Infectious Pepper Mild Mottle Virus and Human Adenoviruses as Viral Indices in Sewage and Water Samples.

The objective of this study was to compare human adenoviruses (HAdVs) genome and infectivity, polyomaviruses (JC and BK) genome (JCPyVs) and (BKPyVs), Pepper Mild Mottle Virus (PMMoV) genome and infectivity, and infectious bacteriophages as viral indices for sewage and water samples. One hundred and forty-four samples were collected from inlets and outlets of water and wastewater treatment plants (WTPs), and WWTPs within Greater Cairo from October 2015 till March 2017. Two methods of viral concentration [Aluminium hydroxide (Al(OH)3) precipitation method and adsorption-elution technique followed by organic flocculation method] were compared to determine which of them was the best method to concentrate viruses from sewage and water. Although samples with only one litre volume were concentrated using Al(OH)3 precipitation method and the same samples with larger volumes (5-20 L) were concentrated using the adsorption-elution technique followed by the organic flocculation method, a non-significant difference was observed between the efficiency of the two methods in all types of samples except for the drinking water samples. Based on the qualitative prevalence of studied viruses in water and wastewater samples, the number of genome copies and infectious units in the same samples, resistance to treatment processes in water and wastewater treatment plants, higher frequency of both adenoviruses and PMMoV genomes as candidate viral indices in treated sewage and drinking water was observed. The problem of having a viral genome as indices of viral pollution is that it does not express the recent viral pollution because of the longer survivability of the viral genome than the infectious units in water and wastewater. Both infectious adenovirus and infectious phiX174 bacteriophage virus showed similar efficiencies as indices for viral pollution in drinking water and treated sewage samples. On the other hand, qualitative detection of infectious PMMoV failed to express efficiently the presence/absence of infectious enteric viruses in drinking water samples. Infectious adenoviruses and infectious bacteriophage phiX174 virus may be better candidates than adenoviruses genome, polyomaviruses genome, and PMMoV genome and infectivity as viral indices for water and wastewater.

Human Coronavirus NL63 Among Other Respiratory Viruses in Clinical Specimens of Egyptian Children and Raw Sewage Samples.

The objective of this study was to investigate human coronavirus NL63 (HCoV-NL63) prevalence among the other respiratory viruses such as parainfluenza, respiratory syncytial virus, and non-enteric adenoviruses in clinical specimens of Egyptian children and raw sewage samples. One hundred clinical specimens were collected from Egyptian children suffering from upper and lower respiratory viral infections in the years 2005-2006 to detect HCoV-NL63 genome using RT-PCR. All the specimens were negative for the virus. Also, a complete absence of HCoV-NL63 genome was observed in the twenty-four raw sewage samples collected from two wastewater treatment plants within Greater Cairo from February 2006 to January 2007. Using nested RT-PCR, parainfluenza virus type 1, respiratory syncytial virus type A, adenovirus type 4, and adenovirus type 7 were detected in 3%, 2%, 5%, and 2% of the clinical specimens, respectively. Of these viruses, only adenovirus type 4 was detected in 1/24 (4.17%) of the raw sewage samples, while a complete absence of the other investigated respiratory viruses was observed in the raw sewage samples. The low percentage of positivity in the clinical specimens, the concentration method of the raw sewage samples, and the indirect routes of transmission may be the reasons for the absence of respiratory viruses in raw sewage samples. On the other hand, enteric adenoviruses were detected in 21/24 (87.5%) of the raw sewage samples with a higher prevalence of adenovirus type 41 than adenovirus type 40. A direct route of transmission of enteric viruses to raw sewage may be the reason for the high positivity percentage of enteric adenoviruses in raw sewage samples.

Assessment and Evaluation of an Integrated Hybrid Anaerobic-Aerobic Sewage Treatment System for the Removal of Enteric Viruses.

The capability of a cost-effective and a small size decentralized pilot wastewater treatment plant (WWTP) to remove enteric viruses such as rotavirus, norovirus genogroup I (GGI), norovirus genogroup II (GGII), Hepatitis E virus (HEV), and adenovirus was studied. This pilot plant is an integrated hybrid anaerobic/aerobic setup which consisted of anaerobic sludge blanket (UASB), biological aerated filter (BAF), and inclined plate settler (IPS). Both the UASB and BAF are packed with a non-woven polyester fabric (NWPF). Results indicated that the overall log10 reductions of enteric viruses' genome copies through the whole system were 3.1 ± 1, 3.3 ± 0.5, and 2.6 ± 0.9 log10 for rotavirus, norovirus GGI, and adenovirus, respectively. Reduction efficiency for both norovirus GGII and HEV after the different treatment steps could not be calculated because there were no significant numbers of positive samples for both viruses. The overall reduction of rotavirus infectious units through the whole system was 2.2 ± 0.8 log10 reduction which is very close to the overall log10 reduction of adenovirus infectious units through the whole system which was 2.1 ± 0.8 log10 reduction. There was no considerable difference in the removal efficiency for different rotavirus G and P types. Adenovirus 41 was the only type detected in the all positive samples. Although the pilot WWTP investigated is cost effective, has a small footprint, does not need a long distance network pipes, and easy to operate, its efficiency to remove enteric viruses is comparable with the conventional centralized WWTPs.

Prevalence of Rotaviruses Groups A and C in Egyptian Children and Aquatic Environment.

The objective of this study is to compare the prevalence of rotaviruses groups A and C in Egyptian children and aquatic environment. From 110 stool specimens of children with acute diarrhea and using RT-PCR, 35 samples (31.8 %) were positive for human rotavirus group A and 15 samples (13.6 %) were positive for human rotavirus group C. From 96 samples collected from Zenin wastewater treatment plant over a 2-year period (November 2009-October 2011) and using RT-PCR, rotavirus group A was detected in (4/24) 16.7 %, (5/24) 20.8 %, (4/24) 16.7 %, and (4/24) 16.7 %, while rotavirus group C was detected in (2/24) 8.3 %, (3/24) 12.5 %, (3/24) 12.5 %, and (0/24) 0 % in raw sewage, after primary sedimentation, after secondary sedimentation, and after final chlorination, respectively. Moreover, from 96 samples collected from El-Giza water treatment plant over a 2-year period (November 2009-October 2011), rotavirus group A was detected in (7/24) 29.2 %, (6/24) 25 %, (5/24) 20.8 %, and (3/24) 12.5 %, while rotavirus group C was detected in (3/24) 12.5 %, (1/24) 4.2 %, (1/24) 4.2 %, and (0/24) 0 % in raw Nile water, after sedimentation, after sand filtration, and after final chlorination, respectively. Using SYBR Green real-time RT-PCR, the number of human rotavirus group A genome or infectious units was higher than rotavirus group C. VP6 sequence analysis of the RT-PCR positive rotavirus group C samples revealed that four clinical specimens and three environmental samples showed similar sequences clustered with Moduganari/Human Nigerian strain AF 325806 with 98 % homology, and two clinical specimens and one environmental sample showed similar sequences clustered with Dhaka CB/Human Bangladesh strain AY 754826 with 97 % homology.

Method validation for norovirus detection in naturally contaminated irrigation water and fresh produce.

Human enteric viruses are shed in extremely high numbers in the feces of infected individuals, becoming environmental contaminants and eventually leading to contamination of a variety of foodstuffs at the pre-harvest stage. Among these foods at risk is fresh produce and irrigation water is a major vehicle for crop contamination. In the present study, a standardized molecular method for quantitative virus assay has been evaluated in different types of fresh produce and in irrigation water for human norovirus (NoV) detection. Two different virus concentration procedures, polyethylene-glycol precipitation (PEG) and organic flocculation (OF), were employed. The procedures were initially assayed in spiked samples and later validated on naturally contaminated samples from the Nile Delta in Egypt. Overall, PEG provided significantly (p<0.05) better virus recoveries than OF for both irrigation water and salad vegetable virus analysis. NoV GI and GII recoveries in spiked irrigation water ranged from 22.0% to 43.3% and from 12.6% to 16.4% with the PEG and OF methods, respectively. In experimentally contaminated salad vegetables, virus recoveries ranged from 28.0% to 48.0% and from 14.0% to 18.8% by PEG precipitation and OF, respectively. Using PEG precipitation, NoV was found in 31.9% of naturally contaminated irrigation water samples. Both NoV GI and GII were detected in these samples with genome copy numbers of around 10(2) per liter. Virus analyses performed in naturally contaminated fresh produce that included green onion, watercress, radish, leek, and lettuce show that NoV GI was present in 20.8%-34.0% of the samples with genome copy numbers of around 10(2) per gram. When OF was employed, NoV was found in 25.0% of the irrigation water samples. Both genogroups could be found in these samples with genome copy numbers of around 10 per liter. In fresh produce, GI was present in 16.0%-25.7% of the samples with genome copy numbers per gram of around 10. Surprisingly, NoV GII was not detected in any salad vegetable despite highly satisfactory virus/nucleic acid extraction and enzyme efficiencies reported in the assays. Available reliable standardized assays for virus detection in food matrices including appropriate quality assurance and quality control measures to assess the efficiency of critical steps in virus analysis open the possibility to produce consistent and accurate exposure data to be used in QMRA (quantitative microbial risk assessment) and at the same time may enable the formulation of guidelines to ensure the virological quality of selected commodities in specific scenarios to reduce the risk of foodborne virus infections.

Persistent gastroenteritis in children infected with astrovirus: association with serotype-3 strains.

The relationship between cases of persistent diarrhoea and the levels and type of human astrovirus was investigated. The potential correlation between human astrovirus excretion levels and the occurrence of protracted gastroenteritis was elucidated after quantifying astroviruses in faecal samples by a competitive RT-PCR. This assay was developed employing an internal RNA standard constructed for this purpose and showed a threshold of positivity of 3.4 x 10(4) genomes per gram of faeces. By this procedure, the levels of astrovirus, belonging to serotypes 1, 2, 3, 4, and 8, in faecal samples could be ascertained to range from 3.4 x 10(8) to 1 x 10(13) per gram of faeces. The mean viral titre in the serotype 3-containing faeces was higher than in any of the other serotype-containing samples. In children with no background disease, persistent gastroenteritis cases were detected in 8.5% of the astrovirus infections, and 37.5% of those were associated with astrovirus type 3 infection. In addition, 42.9% of astrovirus 3 isolates were implicated with persistent cases, some of them lasting for 3 months. Other type 3 isolates, detected in the faeces in very large numbers, caused severe gastroenteritis.

Group A rotavirus in sewage samples from Barcelona and Cairo: emergence of unusual genotypes.

The presence of rotavirus strains in sewage samples from Cairo, Egypt (November 1998 to October 1999), and Barcelona, Spain (November 1998 to December 2002), was investigated by using a generic molecular detection method based on amplification of a VP6 gene fragment. Overall, 85.7 and 66.9% of the sewage samples from Cairo and Barcelona, respectively, were positive. Positive samples were characterized further, and VP7 and VP4 genotypes were determined. Although 30% of the positive samples from Cairo were G untypeable, the distribution of G types in the positive samples was 69.6% G1, 13% G3, 8.7% G4, and 8.7% G9. The percentage of untypeable samples was much higher for the Barcelona samples (56.5%), and the distribution in the positive samples was 56.4% G1, 31.5% G3, 6% G9, 4% G2, and 2% G5. When the P types were examined, 26.7% of the positive samples from Cairo were untypeable, and the distribution of types in the positive samples was 53.3% P[8], 30% P[6], and 16.6% P[4]. In Barcelona, 27.2% of the samples were P untypeable, and the frequencies of the types detected were 49.7% P[8], 37.2% P[4], 8.8% P[6], and 4.2% P[9]. The distribution for strains from Cairo was 38.5% P[8]G1, 27% P[6]G1, 11.5% P[4]G1, 11.5% P[8]G3, 7.7% P[6]G4, and 3.8% P[8]G9. Strikingly, equivalent frequencies of common and uncommon strains were observed for Barcelona samples, and the distribution was 38.8% P[8]G1, 30.6% P[4]G1, 11.6% P[8]G3, 6.6% P[4]G3, 5.8% P[6]G1, 1.6% P[6]G3, 1.6% P[9]G1, 0.8% P[4]G2, 0.8% P[6]G9, 0.8% P[8]G9, and 0.8% P[8]G5. Additionally, two P[-]G5 strains were isolated in Barcelona, and the porcine or human origin of these strains was unclear. Rotavirus variability exhibited not only a geographic pattern but also a temporal pattern.