Tracking Viruses That Contaminate Environments

Transcription

1 Tracking Viruses That Contaminate Environments Using PCR to track stable viruses provides an effective means for monitoring water quality for environmental contaminants Rosina Girones iruses are found in virtually all natural aquatic environments, where V they reach concentrations of approximately 10 8 virus-like particles (VLP) per ml, which are higher than those at which bacteria are found. Even in highly industrialized countries, viruses that infect humans are prevalent throughout the environment, causing public health problems and leading to substantial economic losses. PCR provides sensitive, rapid, and quantitative analytical tools for studying such viruses, including emergent pathogens. These tools can be effective for evaluating the microbiological quality of food and water, for tracing fecal contamination, and for assessing virus removal efficiencies in wastewater treatment plants. Many orally transmitted viruses produce primarily subclinical infections, causing symptoms only in a small proportion of the population. PCR and other rapid quantitative analytical tools can be effective for evaluating the microbiological quality of food and water, for tracing fecal contamination, and for assessing virus removal efficiencies in wastewater treatment plants. Potentially pathogenic viruses in urban sewage include human adenoviruses, human polyomaviruses, several hepatitis viruses, enteroviruses, noroviruses, rotavirus, and astroviruses. Properly controlling viral diseases will depend on regulatory authorities developing surveillance systems with which to monitor and more effectively reduce established and perhaps prevent emergent viral contaminants. However, some viruses may give rise to lifethreatening conditions, such as acute hepatitis infections in adults, as well as severe gastroenteritis in small children and the elderly. The development of disease is related to the infective dose of the viral agent, the age, health, immunological, and nutritional status of the infected individual and the availability of health care. Several viruses that cause gastroenteritis or hepatitis in humans are excreted in high concentrations in feces, while other disease-causing viruses are excreted in lower concentrations in feces or urine. Because such viruses also often infect otherwise healthy individuals, they contribute to the high viral loads that can be detected in urban sewage. Those viruses that are transmitted via contaminated food or water typically are stable because they lack lipid envelopes that render other viruses fragile to environmental agents. Some viruses, such as human polyomaviruses and adenoviruses, infect humans during childhood, thereby establishing persistent infections. In the case of some types of adenovirus, which infects the respiratory tract, viral particles may be excreted in feces for months or even years. Which Viruses Are Commonly Detected in Urban Sewage? Nested-PCR and nucleotide sequence analyses of sewage samples unveiled a large diversity of viruses that are responsible for clinical and subclinical infections in urban populations (see table). Such analyses also help investigators to characterize these viruses genetically, even those that cannot be efficiently cultured in cell lines, including Rosina Girones is tenured professor in the Department of Microbiology, Faculty of Biology, University of Barcelona, Barcelona, Spain. rgirones@ub.edu Volume 1, Number 1, 2006 / Microbe Y 19

2 Table Principal groups of viruses detected in urban sewage and associated diseases. Family Genus Principal viruses Associated diseases Adenoviridae, Mastadenovirus Human Adenovirus A-F Ocular infections, respiratory, urinary, gastroenteritis Polyomaviridae, Polyomavirus JCV Progressive multifocal Leucoencephalopaty BKV Nephropathy associated with polyomaviruses Picornaviridae, Enterovirus Poliovirus Enteroviruses A-D Infections of the CNS, ocular and respiratory systems, myocarditis, diarrhea, congenital heart abnormalities Parechovirus Human Parechovirus Gastroenteritis, respiratory infections Hepatovirus Hepatitis A virus Hepatitis A Caliciviridae Norovirus Norwalk-like viruses Gastroenteritis Sappovirus Sapporo virus Gastroenteritis urban sewage in areas that were considered nonendemic. Human adenoviruses are found in nearly 100% of urban sewage samples that we test, including from cities in Africa, the United States, and Europe. Such viruses also are commonly detected in shellfish, including samples that met current safety standards, which are based on levels of fecal bacteria. Officials at the U.S. Environmental Protection Agency recently added adenoviruses to the list of potentially dangerous contaminants, a move that was based on their high prevalence in urban sewage, ability to cause disease, and to their resistance to water purification treatments, particularly those involving UV irradiation. The human polyomaviruses JC and BK are also detected in most urban sewage samples that we have analyzed. Hepeviridae Hepevirus Hepatitis E virus Hepatitis E Reoviridae Rotavirus Rotavirus groups A and B Gastroenteritis Astroviridae Mamastrovirus Human Astrovirus Gastroenteritis the JC virus (JCV), which is a polyomavirus, hepatitis E virus (HEV), and noroviruses. Genomic amplification techniques also generate more comprehensive information on HAV strains, rotaviruses, and those adenoviruses that remain difficult to isolate in cell cultures. Potentially pathogenic viruses in urban sewage include human adenoviruses and polyomaviruses, the most abundant and commonly found, and enteroviruses, noroviruses, rotavirus, and astroviruses. Hepatitis A virus (HAV) levels vary geographically, but this virus frequently can be detected in urban sewage throughout the year, even in supposedly nonendemic countries with very low numbers of clinical cases. HEV, like HAV, is particularly abundant in countries where sanitation is poor. However, we detected autochthonous strains of HEV in urban sewage in several highly industrialized countries, along with cases of sporadic acute hepatitis caused by these nonimported strains. Strains of HEV also can be detected in sewage from slaughterhouses where pigs are killed and in Diseases Associated with Environmental Viruses Viruses found in the environment are associated with a wide diversity of diseases and symptoms, including meningitis, paralysis, respiratory diseases, diarrhea and vomiting, myocarditis, congenital heart anomalies, acute hepatitis, ocular infections, and possibly cancer. JCV infections, for example, can lead to progressive multifocal leucoencephalopathy (PML). The relationship between colorectal cancer and the human polyomavirus JCV is not fully understood. The most frequently occurring virus-associated pathology remains gastroenteritis, a leading cause of mortality not only in developing countries but also in highly industrialized countries, particularly among the elderly, followed by acute hepatitis. The human polyomaviruses, as well as some adenoviruses excreted in urine or feces, may cause threatening diseases in immunocompromised individuals. Highly efficient sewage treatment facilities can greatly reduce transmissions of bacterial pathogens through the water supply, but typically are not so effective against viruses, which thus remain the principal cause of outbreaks attributable to contaminated water supplies. One notable infectious outbreak associated with 20 Y Microbe / Volume 1, Number 1, 2006

3 For Girones, Research and Life Are Quests in Parallel For Rosina Girones, research is like life itself. In life, we have many questions to answer, she says. We want to know how things are organized around us, how things are working, what people who are important to us are thinking, what we want to do with our lives and how to do it. Some of us also do research, she continues. For me, it is a way to live. Sometimes the answers are not complete. But what is valuable in life, as well as in the laboratory, is learning interesting things and establishing contacts that are positive inputs. Girones teaches Virology in the Department of Microbiology at the University of Barcelona, where her research focuses on the study of water- and foodborne viruses. She frequently collaborates with the Environmental Microbiology group there, and members of her lab are also involved in developing and applying molecular techniques to study adenoviruses and other viruses in the environment, and to identify emergent viral pathogens such as hepatitis E and polyomavirus. There are very clear applied implications in issues related to population safety in improving the control of the microbiological quality of water and food, Girones says. Further, by collaborating with private companies and research foundations, she would like to develop new techniques by which purification plants could better remove viruses from municipal drinking water supplies. Girones was born in Barcelona, where her father worked as an economist for a local company and her mother was a homemaker whose interests included painting and writing. My parents have been deeply supportive and have shown much respect for my decisions, she says. One of the most important things I learned from them was the importance of being positive in the lives of those you are close to, and to have a sense of humor. At 17, an insensitive comment from the director of her high school strengthened her resolve to pursue an ambitious career. Although she was a hard-working, high-achieving, and seriousminded student, he asked: What do you want to be when you are older secretary to a minister? I was very disappointed with the difference in his expectations between boys and girls, she recalls. It must have impressed me a lot because I did not forget it. I think that [this incident] probably helped me to realize that we must work a lot to get some of the things that are important to us. Girones chose a career in science because to learn what happens around us is something I have found deeply stimulating ever since I was a child. I very much like nature. Even though I work in Barcelona, I live in a small town 5,000 inhabitants where life is more relaxed and I can feel close to nature, where we belong, walk through the country and the forest, and feel the different seasons. She received both her undergraduate and doctoral training at the University of Barcelona, and, in 1987, traveled to the United States for postdoctoral work at the National Institute of Allergy and Infectious Diseases. There she studied hepatitis B under the direction of Roger Miller, in the hepatitis viruses section. This was a wonderful period in my life, where I learned a lot about molecular virology, and developed friendships and collaborations for future research projects that have been lasting and even improving during more than 15 years, she says. It really is a great place. Also, I enjoyed the country very much. She continues, It was a very nice experience to live in a place with a really diverse population and diverse cultures, and to have friends from very different areas of the world. Girones is amicably divorced, with two daughters, Rosina, 15, and Marta, 10. She describes them as a great source of energy and stimuli for enjoying and learning about sharing, and about ourselves. At 47, she says, It looks to me that I am just starting everything. Marlene Cimons Marlene Cimons is a freelance writer in Bethesda, Md. environmental fecal contamination is the 1988 Shanghai outbreak, with 300,000 cases of hepatitis A and 25,000 of viral gastroenteritis, following consumption of shellfish cultured in a local estuary beset with fecal contamination. Meanwhile, norovirus outbreaks are also linked to contamination of water and food supplies, including an outbreak in the Netherlands in 2002 when gastroenteritis afflicted about 100 primary schoolchildren who played in a recre- Volume 1, Number 1, 2006 / Microbe Y 21

4 FIGURE 1 Electron micrograph of human polyomaviruses cultured in human glial cells (SVG) isolated from the urine of a healthy individual infected with BKV and JCV. Excreted strains considered archetypal types of JCV and BKV are very difficult to isolate and produce stable infections in cells. ational water fountain. HEV is a cause also of very important outbreaks such as the 1991 HEV outbreak in Kanpur that affected 79,000 people; or more recently, the outbreak in the Sudan region of Darfur during 2004 that led to 6,861 cases and 87 deaths from HEV. Environmental Noro- and Polyomaviruses Are Associated with Several Emergent Diseases Not only are waterborne infectious diseases an important cause of mortality and morbidity worldwide, but the spectrum and incidence of such diseases are increasing. Three groups of viruses that figure in these increases are noroviruses, human polyomaviruses, and HEV. The genus Norovirus belongs to the Caliciviridae family, which contains four genetically and antigenically diverse virus groups that differ in host preference. The human caliciviruses are distributed in two genera, Norovirus and Sapovirus. Norovirus (previously Norwalk-like viruses) and other enteric caliciviruses cause acute gastroenteritis in humans. Although asymptomatic infections are common, noroviruses are the most frequent cause of gastroenteritis in all age groups, albeit with higher incidences in young children. Noroviruses are responsible for sporadic gastroenteritis outbreaks in schools, hospitals, homes for the elderly, hotels, and on cruise ships. Identifying noroviruses can be problematic in part, because these viruses still cannot be cultured in cell lines, and also because of their high degree of genetic diversity. However, they can be detected in environmental and food samples by doing PCR assays with highly degenerate primers, then confirming results by hybridizing amplified sequences with other appropriate probes or by analyzing nucleotide sequences. This genus is distributed in two genogroups that are divided into genotypes, within which new variants and recombinant strains are being identified. The polyomaviruses belong to the Polyomaviridae, a family of small icosahedral viruses, whose members carry a circular, supercoiled double-stranded DNA genome (Fig. 1). Genes that are expressed soon after these viruses infect cells include the T and t antigens, which are potentially oncogenic multifunctional proteins. The polyomaviruses JCV and BKV infect humans, which are the only known hosts and in which these viruses produce severe clinical syndromes, primarily among immunocompromised individuals. However, these polyomaviruses consistently infect approximately 50 90% of the adult population without causing symptoms, suggesting an extended period of coevolution between these viruses and their human host. JCV infections can lead to progressive multifocal leukoencephalopathy (PML), a demyelinating disease that causes death in approximately 4% of all AIDS patients. PML has also been identified as a risk to consider in clinical trials involving promising experimental treatments for either multiple sclerosis or Crohn s disease. JCV infections in humans also are asso- 22 Y Microbe / Volume 1, Number 1, 2006

5 ciated with a higher incidence of several cancers, most notably brain tumors, but also B-cell lymphoma and colorectal cancer. JCV is also linked to chromosomal instability. Furthermore, these viruses induce brain tumors in hamsters and nonhuman primates. Meanwhile, BKV infections may lead to kidney diseases and syndromes, including hemorrhagic cystitis, renal allograft loss, and ureteric stenosis in transplant patients, especially following kidney or bone marrow transplantations. BKV seems to infect humans during infancy and is excreted in urine with lower frequency than the viruses in JCV infections. We analyzed urban sewage looking for human polyomaviruses in Washington, D.C., and in other cities in Spain, France, Sweden, Greece, South Africa, and Egypt. We have constructed a phylogenetic tree showing the genetic relatedness of JCV samples from around the world (Fig. 2). Remarkably, we tested only one sample that was negative for JCV (98% positive samples), while 90% of our tested samples were positive for BKV. Moreover, these same samples test positive for human adenoviruses, demonstrating the applicability of molecular assays for identifying adenoviruses and polyomaviruses in samples containing human fecal contaminants, regardless of where they were collected. The high prevalence of human polyomavirus in urban sewage, as well as the stability and resistance of such viral particles to acidic ph, led us to suggest that the gastrointestinal tract is a major route through which viruses enter host organisms. FIGURE 2 This phylogenetic tree includes 22 JCV reference strains (in red): 123B, 124A, Mad-1 and 402 belong to European Types 1, and belongs to Type 6, a typical strain from West Africa. Strains 311B, 312A and 308A belong to Afro-Asiatic Type B, 223B, GS/K and GS/B belong to Type 2B typical from Eurasia. Strains 230D, 231D and 232D belong to the South Indian Type 2D. Tokyo-1, 224A, 228C, 229C, 225A and 226A belong to Afro-Asiatic Type 2, characteristic of Japanese and Native American individuals. Tai-3 belongs to Afro-Asiatic Type 7, characteristic of South Asia. The other JCV strains were isolated from sewage samples from various cities, except those designated CSF, which were obtained from cerebrospinal fluid samples obtained from hospitals in Barcelona. The NJ tree was constructed using 461 nucleotides (nt 2177 to nt 2637) from the intergenic regions of JCV genomes. The bootstrap confidence levels obtained for 1000 replicates are also shown (only significant values are indicated). (BCN, Barcelona; WASH, Washington). Hepatitis E Virus Also Is Considered Emergent HEV belongs to the Hepeviridae family, whose members contain RNA lineal genomes, and is considered an emergent virus in industrialized countries. Only one serotype is known, although the known strains are distributed in at least four genogroups. While HEV infections often are subclinical, this virus is also the principal cause of sporadic acute hepatitis in many countries with low sanitation levels. In the general population, the lethality index for HEV is 1% because it is associated with liver failure. The death rate nears 20% for HEV-infected pregnant woman, especially during the third trimester. About 80% of U.S. pigs older than 3 months are seropositive for HEV, according to X. J. Meng and collaborators at the Virginia Polytechnic Institute and State University, Blacks- Volume 1, Number 1, 2006 / Microbe Y 23

6 FIGURE 3 We also detected HEV RNA in 53.8% of urban sewage samples collected in Barcelona, and 20-25% of a few samples from France and United States. The stability of the HEV particle in sewage is lower than that of poliovirus type 1, although HEV RNA remains detectable after incubating for one month in urban sewage at 20 C. Based on these samples, we constructed a phylogenetic tree showing the genetic relatedness of these and other strains (Fig. 3). Although many U.S. and European experts regard HEV infections as imported, several autochthonous, or native, strains have been identified, suggesting that endemic infections persist in highly industrialized countries and emphasizing a need for sensitive diagnostic kits and environmental contamination controls that target this virus. Autochthonous HEV strains excreted and detected in urban sewage of industrialized countries also produce sporadic clinical cases. This neighbor-joining tree shows the phylogenetic relationships among representative sequences found in Barcelona (in bold, black, and other colors) and additional isolates from clinical samples in both endemic and non-endemic areas. The bootstrap confidence levels obtained for 1,000 replicates are also shown (only values greater than 60 are indicated). VH represents clinical samples. Other colors in bold letters correspond to strains identified in the urban sewage of Washington (blue) or Barcelona (red). Por1 and Sw are swine strains from the areas of Barcelona (Spain) and the United States, respectively. burg, and this virus is found throughout the global swine population. In Catalonia, Spain, for example, HEV seroprevalence in pigs is approximately 20%. Moreover, we determined that the genomic regions of swine-associated HEV strains are closely related not only to those identified in serum samples from patients with acute hepatitis E, but also to those found in urban sewage samples that were collected in the same geographic area. Investigators in Japan report that humans can become infected with hepatitis E after consuming wild boar and venison. Antibodies against HEV are also found in rats, chickens (avian HEV), cows, goats, cats, and dogs. However, there is no proof that these animals are reservoirs for viral strains that infect humans. Human Viruses Are Primary Indicators of Contamination Current safety standards for determining food and water quality typically do not specify what level of viruses would be considered acceptable. In general, viruses are more stable than common bacterial indicators in the environment, and enteric viruses remain stable for several months in fresh water and longer when associated with solids. Nonetheless, molecular techniques show that enterovirus levels (previously regarded in some regulations as cultivable enteric viruses) do not correlate with those of other viruses. Although some groups of bacteriophages have been suggested as alternative indicators, phage levels do not correlate with the presence of other viruses, including those that are pathogens for humans. Human adenoviruses appear to be effective indicators of viral contaminants of human origin. For one thing, they are prevalent and very stable; for another, they are considered humanspecific and are not detected in animal wastewaters or slaughterhouse sewage. A high proportion of environmental and shellfish samples containing other viral pathogens also contain adenoviruses, which typically are the most abundant viruses in such samples. Moreover, 24 Y Microbe / Volume 1, Number 1, 2006

7 adenoviruses are regularly identified in fecal contamination. As early as 1994, our group began using PCR to determine levels of human-associated or other adenoviruses in specific foods, such as shellfish, in water, and in other environmental samples to trace sources of fecal contamination. Urban sewage typically contains 10 5 enteroviruses and 10 7 human adenoviruses per liter. During the primary treatment phase, settling and retention, few viruses are removed. However, the secondary treatment phase involving activated sludge effectively removes 90 99% of viruses. Treating effluents by chlorination further reduces, but does not eliminate, remaining viruses. Indeed, viruses commonly are detected in wastewater treatment plant effluents as well as in biosolids. For instance, with real-time PCR analysis, we detected human adenoviruses in 100% of the samples of urban sewage that we analyzed, typically at levels of genome copies per ml. When we analyzed treated effluents, the virus concentrations were lower, at 10 5 genome copies per liter. JCV also was present at genomes per ml of urban sewage, which fell to 10 4 per liter in the treated effluents analyzed. Specific Animal Viruses as Indicators of Fecal Contamination Vertebrates are infected by adenoviruses species and probably also by polyomaviruses, which are specific for the animal hosts, making these animal viruses reasonable molecular targets to use when tracing sources of fecal contamination. Other investigators have used either porcine teschovirus or bovine enterovirus. We would suggest also using animal and human DNA viruses because they are highly stable, easy to quantify, abundantly excreted, and associated with specific host species. In our hands, porcine adenoviruses and bovine polyomaviruses prove very useful indicators of porcine and bovine fecal FIGURE 4 Phylogenetic reconstruction of human and porcine adenoviruses based on 310pb amplicons of the hexon gene. Porcine strains were isolated from environmental samples (Por1 9); reference strains (HAd2, HAd3, HAd12, HAd40 and PAV A) are also included. The neighbor-joining tree was constructed using the Phyllip 3.1 program. The phylogenetic distance observed between porcine and human strains supports the use of PCR-based assays in determining whether the source of fecal contamination was of human or porcine origin. contamination, respectively. We detect both viral groups in 100% of sewage samples from slaughterhouses and also in river water samples near a diversity of farms. We plan to refine our assays and to learn more about where these viruses are distributed. Being able to identify sources of fecal contamination quickly and accurately has important implications for protecting environmental water quality and monitoring for potential zoonotic pathogens. Properly controlling viral contamination will depend on regulatory authorities choosing and standardizing effective viral parameters, and then developing surveillance system with which to monitor and more effectively reduce established and perhaps prevent emergent viral diseases. Volume 1, Number 1, 2006 / Microbe Y 25