Volume 2, Number 2, 1997
Microbiology & Pediatric Departments, Peripheral General Hospital "Asklipios", Voula, Athens
Hellenic Virology, 1997, 2(2): 81-86.
Respiratory viral infections by applying immunofluorescence (RIF) assay to samples of nasopharyngeal secretions have been evaluated, using monoclonal antibodies, in 122 children with severe pulmonary infection. During one year period, the specimens were examined for respiratory viruses by RIF, while 81 children 5 years old were examined for Respiratory Syncytial Virus (RSV) by RIF and EIA. RIF was confirmed by serological examination by CF and IFA. A positive diagnosis was detected in 46 children (37.6%). RSV in 15, Pool (Influenzae A, B, Parainfluenzae 1, 2, 3) in 15, Parainfluenzae 3 in 3 and Adenovirus in 9 children. RSV was frequently found in under 5 years old patients. RSV by RIF was positive in 18.5%, while by EIA in 17.5%. Mixed viral infections were diagnosed in 3 children A (Adeno+RSV), B (Pool+RSV), C (P3+Adeno). These results suggest that the rapid detection of respiratory viruses is simple applying the methods above. It is also shown that the direct detection of antigens of respiratory viruses can increase the diagnostic capacity of the laboratory.
The enteroviruses and hepatitis A virus (HAV) are transmitted through the digestive track (oral-faecal route) and can cause a wide range of illnesses. The purpose of this study is the detection of these viruses in samples taken from the environment. We have chosen 25 samples originating from the area of Inofiton and from a biological waste processing plant of the area of Metamorphosis, as well as samples originating from soil and leaves in areas where the waste by-products were used for irrigation or fertilization. The viruses in these samples were concentrated by two different techniques: (a) ultracentrifugation and elution of viral particles in glycine solution, and (b) PEG precipitation and ultrafiltration. For enterovirus detection, two pairs of primers based on the 5' non-translating region and one pair of primers specific for the poliovirus VP1 region were used. For HAV detection we used the pair of A and B primers from the VP1 area of the genome. In order to improve the sensitivity and yield of this technique, we applied Semi-Nested PCR where an internal primer C was used together with A. From all the samples tested, 9 were shown to be positive for enteroviruses and 6 were positive for HAV. The soil and leaf samples were negative.
The aim of the study was the identification of tospoviruses, the investigation of their host range and the study of different thrips species involved in tomato spotted wilt tospovirus (TSWV) epidemiology. Serological studies using monoclonal and polyclonal antibodies showed that TSWV is the only tospovirus present in Greece. TSWV was found in many vegetables (tomato, lettuce, pepper, endive), ornamentals (Anemone sp., Aster ericoides, Diefenbachia sp., Aralia japonica, Dahlia hybrida, Impatiens sp., Cineraria nana hybrida, Iris spp., Cazania sp., Mathiola sp., Callistephus chinensis, Chrysanthemum sp., Pellargonium sp., Ranunculus sp., Gerbera jamesonii, Salvia splendens, Tagetes sp. and Zinnia elegans), arable crops (tobacco, peanut) and some arable weeds (Cirsium arvense, Sillybum marianum and Sonchus sp.) in many areas of Greece. None of the samples was infected with Impatiens necrotic spot virus (INSV). Epidemiological studies in two tobacco producing areas of Macedonia (Kilkis and Drama) during 1995 and 1996 showed that TSWV in tobacco crops is exclusively related to Thrips tabaci. On the other hand, Frankliniella occidentalis specimens were isolated from vegetables and ornamentals suggesting that this species is responsible for TSWV incidence in these crops.
In the present work we have reviewed the tremendous amount of work, accruing exponentially, that accompanied the development and maturation of Quantitative PCR (Q-PCR). The procedure has been critically examined, leading to an understanding of the critical parameters involved in quantitative amplification. Variations in nucleic acid preparation, thermal cycler performance, choice of polymerase and amplification procedure can cause large differences in the final product yield. The first quantitative approach with PCR was semiquantitative and based on the amplification of sample in limiting dilutions. The relative quantitative determination of RNA, as opposed to DNA, is widely applied due to the difficulties in preparing proper RNA standard samples, which are expressed in the same rate and amplified with the same efficiency as the RNA target. The situation is further complicated by the varying efficiency of the reverse transcription step (RNA cDNA). A second approach to quantitation has been based on amplification with known amounts of the external standard, such as a cell line carrying a defined wild-type gene copy number. However, these approaches fail to control tube to tube variation. Alternative strategies including an internal standard have been developed. Internal standards act as controls for amplification efficiency. In quantitative competitive PCR, the gene of interest is coamplified with different concentrations (at least 4) of an added standard. The internal standard is defined to be as closely related as possible to the target, with difference accruing on in size or in restriction pattern. When the sequences of the target and the control are very close, this situation approaches the equivalency. The process of coamplification is truly competitive and PCR can be performed to the plateau. The yield of generated target product can be directly correlated to the internal standard. Alternatively, the technique of competitive PCR mimics can be applied to either DNA or RNA absolute quantitative determination. In this approach, the gene of interest is coamplified either with an endogenous standard, or with a fixed amount of a DNA fragment that carries a heterologous sequence flanked by sequences homologous to the amplification primers. As the sequences and the amounts of the target and control are different, the equivalency is rarely approached, and the amplification process must be stopped during the exponential phase. Finally, quantitative determination of the PCR amplicons can be achieved either by the use of radioactive or non-isotopic techniques, or by gel video analysis systems which can scan and quantitate EtBr-stained gel bands.
Recent data resulting from the application of PCR in studying the pathogenicity and epizootiology of viral infections, the genetic analysis of viruses and the production of recombinant viral proteins of biological interest are presented.
The immunocapture-polymerase chain reaction (IC-PCR) technique with modifications was developed for the detection of satellite RNAs and coat protein gene of cucumber mosaic cucumovirus (CMV) using broad spectrum primers. Initially, the technique consisted of an immunocapture step with antibodies for virus purification and concentration before the reverse transcription of the viral RNA and its further amplification by PCR. Assays involving non specific virus capture, using 5% skimmed milk or bovine serum albumin, were as effective as the immunocapture technique when tobacco samples, a principally experimental virus host, were used. This fact renders the technique independent of immunological reagents. Further simplification was achieved by using tissue prints on Whatmann 3MM paper extracted with 0.5% Triton X100 instead of plant extracts (print-capture PCR, PC-PCR). This kind of modification enables sample preparation directly in the field avoiding at the same time potential contamination problems. PC-PCR with non-specific virus capture from tomato samples, with this host being the most seriously affected by the virus, was again as efficient as immunocapture. The characteristics of this new method are great versatility, simplicity and its potential to be used under routine diagnosis in the field.
To determine the significance of water as a vehicle for enteric virus transmission to humans, convenient and reliable methods including virus concentration from water are needed to detect these viruses. The development of a concentration procedure including adsorption/elution to nitrocellulose membranes and reconcentration by virus precipitation, to recover hepatitis A virus (HAV) from experimentally contaminated distilled, tap, waste and seawater is the purpose of this study. After the determination of the optimum adsorption/elution conditions for HAV concentration (pH, filter type, eluent, cation salts) and the choice of an efficient reconcentration procedure, the concentration of HAV from water was performed as follows: cell culture-adapted HAV (strain CF 53) seeded in 5 to 50 L of water was adsorbed at pH 4.0 with 0.15 M NaCl to two nitrocellulose membranes (1.2 and 0.45 um porosity for distilled and tapwater, or 8.0 and 3.0 um porosity for waste and seawater), then eluted by 3% beef extract at pH 8.5. Eluted virus was further concentrated by polyethylene glycol 6000 (PEG 6000) precipitation. Hepatitis A antigen was detected by a solid-phase radioimmunoassay and quantified by RIA-endpoint titration. Infectious virus was quantified by cell culture titration by using PLC/PRF/5 cell line. Results showed that HAV should be efficiently recovered from waters by this procedure, with a mean average recovery greater than 75% of the initial virus added to the waters. Also, the sensitivity of the method was tested by contaminating 50 L of water with 16 TCID50 only: 69.4% of the initial infectious virus were recovered. The concentration factor obtained by this procedure was approximately 1,700 to 50,000 fold. By using this concentration method, wild strains of HAV were detected in several water samples originated from four European areas after regular water collection. Thus, we recommend the use of the described adsorption/elution and PEG 6000 precipitation method for detecting pollution with viral hepatitis A in samples of drinking, waste and seawater.