Subject(s)
Bothrops , Disease Outbreaks/veterinary , Paramyxoviridae Infections/veterinary , Paramyxovirinae/isolation & purification , Animals , Brazil/epidemiology , DNA, Viral/analysis , Female , Hemagglutination Tests/veterinary , Male , Paramyxoviridae Infections/epidemiology , Paramyxovirinae/genetics , Polymerase Chain Reaction/veterinaryABSTRACT
PCR is the best method for the detection of enteric viruses present at low concentrations in environmental samples. However, some organic and inorganic compounds present in these samples can interfere in the reaction. Many of these substances are cytotoxic, too. The ZP60S filter membranes used in addition to fluorpentane treatment are quite efficient for virus concentration and simultaneous elimination of cytotoxicity from environmental samples. In this study, both procedures were used to promote the elimination of reverse transcriptase PCR (RT-PCR) inhibitors from sewage and sewage-polluted creek water. Samples were subjected separately to each of the following procedures: filtration through electropositive filter membranes (ZP60S), organic extraction with Vertrel XF, and filtration through ZP60S followed by organic extraction. Afterwards, aliquots were experimentally inoculated with rotavirus SA-11 RNA and subjected to RT-seminested PCR for amplification of the VP7 gene. Results showed that the ZP60S membranes efficiently eliminated the RT-PCR inhibitors from water samples. The sample processing method was also applied to 31 in natural sewage and creek water samples for detection of naturally occurring rotavirus. A duplex seminested PCR was used for the quick detection of couples of the four rotavirus genotypes (G1 to G4). Eight samples (25.8%) were positive, and rotavirus sequences were not detected in 23 (74.2%). Results were confirmed by direct immunoperoxidase method. In summary, the use of electropositive filter membrane is appropriate for the elimination of substances that can interfere with RT-PCR, obviating additional sample purification methods.
Subject(s)
Fresh Water/virology , Membranes, Artificial , Reverse Transcriptase Polymerase Chain Reaction , Rotavirus/isolation & purification , Sewage/virology , Animals , Filtration/instrumentation , Filtration/methods , Humans , RNA, Viral/analysis , Rotavirus/genetics , Water PollutionABSTRACT
Zeta plus filter membranes (ZP60S) have been shown to be efficient for rotavirus concentration from wastewater and for the reduction of cytotoxicity for cell cultures. Recently a variability in both properties was observed. In view of the low costs and the high virus recovery rates obtained in the past, we re-evaluated the application of ZP60S filter membranes for virus concentration from environmental samples. Some factors that could interfere with the concentration strategy using ZP60S were also considered and assessed including the type of water to be filtered and the possible release of toxic substances from the membrane matrix during filtration.
Subject(s)
Membranes, Artificial , Rotavirus/isolation & purification , Sewage/virology , Water Microbiology , Cell Culture Techniques , Cytotoxins , Filtration/methods , Sewage/chemistryABSTRACT
Zeta plus filter membranes (ZP60S) have been shown to be efficient for rotavirus concentration from wastewater and for the reduction of cytotoxicity for cell cultures. Recently a variability in both properties was observed. In view of the low costs and the high virus recovery rates obtained in the past, we re-evaluated the application of ZP60S filter membranes for virus concentration from environmental samples. Some factors that could interfere with the concentration strategy using ZP60S were also considered and assessed including the type of water to be filtered and the possible release of toxic substances from the membrane matrix during filtration.
Subject(s)
Membranes, Artificial , Rotavirus/isolation & purification , Sewage/virology , Water Microbiology , Cell Culture Techniques , Cytotoxins , Filtration/methods , Sewage/chemistryABSTRACT
Canine parvovirus (CPV) was first isolated in 1978 in the USA. Analysis of CPV isolates by monoclonal antibodies and restriction enzymes have shown that after the first emergence of CPV (CPV-2) it evolved to give rise to new antigenic types, which were designated CPV type 2a and type 2b. These new types have replaced the original CPV type 2, although the proportions of each of the new antigenic types vary in different countries. In Brazil, CPV-like infections were first observed in 1979, however, there has been no information concerning the antigenic types of CPV prevailing in South America. In this study, we designed a PCR assay to type canine parvovirus strains in fecal samples collected from symptomatic dogs during 1980 through 1986 and 1990 through 1995. Our data showed that the CPV epizootic in Brazil followed the same pattern observed in the USA of emergence of CPV-2 followed by replacement by the variants CPV-2a and 2b. The predominant strain found during 1980 was CPV-2a, which was substantially replaced by CPV-2b from 1990 to 1995.
Subject(s)
Dog Diseases/virology , Parvoviridae Infections/veterinary , Parvovirus, Canine/classification , Animals , Antigenic Variation/genetics , Base Sequence , Brazil , DNA Primers/chemistry , DNA, Viral/chemistry , DNA, Viral/isolation & purification , Dogs , Electrophoresis, Agar Gel/veterinary , Feces/virology , Hemagglutination Tests/veterinary , Molecular Sequence Data , Parvoviridae Infections/virology , Parvovirus, Canine/chemistry , Parvovirus, Canine/genetics , Polymerase Chain Reaction/veterinary , Sensitivity and Specificity , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Nucleic AcidABSTRACT
Simian rotavirus SA-11, experimentally seede, was recovered from raw domestic sewage by a two-step concentration procedure, using filtration through a positively charged microporous filter (Zeta Plus 60 S) followed by ultracentrifugation, effecting an 8000-fold concentration. By this method, a mean recovery of 81 per centñ7.5 of the SA-11 virus was achieved.
Subject(s)
Rotavirus , Wastewater/analysis , Immunoenzyme TechniquesABSTRACT
Simian rotavirus SA-11 experimentally seeded, was recovered from raw domestic sewage by a two-step concentration procedure, using filtration through a positively charged microporous filter (Zeta Plus 60 S) followed by ultracentrifugation, effecting an 8,000-fold concentration. By this method, a mean recovery of 81% +/- 7.5 of the SA-11 virus, was achieved.
Subject(s)
Rotavirus Infections/diagnosis , Rotavirus Infections/prevention & control , Rotavirus/isolation & purification , Sewage/virology , Water Microbiology , Cell Culture Techniques , Fresh Water , Virology/methodsABSTRACT
A total of 22 (65%) of 34 representative rotavirus-positive specimens from infants with acute gastroenteritis were electropherotyped (RNA genome segments) and serotyped using an enzyme immunoassay with monoclonal antibodies (ELISA with MAbs). Serotype 3 was predominant during the 10-month study period (41%), followed by serotype 1 (27%) and serotype 4 (9%). Serotype 2 was not found. Rotavirus strains were grouped into 3 major electropherotypes designated V, W and Z, each corresponding to a single serotype, i.e., serotypes 1, 3 and 4, respectively. Three strains that could not be typed by the serologic technique showed the W electrophoretic profile. The relative migration of the gene segments 7-9 was the main feature distinguishing the predominant serotype 3 from the other serotypes. The migration of segments 2 and 5 was also important for differentiating serotype 4 strains. The present study strengthens the view that electropherotyping, when used in conjunction with serotyping, can help characterize atypical and unusual strains, as well as rotaviruses that cannot be typed by the serologic technique.
Subject(s)
Rotavirus Infections/virology , Rotavirus/classification , Acute Disease , Antibodies, Monoclonal , Brazil , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Feces/virology , Gastroenteritis/microbiology , Humans , Infant , RNA, Viral/analysis , Rotavirus/genetics , Rotavirus/immunology , Rotavirus/isolation & purification , Serotyping , Time FactorsABSTRACT
A total of 22 (65) of 34 representative rotavirus-positive specimens from infants with acute gastroenteritis were electropherotyped (RNA genome segments) and serotyped using an enzyme immunoassay with monoclonal antibodies (ELISA with MAbs). Serotype 3 was predominant during the 10-month study period (41), followed by serotype 1 (27) and serotype 4 (9). Serotype 2 was not found. Rotavirus strains were grouped into 3 major electropherotypes designated V, W and Z, each corresponding to a single serotype, i.e., serotypes 1, 3 and 4, respectively. Three strains that could not be typed by the serologic technique showed the W electrophoretic profile. The relative migration of the gene segments 7-9 was the main feature distinguishing the predominant serotype 3 from the other serotypes. The migration of segments 2 and 5 was also important for differentiating serotype 4 strains. The present study strengthens the view that electropherotyping, when used in conjunction with serotyping, can help characterize atypical and unusual strains, as well as rotaviruses that cannot be typed by the serologic technique.
Subject(s)
Humans , Infant , Rotavirus Infections/virology , Rotavirus , Acute Disease , Antibodies, Monoclonal , Brazil , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Feces , Gastroenteritis , RNA, Viral , Rotavirus , Serotyping , Time FactorsABSTRACT
Rotaviruses were concentrated from 8-liter samples of raw domestic sewage and sewage-polluted creek water by adsorption to and elution from positively charged microporous filters (Zeta Plus 60S), followed by ultracentrifugation of the filter eluates. Indirect immunofluorescence and direct immunoperoxidase methods allowed detection and enumeration of rotavirus in 6 (20.6%) of 29 sewage samples and in 19 (34.5%) of 55 creek water samples. Levels of rotaviruses ranged from < 3 to 63 focus-forming units (FFU)/liter, and the geometric means were 2.2 FFU/liter in sewage, 2.9 FFU/liter at creek Tremembé, and 2.6 FFU/liter at creek Pirajussara. Wastewater samples examined during autumn and winter months showed a higher rate positivity for rotavirus than those collected in spring and summer, corresponding to the seasonal variation of rotaviral diarrhea in the city of São Paulo.