A multi-marker assessment of sewage contamination in streams using human-associated indicator bacteria, human-specific viruses, and pharmaceuticals.

Human sewage contaminates waterways, delivering excess nutrients, pathogens, chemicals, and other toxic contaminants. Contaminants and various sewage indicators are measured to monitor and assess water quality, but these analytes vary in their representation of sewage contamination and the inferences about water quality they support. We measured the occurrence and concentration of multiple microbiological (n = 21) and chemical (n = 106) markers at two urban stream locations in Milwaukee, Wisconsin, USA over two years. Five-day composite water samples (n = 98) were collected biweekly, and sewage influent samples (n = 25) were collected monthly at a Milwaukee, WI water reclamation facility. We found the vast majority of markers were not sensitive enough to detect sewage contamination. To compare analytes for monitoring applications, five consistently detected human sewage indicators were used to evaluate temporal patterns of sewage contamination, including microbiological (pepper mild mottle virus, human Bacteroides, human Lachnospiraceae) and chemical (acetaminophen, metformin) markers. The proportion of human sewage in each stream was estimated using the mean influent concentration from the water reclamation facility and the mean concentration of all stream samples for each sewage indicator marker. Estimates of instream sewage pollution varied by marker, differing by up to two orders of magnitude, but four of the five sewage markers characterized Underwood Creek (mean proportions of human sewage ranged 0.0025 % - 0.075 %) as less polluted than Menomonee River (proportions ranged 0.013 % - 0.14 %) by an order of magnitude more. Chemical markers correlated with each other and yielded higher estimates of sewage pollution than microbial markers, which exhibited greater temporal variability. Transport, attenuation, and degradation processes can influence chemical and microbial markers differently and cause variation in human sewage estimates. Given the range of potential human and ecological health effects of human sewage contamination, robust characterization of sewage contamination that uses multiple lines of evidence supports monitoring and research applications.

Environmental health of water bodies from a Brazilian Amazon Metropolis based on a conventional and metagenomic approach.

AIMS: The present study aimed to use a conventional and metagenomic approach to investigate the microbiological diversity of water bodies in a network of drainage channels and rivers located in the central area of the city of Belém, northern Brazil, which is considered one of the largest cities in the Brazilian Amazon. METHODS AND RESULTS: In eight of the analyzed points, both bacterial and viral microbiological indicators of environmental contamination-physical-chemical and metals-were assessed. The bacterial resistance genes, drug resistance mechanisms, and viral viability in the environment were also assessed. A total of 473 families of bacteria and 83 families of viruses were identified. Based on the analysis of metals, the levels of three metals (Cd, Fe, and Mn) were found to be above the recommended acceptable level by local legislation. The levels of the following three physicochemical parameters were also higher than recommended: biochemical oxygen demand, dissolved oxygen, and turbidity. Sixty-three bacterial resistance genes that conferred resistance to 13 different classes of antimicrobials were identified. Further, five mechanisms of antimicrobial resistance were identified and viral viability in the environment was confirmed. CONCLUSIONS: Intense human actions combined with a lack of public policies and poor environmental education of the population cause environmental degradation, especially in water bodies. Thus, urgent interventions are warranted to restore the quality of this precious and scarce asset worldwide.

Delving into the Aftermath of a Disease-Associated Near-Extinction Event: A Five-Year Study of a Serpentovirus (Nidovirus) in a Critically Endangered Turtle Population.

Bellinger River virus (BRV) is a serpentovirus (nidovirus) that was likely responsible for the catastrophic mortality of the Australian freshwater turtle Myuchelys georgesi in February 2015. From November 2015 to November 2020, swabs were collected from turtles during repeated river surveys to estimate the prevalence of BRV RNA, identify risk factors associated with BRV infection, and refine sample collection. BRV RNA prevalence at first capture was significantly higher in M. georgesi (10.8%) than in a coexisting turtle, Emydura macquarii (1.0%). For M. georgesi, various risk factors were identified depending on the analysis method, but a positive BRV result was consistently associated with a larger body size. All turtles were asymptomatic when sampled and conjunctival swabs were inferred to be optimal for ongoing monitoring. Although the absence of disease and recent BRV detections suggests a reduced ongoing threat, the potential for the virus to persist in an endemic focus or resurge in cyclical epidemics cannot be excluded. Therefore, BRV is an ongoing potential threat to the conservation of M. georgesi, and strict adherence to biosecurity principles is essential to minimise the risk of reintroduction or spread of BRV or other pathogens.

SARS-CoV-2 in a stream running through an underprivileged, underserved, urban settlement in São Paulo, Brazil: A 7-month follow-up.

COVID-19 pandemic has led to concerns on the circulation of SARS-CoV-2 in the environment, its infectivity from the environment and, the relevance of transmission via environmental compartments. During 31 weeks, water samples were collected from a heavily contaminated stream going through an urban, underprivileged community without sewage collection. Our results showed a statistically significant correlation between cases of COVID-19 and SARS in the community, and SARS-CoV-2 concentrations in the water. Based on the model, if the concentrations of SARS-CoV-RNA (N1 and N2 target regions) increase 10 times, there is an expected increase of 104% [95%CI: (62-157%)] and 92% [95%CI: (51-143%)], respectively, in the number of cases of COVID-19 and SARS. We believe that differences in concentration of the virus in the environment reflect the epidemiological status in the community, which may be important information for surveillance and controlling dissemination in areas with vulnerable populations and poor sanitation. None of the samples were found infectious based cultures. Our results may be applicable globally as similar communities exist worldwide.

Probable Nipa Palm Wine-Associated Hepatitis A Outbreak after Attending a Funeral Ceremony in Sabah.

Foodborne outbreaks of hepatitis A virus (HAV) are most commonly associated with fresh and frozen produce and with various types of shellfish. Alcoholic beverage-borne outbreaks of hepatitis A are extremely rare. Here, we report an outbreak of hepatitis A associated with the consumption of a traditional wine at a funeral ceremony in the Sabah state of Malaysian Borneo. Confirmed cases were determined by serum anti-HAV immunoglobulin M and/or for fecal HAV by reverse transcription polymerase chain reaction (RT-PCR). The amplicons of RT-PCR were subjected to nucleotide sequencing followed by phylogenetic analysis. We conducted a 1:2 case-control study to identify the possible exposure that led to the outbreak. Sixteen patients met the case definition, they were 18 to 58 years old and 90% of them were males. The case-control study showed that the consumption of nipa palm wine during the ceremony was significantly associated (P = 0.0017) with hepatitis A infection (odds ratio, 5.44; 95% CI, 1.80-16.43). Untreated river water was used to dilute the traditional wine, which was assumed to be the source of the infection. Phylogenetically, these viruses belonged to genotype IA and formed an independent cluster with strains from Taiwan, Japan, and the Philippines. This strain might be an emerging HAV in Asian countries. Environmental assessments were performed and environmental samples were negative for HAV. The incidence of hepatitis A in Sabah was also determined and it was 0.795/100,000 population. Strict monitoring of traditional wine production should be implemented by the local authority to prevent future outbreaks.

Environmental stability of porcine respiratory coronavirus in aquatic environments.

Coronaviruses (CoVs) are a family of viruses that are best known as the causative agents of human diseases like the common cold, Middle East Respiratory Syndrome (MERS), Severe Acute Respiratory Syndrome (SARS) and COVID-19. CoVs spread by human-to-human transmission via droplets or direct contact. There is, however, concern about potential waterborne transmission of SARS-CoV-2, the virus responsible for COVID-19, as it has been found in wastewater facilities and rivers. To date, little is known about the stability of SARS-CoV-2 or any other free coronavirus in aquatic environments. The inactivation of terrestrial CoVs in seawater is rarely studied. Here, we use a porcine respiratory coronavirus (PRCV) that is commonly found in animal husbandry as a surrogate to study the stability of CoVs in natural water. A series of experiments were conducted in which PRCV (strain 91V44) was added to filtered and unfiltered fresh- and saltwater taken from the river Scheldt and the North Sea. Virus titres were then measured by TCID50-assays using swine testicle cell cultures after various incubation times. The results show that viral inactivation of PRCV in filtered seawater can be rapid, with an observed 99% decline in the viral load after just two days, which may depend on temperature and the total suspended matter concentration. PRCV degraded much slower in filtered water from the river Scheldt, taking over 15 days to decline by 99%, which was somewhat faster than the PBS control treatment (T99 = 19.2 days). Overall, the results suggest that terrestrial CoVs are not likely to accumulate in marine environments. Studies into potential interactions with exudates (proteases, nucleases) from the microbial food web are, however, recommended.

Impacts of detritivore diversity loss on instream decomposition are greatest in the tropics.

The relationship between detritivore diversity and decomposition can provide information on how biogeochemical cycles are affected by ongoing rates of extinction, but such evidence has come mostly from local studies and microcosm experiments. We conducted a globally distributed experiment (38 streams across 23 countries in 6 continents) using standardised methods to test the hypothesis that detritivore diversity enhances litter decomposition in streams, to establish the role of other characteristics of detritivore assemblages (abundance, biomass and body size), and to determine how patterns vary across realms, biomes and climates. We observed a positive relationship between diversity and decomposition, strongest in tropical areas, and a key role of abundance and biomass at higher latitudes. Our results suggest that litter decomposition might be altered by detritivore extinctions, particularly in tropical areas, where detritivore diversity is already relatively low and some environmental stressors particularly prevalent.

Sewage and sewage-contaminated environments are the most prominent sources to isolate phages against Pseudomonas aeruginosa.

BACKGROUND: P. aeruginosa is the primary source of hospital-acquired infections. Unfortunately, antibiotic resistance is growing to precariously high levels, making the infections by this pathogen life-threatening and hard to cure. One possible alternative to antibiotics is to use phages. However, the isolation of phages suitable for phage therapy- be lytic, be efficient, and have a broad host range -against some target bacteria has proven difficult. To identify the best places to look for these phages against P. aeruginosa we screened hospital sewages, soils, and rivers in two cities. RESULTS: We isolated eighteen different phages, determined their host range, infection property, and plaque morphology. We found that the sewage and sewage-contaminated environments are the most reliable sources for the isolation of Pseudomonas phages. In addition, phages isolated from hospital sewage showed the highest efficiency in lysing the bacteria used for host range determination. In contrast, phages from the river had larger plaque size and lysed bacteria with higher levels of antibiotic resistance. CONCLUSIONS: Our findings provided additional support for the importance of sewage as the source of phage isolation.

Virulence and Infectivity of UC, MD, and L Strains of Infectious Hematopoietic Necrosis Virus (IHNV) in Four Populations of Columbia River Basin Chinook Salmon.

Infectious Hematopoietic Necrosis Virus (IHNV) infects juvenile salmonid fish in conservation hatcheries and aquaculture facilities, and in some cases, causes lethal disease. This study assesses intra-specific variation in the IHNV susceptibility of Chinook salmon (Oncorhynchus tshawytscha) in the Columbia River Basin (CRB), in the northwestern United States. The virulence and infectivity of IHNV strains from three divergent virus genogroups are measured in four Chinook salmon populations, including spring-run and fall-run fish from the lower or upper regions of the CRB. Following controlled laboratory exposures, our results show that the positive control L strain had significantly higher virulence, and the UC and MD strains that predominate in the CRB had equivalently low virulence, consistent with field observations. By several experimental measures, there was little variation in host susceptibility to infection or disease. However, a small number of exceptions suggested that the lower CRB spring-run Chinook salmon population may be less susceptible than other populations tested. The UC and MD viruses did not differ in infectivity, indicating that the observed asymmetric field prevalence in which IHNV detected in CRB Chinook salmon is 83% UC and 17% MD is not due to the UC virus being more infectious. Overall, we report little intra-species variation in CRB Chinook salmon susceptibility to UC or MD IHNV infection or disease, and suggest that other factors may instead influence the ecology of IHNV in the CRB.

Characterization of a Lytic Bacteriophage against Pseudomonas syringae pv. actinidiae and Its Endolysin.

Pseudomonas syringae pv. actinidiae (Psa) is a phytopathogen that causes canker in kiwifruit. Few conventional control methods are effective against this bacterium. Therefore, alternative approaches, such as phage therapy are warranted. In this study, a lytic bacteriophage (PN09) of Psa was isolated from surface water collected from a river in Hangzhou, China in 2019. Morphologically, PN09 was classified into the Myoviridae family, and could lyse all 29 Psa biovar 3 strains. The optimal temperature and pH ranges for PN09 activity were determined as 25 to 35 ∘C and 6.0 to 9.0, respectively. The complete genome of PN09 was found to be composed of a linear 99,229 bp double-stranded DNA genome with a GC content of 48.16%. The PN09 endolysin (LysPN09) was expressed in vitro and characterized. LysPN09 was predicted to belong to the Muraidase superfamily domain and showed lytic activity against the outer-membrane-permeabilized Psa strains. The lytic activity of LysPN09 was optimal over temperature and pH ranges of 25 to 40 ∘C and 6.0 to 8.0, respectively. When recombinant endolysin LysPN09 was combined with EDTA, Psa strains were effectively damaged. All these characteristics demonstrate that the phage PN09 and its endolysin, LysPN09, are potential candidates for biocontrol of Psa in the kiwifruit industry.

Improving the Inhibitory Effect of Phages against Pseudomonas aeruginosa Isolated from a Burn Patient Using a Combination of Phages and Antibiotics.

Antibiotic resistance causes around 700,000 deaths a year worldwide. Without immediate action, we are fast approaching a post-antibiotic era in which common infections can result in death. Pseudomonas aeruginosa is the leading cause of nosocomial infection and is also one of the three bacterial pathogens in the WHO list of priority bacteria for developing new antibiotics against. A viable alternative to antibiotics is to use phages, which are bacterial viruses. Yet, the isolation of phages that efficiently kill their target bacteria has proven difficult. Using a combination of phages and antibiotics might increase treatment efficacy and prevent the development of resistance against phages and/or antibiotics, as evidenced by previous studies. Here, in vitro populations of a Pseudomonas aeruginosa strain isolated from a burn patient were treated with a single phage, a mixture of two phages (used simultaneously and sequentially), and the combination of phages and antibiotics (at sub-minimum inhibitory concentration (MIC) and MIC levels). In addition, we tested the stability of these phages at different temperatures, pH values, and in two burn ointments. Our results show that the two-phages-one-antibiotic combination had the highest killing efficiency against the P. aeruginosa strain. The phages tested showed low stability at high temperatures, acidic pH values, and in the two ointments. This work provides additional support for the potential of using combinations of phage-antibiotic cocktails at sub-MIC levels for the treatment of multidrug-resistant P. aeruginosa infections.

Pepper Mild Mottle Virus as Indicator of Pollution: Assessment of Prevalence and Concentration in Different Water Environments in Italy.

Pepper mild mottle virus (PMMoV), a plant pathogenic virus belonging to the family Virgoviridae, has been proposed as a potential viral indicator for human faecal pollution in aquatic environments. The present study investigated the occurrence, amount and diversity of PMMoV in water environments in Italy. A total of 254 water samples, collected between 2017 and 2019 from different types of water, were analysed. In detail, 92 raw sewage, 32 treated sewage, 16 river samples, 9 estuarine waters, 20 bathing waters, 67 groundwater samples and 18 drinking waters were tested. PMMoV was detected in 79% and 75% of untreated and treated sewage samples, respectively, 75% of river samples, 67% and 25% of estuarine and bathing waters and 13% of groundwater samples. No positive was detected in drinking water. The geometric mean of viral concentrations (genome copies/L) was ranked as follows: raw sewage (2.2 × 106) > treated sewage (2.9 × 105) > river waters (6.1 × 102) > estuarine waters (4.8 × 102) > bathing waters (8.5 × 101) > groundwater (5.9 × 101). A statistically significant variation of viral loads could be observed between raw and treated sewage and between these and all the other water matrices. PMMoV occurrence and viral loads did not display seasonal variation in raw sewage nor correlation with faecal indicator bacteria in marine waters and groundwater. This study represents the first report on the occurrence and quantification PMMoV in different water environments in Italy. Further studies are required to evaluate the suitability of PMMoV as a viral indicator for human faecal pollution and for viral pathogens in waters.

Occurrence of Human Enteric Viruses in Water Sources and Shellfish: A Focus on Africa.

Enteric viruses are a diverse group of human pathogens which are primarily transmitted by the faecal-oral route and are a major cause of non-bacterial diarrhoeal disease in both developed and developing countries. Because they are shed in high numbers by infected individuals and can persist for a long time in the environment, they pose a serious threat to human health globally. Enteric viruses end up in the environment mainly through discharge or leakage of raw or inadequately treated sewage into water sources such as springs, rivers, dams, or marine estuaries. Human exposure then follows when contaminated water is used for drinking, cooking, or recreation and, importantly, when filter-feeding bivalve shellfish are consumed. The human health hazard posed by enteric viruses is particularly serious in Africa where rapid urbanisation in a relatively short period of time has led to the expansion of informal settlements with poor sanitation and failing or non-existent wastewater treatment infrastructure, and where rural communities with limited or no access to municipal water are dependent on nearby open water sources for their subsistence. The role of sewage-contaminated water and bivalve shellfish as vehicles for transmission of enteric viruses is well documented but, to our knowledge, has not been comprehensively reviewed in the African context. Here we provide an overview of enteric viruses and then review the growing body of research where these viruses have been detected in association with sewage-contaminated water or food in several African countries. These studies highlight the need for more research into the prevalence, molecular epidemiology and circulation of these viruses in Africa, as well as for development and application of innovative wastewater treatment approaches to reduce environmental pollution and its impact on human health on the continent.

Complete genome sequence of Sikte (Sitke) waterborne virus, a member of the genus Tombusvirus.

The 4704-nt genome sequence of Sikte waterborne virus (SWV), determined by fragmented and primer ligated dsRNA sequencing and by direct Sanger sequencing, is linear, nonsegmented and has the five ORFs of other tombusviruses. The 5' and 3' untranslated regions (UTRs) are 150 and 335 nt long, respectively. Phylogenetic analysis of the coat protein revealed that SWV is related to CymRSV and PNSV, but that of the SWV replicase protein, the p92 readthrough protein, indicated a close relationship to CNV. These phylogenetic analyses suggest the occurrence of recombination events in SWV, as reported previously for other tombusviruses.

Persistent Detection of Cosavirus and Saffold Cardiovirus in Riachuelo River, Argentina.

Cosaviruses (CoSV) and Saffold cardiovirus (SAFV) are novel members of the Picornaviridae family. The Matanza-Riachuelo river basin covers a total area of 2200 km2 with approximately 60 km long. Its last section is called Riachuelo River. The aim of this study was to describe the circulation of both picornaviruses and their relationship with the environmental situation of the Riachuelo River using 274 samples collected from 2005 to 2015. CoSV and SAFV were investigated in samples available by two periods: 2005-2006 and 2014-2015 (103 and 101, respectively). Physicochemical and bacteriological parameters confirmed very high levels of human fecal contamination during the 11 years evaluated. CoSV was detected in 85.7% (66/77) and 65.4% (17/26) of the samples collected in 2005-2006 and 2014-2015 periods, respectively. Species A and D were identified, the first one being widely predominant: 74.1% (20/27) and 75.0% (3/4) in both periods. SAFV virus was detected in 47.1% (32/68) and 52.6% (10/19) in periods 2005-2006 and 2014-2015, respectively. SAFV-6 was the most identified genotype in the entire study, while SAFV-3 was predominant in 2005-2006. The contribution of genotypes 1, 2, 4 and 8 was minor. The high prevalence of CoSV and SAFV suggests that both viruses have been circulating in Argentina at least since 2005. Our results show that a watercourse with high rates of human fecal contamination can become a persistent source of new viruses which capacity to produce human diseases is unknown.

Prevalence and Genetic Characterization of Aichivirus in Environmental Waters in Thailand.

Aichivirus 1 (AiV-1) is an enteric virus that has been documented to be the causative agent of diarrhea in humans. It is transmitted by fecal-oral route, through person-to-person contact, consumption of contaminated food or water, or recreation of contaminated water. AiV-1 is highly prevalent in water samples and has been proposed as a potential indicator of fecal contamination in water reservoirs. This study aimed to investigate the prevalence and genetic diversity of AiV-1 in environmental water samples in Thailand. A total of 126 samples were collected monthly from November 2016 to July 2018 from various sources of environmental water including irrigation water, reservoir, river, and wastewater. The presence of AiV-1 was detected by RT-nested PCR of the 3CD region and further analyzed by phylogenetic analysis. The AiV-1 was detected in 28 out of 126 (22.2%) of tested samples. A high frequency of AiV-1 detection was in wastewater (52.4%). All 28 AiV-1 strains detected in this study belonged to the genotype B and were closely related to AiV strains detected previously in environmental waters and in humans worldwide. This study demonstrated, for the first time, the contamination of AiV-1 in various sources of water samples in Thailand and provided a better insight into the prevalence of AiV-1 in environmental waters and its potential risk of human health.

Genetic diversity of species A rotaviruses detected in clinical and environmental samples, including porcine-like rotaviruses from hospitalized children in the Philippines.

Rotaviruses are the major cause of severe acute diarrhea in infants and young children. Rotaviruses exhibit zoonosis and thereby infect both humans and animals. Viruses detected in urban rivers possibly reflect the presence of circulating viruses in the catchment. The present study investigates the genetic diversity of species A rotaviruses detected from river water and stool of hospitalized children with acute diarrhea in Tacloban City, the Philippines. Species A rotaviruses were detected by real-time RT-PCR and their genotypes were identified by multiplex PCR and sequencing of partial regions of VP7 and VP4. Rotaviruses were detected in 85.7% (30/35) of the river water samples and 62.7% (151/241) of the clinical samples. Genotypes of VP7 in the river water samples were G1, G2, G3, G4, G5, and G9, and those of VP4 were P[3], P[4], P[6], P[8], and P[13]. Genotypes of viruses from the clinical samples were G2P[4], G1P[8], G3P[8], G4P[6], G5P[6], and G9P[8]. Among those, G2P[4] in clinical samples (77.9%, 81/104) and P[4] of VP4 in river water samples (67.5%, 56/83)) were the most frequently detected rotavirus genotypes. However, G5 was the more frequently detected than G2 in the river water samples (42% vs. 13%) which may be originated from porcine rotavirus. Sequence analyses of eleven gene segments revealed one G5P[6] and two G4P[6] rotaviruses in the clinical samples, wherein, several gene segments were closely related to porcine rotaviruses. The constellation of these rotavirus genes suggests the emergence of reassortment between human and porcine rotavirus due to interspecies transmission. Although two commercial rotavirus vaccines are available now, these vaccines are designed to confer immunity against the major human rotaviruses. Constant monitoring of viral variety in populated areas where humans and domestic animals live in close proximity provides vital information related to the diversity of rotaviruses in a human population.

Quantitative Detection of Human Adenovirus and Human Rotavirus Group A in Wastewater and El-Rahawy Drainage Canal Influencing River Nile in the North of Giza, Egypt.

Environmental monitoring is critical in a developing country like Egypt where there is an insufficient framework for recording and tracking outbreaks. In this study, the prevalence of human adenovirus (HAdV), rotavirus group A (RVA) was determined in urban sewage, activated sludge, drainage water, drainage sediment, Nile water, and Nile sediment, using quantitative polymerase chain reaction (qPCR) analysis. HAdV was detected in 50% of urban sewage with viral concentrations ranging from 103 to 107 genome copies/liter (GC/L), 33% of activated sludge with viral concentrations ranging from 103 to 107 GC/kilogram (GC/kg), 95% of drainage water with viral concentrations ranging from 103 to 107 GC/L, 75% of drainage sediment with viral concentrations ranging from 103 to 107 GC/L, 50% of Nile water with viral concentrations ranging from 103 to 107 GC/L, and 45% of Nile sediment with viral concentrations ranging from 103 to 107 GC/kg. RVA was detected in 50% of urban sewage with viral concentrations ranging from 103 to 107 GC/L, 75% of activated sludge with viral concentrations ranging from 103 to 107 GC/L, 58% of drainage water with viral concentrations ranging from 103 to 107 GC/L, 50% of drainage sediment with viral concentrations ranging from 103 to 107 GC/L, and 45% of Nile water with viral concentrations ranging from 103 to 107 GC/kg. In conclusion, Abu-Rawash WWTP acts as a source of HAdV and RVA, releasing them into El-Rahawy drain then to the River Nile Rosetta branch.

G3 and G9 Rotavirus genotypes in waste water circulation from two major metropolitan cities of Pakistan.

Rotavirus A (RVA) is a diarrheal pathogen affecting children under age five, particularly in developing and underdeveloped regions of the world due to malnutrition, poor healthcare and hygienic conditions. Water and food contamination are found to be major sources of diarrheal outbreaks. Pakistan is one of the countries with high RVA related diarrhea burden but with insufficient surveillance system. The aim of this study was to gauge the RVA contamination of major open sewerage collecting streams and household water supplies in two major metropolitan cities of Pakistan. Three concentration methods were compared using RNA purity and concentration as parameters, and detection efficiency of the selected method was estimated. Water samples were collected from 21 sites in Islamabad and Rawalpindi in two phases during the year 2014-2015. Meteorological conditions were recorded for each sampling day and site from Pakistan Meteorological Department (PMD). Nested PCR was used to detect the presence of RVA in samples targeting the VP7 gene. Logistic regression was applied to assess the association of weather conditions with RVA persistence in water bodies. Statistical analysis hinted at a temporal and seasonal pattern of RVA detection in water. Phylogenetic analysis of selected isolates showed a close association of environmental strains with clinical RVA isolates from hospitalized children with acute diarrhea during the same period. This is the first scientific report cataloging the circulating RVA strains in environmental samples from the region. The study highlights the hazards of releasing untreated sewerage containing potentially infectious viral particles into collecting streams, which could become a reservoir of multiple pathogens and a risk to exposed communities. Moreover, routine testing of these water bodies can present an effective surveillance system of circulating viral strains in the population.

Molecular detection of a novel cyprinid herpesvirus in roach (Rutilus rutilus) and asp (Leuciscus aspius) showing typical signs of carp pox disease.

In the early spring of 2018, in Lake Balaton (Hungary), a roach (Rutilus rutilus) and an asp (Leuciscus aspius) were found in an fish trap at the outlet of the river Sió showing typical signs of the so-called carp pox disease, such as foci of epidermal hyperplasia on the head and the whole body surface, including the fins. Molecular tests revealed the presence of the DNA of an unknown fish herpesvirus. Three genes encoding the DNA-dependent DNA polymerase, major capsid protein and ATPase subunit of terminase were amplified and sequenced from the alloherpesviral genome. The gene sequences of the viruses obtained from the two different fish species shared 94.4% nucleotide sequence identity (98.1% amino acid sequence identity), suggesting that they belong to the same virus species. Phylogenetic analysis based on the DNA polymerase (and the concatenated sequences of the amplified genes, as well) implied that the detected virus belongs to the genus Cyprinivirus within the family Alloherpesviridae. The sequences of the novel alloherpesvirus diverge from those of the five cyprinivirus species described previously, so it putatively represents the sixth virus species in the genus.