Enteroviruses In Water Samples Research Articles

Identification of enteroviruses along Lake Victoria shoreline - a potential indicator of sewage pollution.

E n t e r i c v i r u s e s a r e m a i n l y t r a n s m i t t e d b y t h e f a e c a l - o r a l r o u t e a n d h a v e b e e n l i n k e d t o s e v e r a l d i s e a s e s i n c l u d i n g g a s t r o e n t e r i t i s a n d r e s p i r a t o r y i n f e c t i o n s . T h e i r p r e s e n c e i n s u r f a c e w a t e r s h a s b e e n exacerbated by p o l l u t i o n f r o m a v a r i e t y o f p o i n t s o u r c e s s u c h a s s e w a g e d i s c h a r g e . W e s t u d i e d t h e occurrence o f e n t e r o v i r u s e s i n w a t e r s a m p l e s f r o m L a k e V i c t o r i a i n K e n y a t o i n v e s t i g a t e i f t h e r e w a s a l i n k b e t w e e n s e w a g e p o l l u t i o n a n d d e t e c t i o n o f e n t e r o v i r u s e s ( E V s ) t o b u i l d a b a s e l i n e f o r a n enteric viruses monitoring platform for this region. We analysed 216 samples collected over 6 months from six different locations along the Homa Bay Pier. The six sampling locations comprised of three sites (P3, P5, P6) located <500 m from a local sewage treatment plant and pit latrines while three other sites (P1, P2, P4) were located at approximately 0.5 to 3 Km. EVs were concentrated using glass wool adsorption elution protocol and identified using the nested reverse transcription-polymerase chain reaction. The odds ratio was performed to determine whether the location of the sources of sewage pollution near the lake was associated with the EVs contamination. Five out of 108 (5 %) samples collected from the sites (P3, P5 and P6 were EV positive, while 2 % (2/108) of samples from P1, P2 and P4 were EV positive. The presence of the EVs was associated with the distance from the possible sources of faecal contamination (odds ratio 20.28 and 4.86, confidence interval 2.42, and 0.95) for pit latrines and the sewage treatment plant respectively. The result from this study indicates that sewage discharge at the shoreline of Lake Victoria may have been the source of EVs contamination. Data from this study could significantly contribute to informing risk management on sewage pollution in Lake Victoria and it is important to continue monitoring this lake for potentially pathogenic enteric viruses.

Experimental and Theoretical Substantiation of the Express Method Development for Detection of Enteroviruses in Water by Surface Plasmon Resonance Method

Background. As a result of anthropogenic pollution and number of other factors, in drinking water can be present some viruses such as enterovirus and other pathogens that can be transmitted by water. Enteroviruses are capable of causing lesions of organs and systems of varying severity in humans, manifested by a number of clinical symptoms. For the detection of viruses appropriate research methods were developed, but they take a lot of time and require special reagents and equipment, that is significant drawback of sanitary-virological studies of drinking water quality. Objective. The aim of the paper is to study the general possibility of using a surface plasmon resonance (SPR) phenomenon for detection of enteroviruses in water samples (swine Teschovirus A as an example) and to create on its basis a new express diagnostic method. Methods. For implementation of a method for rapid detection of viral diseases pathogens in water by using SPR, the device was developed at the Institute of V.E. Lashkaryov Institute of Semiconductor Physics NAS of Ukraine. The research on the interaction of antigen and specific serum in various buffers was held at the State Scientific Control Institute of Biotechnology and Strains of Microorganisms. Results. A general possibility of using SPR to detect enteroviruses in distilled and drinking water (swine Teschovirus A as an example) was theoretically substantiated and experimentally proved, and the express method for their detection was developed. It is shown that upon detection of pathogen in distilled water, the response of the SPR sensor is 1.57 times higher than in drinking water, which may be due to the influence of ionic composition in water on the interaction of the antigen with the antibody. The developed method allows real-time monitoring of changes in the test environment, significantly reduces the duration of enteroviruses detection and does not require the use of expensive nutrient media and reagents. Сonclusions. The possibility of using the SPR method and the Plasmon-6 device implementing this method for express detection of enteroviruses in distilled and drinking water was theoretically substantiated and experimentally proved.

Evaluation of MinION nanopore sequencing for rapid enterovirus genotyping

Enteroviruses (EV) are associated with a range of serious infections, including aseptic meningitis, hand foot and mouth disease, myocarditis, acute flaccid paralysis and encephalitis. Improved methods for assessing EV genotypic diversity could assist molecular epidemiology studies, clinical diagnosis and environmental surveillance. We report new methods for EV genome amplification, and subsequent genotyping using the miniaturised MinION sequencing device. Importantly, this next-generation sequencer enabled correct strain-level assignment of identity for the EV-A71 isolate assessed, where strains with up to 99.7% similarity were discriminated. In addition, an accurate consensus sequence was produced for EV-A71 isolate RNA, with 99.3–99.6% similarity to the reference sequence. Thus, the long nanopore reads enabled rapid whole genome sequencing and strain level identification of EV- A71 isolate RNA. We also demonstrated potential for using MinION sequencing for direct detection of EV in water samples, which may have application for diversity analysis, water quality monitoring, and environmental surveillance.

Detection of Enteroviruses in Water Samples from Yopougon, Côte d’Ivoire by Cell Culture and Polymerase Chain Reaction

The objective of this study was to compare sensitivities of enterovirus isolation from wastewater in different cell lines as well as to compare the sensitivity and specificity of isolation in cell culture with direct detection by reverse transcription polymerase chain reaction (RT-PCR). Sixty-eight samples of wastewaters were collected between September 2008 and January 2009 in Yopougon, Abidjan. Enteroviruses were concentrated according to World Health Organization recommendations. Viruses were inoculated into various cell lines while direct RT-PCR was performed on water concentrates. The buffalo green monkey kidney cell line was the most sensitive with 58.8% of viral isolation. This was followed by the rhabdomyosarcoma cell line with sensitivity of 51.6%, with human epidermoid carcinoma cell line showing sensitivity of 50% and fibroblastic cells derived from transgenic mice LTK-1 (L20B) cell showing 23.50% sensitivity. However, a lower specificity of 2.9% was observed with the L20B cell line. 44.1% of the samples were positive by direct RT-PCR detection while 51.47% samples were positive by using RT-PCR on infected cell cultures. No difference in percentage positivity was observed using RT-PCR on infected tissue culture isolates or using RT-PCR directly on wastewater samples.

In search of enteroviruses in water media in Marrakech

Enteroviruses are among the most common viruses infecting the human intestine; they are very widespread in nature and resistant to external agents. They are eliminated in the faeces and contaminate water and food. These viruses cause various clinical syndromes and constitute a big public health problem. The aim of our study was to search for enteroviruses in the water samples. For this, a total of 225 L of tap water coming from the national network office for drinking water (ONEP) and 18 samples of waste water originating from Marrakech city were studied. These samples were concentrated, treated with polyethylene glycol 6000 and then analyzed by RT-PCR. Only two samples were found to be positive for enteroviruses by RT-PCR among the 18 waste water samples analyzed, which gave a rate of 11.11%. On the other hand, no positive samples were found in the tap water. This study made it possible on the one hand to apply for the first time RT-PCR for the detection of enteroviruses in water samples originating from Marrakech city, and on the other hand to show that tap water of this city does not present any risk of contamination by this type of germ. Key words: Drinking water, waste water, enterovirus, RT-PCR, Marrakech.

Reverse transcription PCR to detect enteroviruses in surface water.

We have developed a simple, fast, and efficient procedure to detect enteroviruses in water samples. Aliquots of water are subjected to two-step filtration, with the second filter containing a positively charged nylon membrane that holds back virus particles. Viruses thus adsorbed are directly lysed, and RNA is isolated by hybridization to specific oligonucleotides bound to magnetic beads. The solution used contains guanidine thiocyanate, which lyses virus particles, inactivates enzymes, e.g., RNases, allows mild hybridization conditions, and does not influence biotin-streptavidin interaction on magnetic beads. Detection and specific identification are accomplished by reverse transcription PCR of the highly conserved noncoding region at the 5' end of virus RNA combined with Southern hybridization. The system was tested with tap water artificially spiked with poliovirus vaccine and yielded a detection limit of 20 50% tissue culture infective doses per liter. We used the same procedure to investigate the water quality of surface water at public beaches by rivers and lakes. Of 40 samples tested, 7 were positive for enteroviruses. A comparison with enterobacterial contamination determined by PCR and classical microbiological methods in parallel showed that enteroviruses were found only in samples also positive for Escherichia coli. In conclusion, this procedure can easily be adapted to test large water samples and is simple enough to be used for routine determinations of water quality in terms of virus contamination.

Detection of hepatitis A virus and other enteroviruses in water by ssRNA probes

Sensitive and specific methods are needed to detect hepatitis A virus (HAV) and other human enteroviruses in environmental samples such as drinking water and foods. Clones of cDNA encoding the 5'-most 1 kb of the HAV and coxsackievirus B3 (CB3) genomes were subcloned into T7/SP6 RNA transcription vectors. In vitro transcribed RNA from the T7 promoter detected their respective HAV or CB3 genomic RNA. Conversely, SP6 transcripts detected viral negative-stranded RNA but not the genome. When both ssRNA probes were tested at high temperature (65°C), they did not hybridize with intracellular RNAs from 6 primate cell cultures used for isolation of HAV and other enteroviruses. The HAV probe did not hybridize with 13 different enteroviruses but detected as little as 500–1000 infectious units of the 7 strains of HAV tested. Conversely, the CB3 probe showed strong homology with all 13 enteroviruses tested but not HAV. The probes were used to detect HAV and other enteroviruses in water samples after virus amplification in cell culture. HAV was detected in water samples obtained during a waterborne hepatitis outbreak using the ssRNA probe. These samples were negative for HAV by direct solid phase radioimmunoassay and were not positive by immunoassays of inoculated cell cultures until several weeks of propagation. The CB3 ssRNA probe detected enteroviruses in samples of surface water and drinking water that were negative for cytopathic effects in inoculated cell cultures.