequently detected in cases of primary infection (54%) than in cases of superinfection (16%). Despite the limitations imposed by immunological interference, the antigen capture RIA appears useful as a rapid diagnostic technique for dengue surveillance.

Aedes↗
PubMed1994

A PCR-microplate hybridization method for plant virus detection.

A sensitive nonradioactive method for detection of plant viruses was evaluated. A cDNA fragment from the coat protein coding region of the potato virus Y (PVY) RNA genome amplified by reverse transcription (RT) followed by polymerase chain reaction (PCR), was directly adsorbed onto polystyrene microplate wells after heat denaturation. The adsorbed cDNA was hybridized with a digoxigenin (DIG)-labelled cDNA probe without a prehybridization step. The probe was also prepared by PCR using the same pair of primers to amplify the target sequence from a cDNA clone of part of the viral genome. The hybrid of the adsorbed cDNA and DIG-labelled probe was reacted with alkaline phosphatase-conjugated anti-DIG antibody. The enzyme activity was then detected by hydrolysis of a substrate, and the absorbance values were measured using a microplate reader. Highest absorbance values were obtained when the amplified DNA fragments were diluted 100 or 125-fold in 10 x SSC (1.5 M NaCl, 0.15 M sodium citrate) for adsorption. Highly concentrated DNA gave lower absorbance values. The absorbance values differed depending on the microplates used, and the highest value was obtained using Nunc Immunoplate II-Maxisorp microplates. DNA fragments longer than 300 bp all gave similar absorbance values, which were twice as high as those obtained with shorter fragments. When the amplified DNA was diluted 100-fold, 10 fg of PVY genomic RNA could be detected by this method, which is called PCR-microplate hybridization. This is about 10,000 times more sensitive than enzyme-linked immunosorbent assay (ELISA). By this method, PVY was detected from five field potato samples showing to be free from PVY by ELISA. PCR-microplate hybridization is nucleic acid-based ELISA-like highly sensitive diagnostic method, and may be generally applicable for detection of plant viruses, viroids, and possibly other plant pathogens.

Adsorption↗