Eukaryotic Viruses in Wastewater Samples from the United States

Human fecal matter contains a large number of viruses, and current bacterial indicators used for monitoring water quality do not correlate with the presence of pathogenic viruses. Adenoviruses and enteroviruses have often been used to identify fecal pollution in the environment; however, other viruses shed in fecal matter may more accurately detect fecal pollution. The purpose of this study was to develop a baseline understanding of the types of viruses found in raw sewage. PCR was used to detect adenoviruses, enteroviruses, hepatitis B viruses, herpesviruses, morbilliviruses, noroviruses, papillomaviruses, picobirnaviruses, reoviruses, and rotaviruses in raw sewage collected throughout the United States. Adenoviruses and picobirnaviruses were detected in 100% of raw sewage samples and 25% and 33% of final effluent samples, respectively. Enteroviruses and noroviruses were detected in 75% and 58% of raw sewage samples, respectively, and both viral groups were found in 8% of final effluent samples. This study showed that adenoviruses, enteroviruses, noroviruses, and picobirnaviruses are widespread in raw sewage. Since adenoviruses and picobirnaviruses were detected in 100% of raw sewage samples, they are potential markers of fecal contamination. Additionally, this research uncovered previously unknown sequence diversity in human picobirnaviruses. This baseline understanding of viruses in raw sewage will enable educated decisions to be made regarding the use of different viruses in water quality assessments.Millions of viruses and bacteria are excreted in human fecal matter (5, 17, 82), and current methods of sewage treatment do not always effectively remove these organisms (74, 76-78). The majority of treated wastewater, as well as untreated sewage, drains into the marine environment (1) and has the potential to threaten environmental (e.g., nutrients and chemicals) (45) and public (e.g., pathogen exposure via swimming and seafood consumption) (1, 24, 28, 29, 33, 44, 57, 63) health. Currently, the U.S. Environmental Protection Agency (EPA) mandates the use of bacterial indicators such as fecal coliforms and enterococci to assess water quality (75). Although monitoring of these bacteria is simple and inexpensive, it has been shown that fecal-associated bacteria are not ideal indicators of fecal pollution.Since fecal-associated bacteria are able to live in sediments in the absence of fecal pollution (18, 32, 55), their resuspension into the water column can result in false-positive results and mask correlations between their concentrations and the extent of recent fecal pollution. Another unfavorable characteristic of current bacterial indicators is their inability to predict or correlate with the presence of pathogenic viruses (25, 40, 41, 64, 80). Human-pathogenic viruses associated with feces are generally more robust than enteric bacteria and are not as easily eliminated by current methods of wastewater treatment (43, 80). For example, adenoviruses are more resilient to tertiary wastewater treatment and UV disinfection than are bacterial indicators of fecal pollution (74). Since bacterial indicators cannot accurately depict the risks to human health from fecal pollution, several studies have proposed the use of a viral indicator of wastewater contamination (35, 41, 61).While it is impractical to monitor the presence of all viral pathogens related to wastewater pollution, the development of an accurate viral indicator of sewage contamination is needed for enhanced water quality monitoring. Enteric viruses (including viruses belonging to the families Adenoviridae, Caliciviridae, Picornaviridae, and Reoviridae) are transmitted via the fecal-oral route and are known to be abundant in raw sewage. These viruses have been used to identify fecal pollution in coastal environments throughout the world (27, 35, 39, 40, 48, 50, 56, 57, 63, 64, 67-69, 71, 80). To determine which viruses are effective indicators of fecal pollution, it is first necessary to establish a broad, baseline understanding of the many diverse groups of eukaryotic viruses in raw sewage. Several studies have identified adenoviruses, noroviruses, reoviruses, rotaviruses, and other enteroviruses (e.g., polioviruses, coxsackie viruses, and echoviruses) in raw sewage in Australia, Europe, and South Africa (30, 47, 58, 76-78). However, no broad baseline data on the presence of eukaryotic viruses in raw sewage in the United States currently exist.This study determined the presence of 10 viral groups (adenoviruses, enteroviruses, hepatitis B viruses, herpesviruses, morbilliviruses, noroviruses, papillomaviruses, picobirnaviruses, reoviruses, and rotaviruses) in raw sewage samples collected throughout the United States. All viral groups that were detected in raw sewage were then examined further to determine if they were also present in final treated wastewater effluent. These 10 viral groups were chosen because of their potential to be transmitted via the fecal-oral route, suggesting that they might be found in raw sewage. Many of these viruses (excluding adenoviruses, enteroviruses, noroviruses, reoviruses, and rotaviruses) have not been studied in sewage despite their likely presence. Picobirnaviruses have been detected in individual fecal samples (12, 70, 79, 82); however, their presence has never been analyzed in collective waste, nor have they been proposed to be potential markers of fecal pollution. This study identified potential viral indicators of fecal pollution and will have important applications to water quality monitoring programs throughout the country.     

Pepper Mild Mottle Virus as an Indicator of Fecal Pollution

Accurate indicators of fecal pollution are needed in order to minimize public health risks associated with wastewater contamination in recreational waters. However, the bacterial indicators currently used for monitoring water quality do not correlate with the presence of pathogens. Here we demonstrate that the plant pathogen Pepper mild mottle virus (PMMoV) is widespread and abundant in wastewater from the United States, suggesting the utility of this virus as an indicator of human fecal pollution. Quantitative PCR was used to determine the abundance of PMMoV in raw sewage, treated wastewater, seawater exposed to wastewater, and fecal samples and/or intestinal homogenates from a wide variety of animals. PMMoV was present in all wastewater samples at concentrations greater than 1 million copies per milliliter of raw sewage. Despite the ubiquity of PMMoV in human feces, this virus was not detected in the majority of animal fecal samples tested, with the exception of chicken and seagull samples. PMMoV was detected in four out of six seawater samples collected near point sources of secondary treated wastewater off southeastern Florida, where it co-occurred with several other pathogens and indicators of fecal pollution. Since PMMoV was not found in nonpolluted seawater samples and could be detected in surface seawater for approximately 1 week after its initial introduction, the presence of PMMoV in the marine environment reflects a recent contamination event. Together, these data demonstrate that PMMoV is a promising new indicator of fecal pollution in coastal environments.Existing wastewater treatment practices are not always effective at removing the large number of pathogens (bacteria, protists, and viruses) present in human feces (17, 42, 47-49, 51). Therefore, wastewater discharges into the environment can have a negative impact on human health. Recreational waters throughout the United States are monitored for the presence of fecal pollution as a means of limiting public exposure to pathogens in areas impacted by wastewater discharges (44). The presence of pathogenic viruses in aquatic environments is an important parameter to consider in the evaluation of water quality. However, the bacterial indicators currently used to detect fecal contamination, such as fecal coliforms and enterococci, often do not correlate with the presence of feces-associated viruses and other pathogens (5, 10, 26, 33, 37, 51). In response, several researchers have proposed the use of viral indicators as a more effective method for monitoring wastewater contamination and the associated risks to public health (11, 14, 31).To date, the majority of the proposed viral indicators of fecal pollution are enteric viruses transmitted via the fecal-oral route (4). Enteric viruses present in raw sewage (including members of the families Adenoviridae, Caliciviridae, Picornaviridae, and Reoviridae and of the genus Anellovirus) have been used in several previous studies to identify fecal pollution in the environment (7, 8, 11, 12, 13, 18, 19, 27, 28, 32-36, 38, 50, 51). Of the enteric viruses that have been used as indicators, only the adenoviruses were ubiquitously found in raw sewage samples collected throughout the United States (41). Picobirnaviruses and Torque teno virus are abundant in raw sewage from some regions and thus have also been proposed as indicator viruses (15, 41). However, one potential problem with the use of human viruses as indicators is that their abundance in wastewater depends on the degree of infection and shedding in the human population at any given time.In addition to viruses infecting humans, other viruses shed in feces may be useful for indicating wastewater pollution. The plant pathogen Pepper mild mottle virus (PMMoV) was the most abundant virus found in a metagenomic survey of RNA viruses from human feces (52). PMMoV is a positive-sense, single-stranded RNA virus that belongs to the Tobamovirus genus and infects hot, bell, and ornamental peppers (Capsicum spp.) (9). The nonenveloped, rod-shaped PMMoV virions are extremely stable (9) and have been demonstrated to retain their infectivity for plants after passage through the human gut (52). PMMoV originates from processed pepper products (e.g., hot sauce and curry) and is excreted in human feces at concentrations of 1 million to 1 billion viruses per g (dry weight) (52). Since the presence of PMMoV in human feces is dietary in origin, this plant pathogen may be more abundant in the healthy human population than viruses that cause human disease.This study analyzed the presence of PMMoV in raw sewage and treated wastewater samples collected from wastewater treatment facilities throughout the coastal United States. To determine if PMMoV is a human-specific indicator useful for tracking the source of fecal pollution, fecal samples from numerous animals were tested for this virus. Finally, the presence of PMMoV in marine environments exposed to wastewater was determined and compared to that of other microbial indicators. The results of this work demonstrate that PMMoV is a promising indicator of fecal pollution.     

Investigation of Clade B New World Arenavirus Tropism by Using Chimeric GP1 Proteins

Clade B of the New World arenaviruses contains both pathogenic and nonpathogenic members, whose surface glycoproteins (GPs) are characterized by different abilities to use the human transferrin receptor type 1 (hTfR1) protein as a receptor. Using closely related pairs of pathogenic and nonpathogenic viruses, we investigated the determinants of the GP1 subunit that confer these different characteristics. We identified a central region (residues 85 to 221) in the Guanarito virus GP1 that was sufficient to interact with hTfR1, with residues 159 to 221 being essential. The recently solved structure of part of the Machupo virus GP1 suggests an explanation for these requirements.Arenaviruses are bisegmented, single-stranded RNA viruses that use an ambisense coding strategy to express four proteins: NP (nucleoprotein), Z (matrix protein), L (polymerase), and GP (glycoprotein). The viral GP is sufficient to direct entry into host cells, and retroviral vectors pseudotyped with GP recapitulate the entry pathway of these viruses (5, 13, 24, 31). GP is a class I fusion protein comprising two subunits, GP1 and GP2, cleaved from the precursor protein GPC (4, 14, 16, 18, 21). GP1 contains the receptor binding domain (19, 28), while GP2 contains structural elements characteristic of viral membrane fusion proteins (8, 18, 20, 38). The N-terminal stable signal peptide (SSP) remains associated with the mature glycoprotein after cleavage (2, 39) and plays a role in transport, maturation, and pH-dependent fusion (17, 35, 36, 37).The New World arenaviruses are divided into clades A, B, and C based on phylogenetic relatedness (7, 9, 11). Clade B contains the human pathogenic viruses Junin (JUNV), Machupo (MACV), Guanarito (GTOV), Sabia, and Chapare, which cause severe hemorrhagic fevers in South America (1, 10, 15, 26, 34). Clade B also contains the nonpathogenic viruses Amapari (AMAV), Cupixi, and Tacaribe (TCRV), although mild disease has been reported for a laboratory worker infected with TCRV (29).Studies with both viruses and GP-pseudotyped retroviral vectors have shown that the pathogenic clade B arenaviruses use the human transferrin receptor type 1 (hTfR1) to gain entry into human cells (19, 30). In contrast, GPs from nonpathogenic viruses, although capable of using TfR1 orthologs from other species (1), cannot use hTfR1 (1, 19) and instead enter human cells through as-yet-uncharacterized hTfR1-independent pathways (19). In addition, human T-cell lines serve as useful tools to distinguish these GPs, since JUNV, GTOV, and MACV pseudotyped vectors readily transduce CEM cells, while TCRV and AMAV GP vectors do not (27; also unpublished data). These properties of the GPs do not necessarily reflect a tropism of the pathogenic viruses for human T cells, since viral tropism is influenced by many factors and T cells are not a target for JUNV replication in vivo (3, 22, 25).     

Reassortment between Avian H5N1 and Human H3N2 Influenza Viruses in Ferrets: a Public Health Risk Assessment

This study investigated whether transmissible H5 subtype human-avian reassortant viruses could be generated in vivo. To this end, ferrets were coinfected with recent avian H5N1 (A/Thailand/16/04) and human H3N2 (A/Wyoming/3/03) viruses. Genotype analyses of plaque-purified viruses from nasal secretions of coinfected ferrets revealed that approximately 9% of recovered viruses contained genes from both progenitor viruses. H5 and H3 subtype viruses, including reassortants, were found in airways extending toward and in the upper respiratory tract of ferrets. However, only parental H5N1 genotype viruses were found in lung tissue. Approximately 34% of the recovered reassortant viruses possessed the H5 hemagglutinin (HA) gene, with five unique H5 subtypes recovered. These H5 reassortants were selected for further studies to examine their growth and transmissibility characteristics. Five H5 viruses with representative reassortant genotypes showed reduced titers in nasal secretions of infected ferrets compared to the parental H5N1 virus. No transmission by direct contact between infected and naïve ferrets was observed. These studies indicate that reassortment between H5N1 avian influenza and H3N2 human viruses occurred readily in vivo and furthermore that reassortment between these two viral subtypes is likely to occur in ferret upper airways. Given the relatively high incidence of reassortant viruses from tissues of the ferret upper airway, it is reasonable to conclude that continued exposure of humans and animals to H5N1 alongside seasonal influenza viruses increases the risk of generating H5 subtype reassortant viruses that may be shed from upper airway secretions.Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype have caused devastating outbreaks in avian species during the past decade. After emerging in the Guangdong province of China in 1996, H5N1 viruses have extended their geographic distribution from Asia into Europe and Africa (45, 51). Sporadic transmission of H5N1 viruses from infected birds to humans has resulted in over 380 laboratory-confirmed infections and a case fatality rate of ∼60% since 2003 (48). Currently circulating H5N1 viruses lack the ability to undergo efficient and sustained transmission among humans although instances of limited human-to-human transmission have been reported (13, 41). If H5N1 viruses were to acquire genetic changes that confer efficient transmissibility among humans, then another pandemic would likely occur.The pandemics of 1957 and 1968 highlight the importance of genetic reassortment between avian and human influenza viruses as a mechanism for the generation of human pandemic strains (15, 46, 47). The structural separation of the influenza virus genome into eight independent genes allows formation of hybrid progeny viruses during coinfections. The 1957 H2N2 and 1968 H3N2 pandemic viruses acquired the hemagglutinin (HA) and PB1 genes, with or without the neuraminidase (NA) gene, respectively, from an avian virus progenitor (14, 33). The remaining genes of these pandemic reassortants were derived from a contemporary human virus (14, 33). The host species in which such human pandemic strains were generated by reassortment between human and avian viruses is not known. However, coinfection of the same cell with both human and avian viruses must have occurred, even though human and avian influenza viruses have preferences for different sialic acid receptor structures present on cell surface glycoproteins and glycolipids (20, 30). The HA of human viruses preferentially binds α(2,6)-linked sialic acids while that of avian viruses preferentially bind α(2,3)-linked sialic acids (3, 12). Cells possessing both of these receptors could support coinfection of avian and human viruses, leading to reassortment.Human respiratory tract epithelial cells can possess surface glycans with α(2,3)- and α(2,6)-linked sialic acids and as such represent a potential host for the generation of avian-human reassortant viruses (24, 35). The general distribution of surface α(2,3)- and α(2,6)-linked sialic acids varies among cells of the human upper and lower respiratory tracts, which are anatomically separated by the larynx. Recent studies have shown that α(2,3)-linked sialic acids are present in tissues of the human lower respiratory tract (i.e., lung alveolar cells) (24, 35) as well as tissues of the human upper respiratory tract (24). Consistent with these findings, HPAI H5N1 viruses have been shown to attach to and infect tissues belonging to the lower respiratory tract (i.e., trachea, bronchi, and lung) (5, 25, 35, 40, 42, 43) as well as tissues belonging to the upper respiratory tract (i.e., nasopharyngeal, adenoid, and tonsillar) (25). Glycans with α(2,6)-linked sialic acids are more widespread on epithelial cells of the upper airways than lung alveoli (24, 35). In accordance, human seasonal influenza viruses preferentially attach to and infect cells of the upper respiratory tract (6, 25, 35, 43). If cells with both types of receptors are present in the human respiratory tract, simultaneous infection of a person with both human and avian viruses could generate reassortant viruses.Although viruses derived by reassortment between avian H5N1 and human H3N2 progenitors have been generated in vitro (17), reassortment between these avian and human strains in a coinfected mammalian host has not been shown. Furthermore, our knowledge of the genetic and phenotypic repertoire of such reassortants generated in vivo and their potential for transmission to uninfected hosts is limited (2, 17). In the present study, we used the ferret model to better understand the generation of reassortant viruses in a host coinfected with contemporary avian (H5N1) and human (H3N2) viruses and the extent to which such reassortants replicate and transmit from animal to animal. The domestic ferret (Mustela putoris) serves as an ideal small-animal model for influenza because ferrets are susceptible to human and avian influenza viruses, including HPAI H5N1 viruses, and reflect the relative transmissibility of human and avian influenza viruses in humans (9, 17, 18, 31, 36, 39, 53). Our study revealed that coinfection of ferrets reproducibly generated reassortant viruses that could be recovered from tissues within and extending toward the upper respiratory tract. Although H5 reassortant viruses were recovered from the upper airways, they displayed no transmissibility to contact ferrets, suggesting that additional functional changes are required for these viral subtypes to become pandemic within human populations.     

Bat Guano Virome: Predominance of Dietary Viruses from Insects and Plants plus Novel Mammalian Viruses

Bats are hosts to a variety of viruses capable of zoonotic transmissions. Because of increased contact between bats, humans, and other animal species, the possibility exists for further cross-species transmissions and ensuing disease outbreaks. We describe here full and partial viral genomes identified using metagenomics in the guano of bats from California and Texas. A total of 34% and 58% of 390,000 sequence reads from bat guano in California and Texas, respectively, were related to eukaryotic viruses, and the largest proportion of those infect insects, reflecting the diet of these insectivorous bats, including members of the viral families Dicistroviridae, Iflaviridae, Tetraviridae, and Nodaviridae and the subfamily Densovirinae. The second largest proportion of virus-related sequences infects plants and fungi, likely reflecting the diet of ingested insects, including members of the viral families Luteoviridae, Secoviridae, Tymoviridae, and Partitiviridae and the genus Sobemovirus. Bat guano viruses related to those infecting mammals comprised the third largest group, including members of the viral families Parvoviridae, Circoviridae, Picornaviridae, Adenoviridae, Poxviridae, Astroviridae, and Coronaviridae. No close relative of known human viral pathogens was identified in these bat populations. Phylogenetic analysis was used to clarify the relationship to known viral taxa of novel sequences detected in bat guano samples, showing that some guano viral sequences fall outside existing taxonomic groups. This initial characterization of the bat guano virome, the first metagenomic analysis of viruses in wild mammals using second-generation sequencing, therefore showed the presence of previously unidentified viral species, genera, and possibly families. Viral metagenomics is a useful tool for genetically characterizing viruses present in animals with the known capability of direct or indirect viral zoonosis to humans.Bats belong to one of the most diverse, abundant, and widely distributed group of mammals. More than 1,100 bat species belong to the order of Chiroptera, representing approximately 20% of all mammalian species (54). Most bat species feed on insects and other arthropods, while others feed on fruit nectar, bird or mammal blood, and small vertebrates such as fish, frogs, mice, and birds (30). Of the 47 species of bats reported in the United States, most of them are insectivorous (http://www.batcon.org/).Bats are considered the natural reservoir of a large variety of zoonotic viruses causing serious human diseases such as lyssaviruses, henipaviruses, severe acute respiratory syndrome coronavirus, and Ebola virus (6, 38, 46, 59, 63, 65). Characteristics of bats, including their genetic diversity, broad geological distribution, gregarious habits, high population density, migratory habits, and long life span (30, 58), likely endow them with the ability to host diverse viruses, some of which are also able to infect humans and other mammals (41, 63).More than 80 virus species have been isolated or detected in bats using nucleic acid-based methods (6, 38, 59, 65). Viruses that have been recently discovered in bats include astroviruses, adeno-associated viruses (AAVs), adenoviruses, herpesviruses, and polyomavirus (8, 9, 13, 31, 32, 35, 37, 39, 40, 42, 61, 62, 68). For example, it was recently reported that a newly identified adenovirus isolated from bat guano was capable of infecting various vertebrate cell lines, including those of humans, monkeys, dogs, and pigs (35). With increasing human populations in previously wild areas, contact of bats with humans and with wild and domestic animals has increased, providing greater opportunities for cross-species transmissions of potentially pathogenic bat viruses. To better understand the range of viruses carried by bats, we undertook an initial characterization of the guano viromes of several common bat species in the United States.The development of massively parallel sequencing technology makes is possible to reveal uncultured viral assemblages within biological or environmental samples (11, 28). To date, this approach has been used to characterize viruses in equine feces (7), human blood (5), tissue (14), human feces (3, 4, 15, 45, 60, 67), and human respiratory secretions (64), which in turn has facilitated the discovery of many novel viruses (18, 20, 25, 33, 47, 50). In the present study, we analyzed the viruses present in guano from several bat species in California and Texas, using sequence-independent PCR amplification, pyrosequencing, and sequence similarity searches.     

Molecular Indicators Used in the Development of Predictive Models for Microbial Source Tracking

A number of chemical, microbial, and eukaryotic indicators have been proposed as indicators of fecal pollution sources in water bodies. No single one of the indicators tested to date has been able to determine the source of fecal pollution in water. However, the combined use of different indicators has been demonstrated to be the best way of defining predictive models suitable for determining fecal pollution sources. Molecular methods are promising tools that could complement standard microbiological water analysis. In this study, the feasibility of some proposed molecular indicators for microbial source tracking (MST) was compared (names of markers are in parentheses): host-specific Bacteroidetes (HF134, HF183, CF128, and CF193), Bifidobacterium adolescentis (ADO), Bifidobacterium dentium (DEN), the gene esp of Enterococcus faecium, and host-specific mitochondrial DNA associated with humans, cattle, and pigs (Humito, Bomito, and Pomito, respectively). None of the individual molecular markers tested enabled 100% source identification. They should be combined with other markers to raise sensitivity and specificity and increase the number of sources that are identified. MST predictive models using only these molecular markers were developed. The models were evaluated by considering the lowest number of molecular indicators needed to obtain the highest rate of identification of fecal sources. The combined use of three molecular markers (ADO, Bomito, and Pomito) enabled correct identification of 75.7% of the samples, with differentiation between human, swine, bovine, and poultry sources. Discrimination between human and nonhuman fecal pollution was possible using two markers: ADO and Pomito (84.6% correct identification). The percentage of correct identification increased with the number of markers analyzed. The best predictive model for distinguishing human from nonhuman fecal sources was based on 5 molecular markers (HF134, ADO, DEN, Bomito, and Pomito) and provided 90.1% correct classification.Fecal pollution represents a serious public health problem. Pathogens from infected animals and humans can be introduced into the environment through feces and cause health risks, environmental degradation, and economic losses. In recent years, water authorities'' environmental and sanitary regulations have focused on the total fecal load that can be held by a water body and on determining the source of fecal pollution. Accurate and reliable methods for detecting fecal pollution are needed to reduce its occurrence, prevent future spills, decrease economic losses, and take legal measures.Total coliforms, fecal coliforms, enterococci, and Escherichia coli have traditionally been used as microbial fecal indicators in water. These microorganisms are easy to enumerate by cultivation methods. However, they do not identify the source of fecal pollution.Fecal pollution of surface waters comes from point or diffuse sources, including municipal sewage, slaughterhouse wastewater, manure and biosolid disposal, wildlife, and undetermined runoff. Reliable microbial source tracking (MST) methods can provide efficient and rapid fecal source determination and facilitate cost-effective remediation. In recent years, various MST methods have been developed that are based on library-dependent or -independent methods and analyze phenotypic and/or genomic characteristics (39, 54, 55). Library-dependent methods (LDM) require a comprehensive library of isolates from known sources. Isolates from unknown sources are classified by correspondence with those from the library (57). LDM include antibiotic resistance analysis, carbon source utilization, repetitive PCR, and ribotyping. However, the geographic and temporal stability and the numerical methods used for these LDM have been questioned (26, 44). Some cultivation-based methods have already been described, such as the detection of specific enterotoxins of E. coli strains (30, 31, 43), the differentiation and enumeration of sorbitol-fermenting bifidobacteria (10, 37), and the enumeration of phages that infect host-specific Bacteroides spp. (8, 45). Cultivation methods detect only viable bacteria, may give a biased picture of the populations, and thus misrepresent the bacterial diversity (60). The use of PCR-based methods allows the detection of bacteria that are difficult to grow, such as anaerobes, including the genera Bacteroides and Bifidobacterium (5, 9, 14, 63), Rhodococcus coprophilus (51), methanogenic archaeal bacteria (59), and viruses (27). More detailed information on MST methods can be found in several technical reviews (8, 17, 54, 55, 57).Bifidobacterium and Bacteroides have been proposed as possible source-tracking indicators for waterborne fecal pathogens (3, 18, 33, 37, 41, 48). Several Bifidobacterium species are thought to be human host specific, such as Bifidobacterium adolescentis, Bifidobacterium dentium, and Bifidobacterium longum (58). Meanwhile, others have been linked to certain domestic animals (20, 61). A multiplex PCR has been developed to detect human fecal pollution by analyzing the presence of B. dentium and B. adolescentis in water (9). Bacteroidetes markers are mainly based on the definition of host-specific oligonucleotides (for example, to detect human, ruminant, and swine pollution) that are associated with some uncultured populations (5, 15, 32, 46, 47). Geographical differences in host specificity have been observed when these markers are applied in different world regions (1, 2, 11, 21, 22, 24, 40, 42). The detection of the gene esp, which encodes an enterococcal surface protein, has also been proposed as an indicator of human fecal pollution (53). This gene has been associated with the virulence, colonization and biofilm formation found in Enterococcus faecium and Enterococcus faecalis (25). However, recent studies have indicated that the detection of esp may not always be related to human fecal pollution (12, 35). Other MST indicators have been developed for eukaryotic molecular markers. Martellini et al. (38) designed a PCR protocol that targets eukaryotic genetic markers as a fecal source tracking method for differentiating human, porcine, bovine, and ovine fecal pollution. This protocol consists of nested PCRs, based on the amplification of mitochondrial DNA from the host cells. Multiplex and real-time PCR methods for mitochondrial MST indicators have also been developed (4, 13, 52).It has been shown that no single microbial or chemical MST indicator can determine the source of fecal pollution. Therefore, a selection of indicators is required (7, 8, 22, 24). Predictive models to distinguish between human and nonhuman pollution have been developed by combining indicators. These models have achieved a 100% likelihood of success (7, 24, 56). However, they are mostly based on culture-dependent methods, and discernment among different animal sources should be attained. In this study, microbial and eukaryotic molecular markers were compared for use as MST indicators. Potential combinations were also evaluated. Finally, MST predictive models using only these molecular markers were developed using a number of established statistical methods. The models were evaluated by considering the lowest number of molecular indicators needed to obtain the highest rate of discrimination among fecal sources.     

A Novel Genotype H9N2 Influenza Virus Possessing Human H5N1 Internal Genomes Has Been Circulating in Poultry in Eastern China since 1998

Many novel reassortant influenza viruses of the H9N2 genotype have emerged in aquatic birds in southern China since their initial isolation in this region in 1994. However, the genesis and evolution of H9N2 viruses in poultry in eastern China have not been investigated systematically. In the current study, H9N2 influenza viruses isolated from poultry in eastern China during the past 10 years were characterized genetically and antigenically. Phylogenetic analysis revealed that these H9N2 viruses have undergone extensive reassortment to generate multiple novel genotypes, including four genotypes (J, F, K, and L) that have never been recognized before. The major H9N2 influenza viruses represented by A/Chicken/Beijing/1/1994 (Ck/BJ/1/94)-like viruses circulating in poultry in eastern China before 1998 have been gradually replaced by A/Chicken/Shanghai/F/1998 (Ck/SH/F/98)-like viruses, which have a genotype different from that of viruses isolated in southern China. The similarity of the internal genes of these H9N2 viruses to those of the H5N1 influenza viruses isolated from 2001 onwards suggests that the Ck/SH/F/98-like virus may have been the donor of internal genes of human and poultry H5N1 influenza viruses circulating in Eurasia. Experimental studies showed that some of these H9N2 viruses could be efficiently transmitted by the respiratory tract in chicken flocks. Our study provides new insight into the genesis and evolution of H9N2 influenza viruses and supports the notion that some of these viruses may have been the donors of internal genes found in H5N1 viruses.Wild birds, including wild waterfowls, gulls, and shorebirds, are the natural reservoirs for influenza A viruses, in which they are thought to be in evolutionary stasis (2, 33). However, when avian influenza viruses are transmitted to new hosts such as terrestrial poultry or mammals, they evolve rapidly and may cause occasional severe systemic infection with high morbidity (20, 29). Despite the fact that avian influenza virus infection occurs commonly in chickens, it is unable to persist for a long period of time due to control efforts and/or a failure of the virus to adapt to new hosts (29). In the past 20 years, greater numbers of outbreaks in poultry have occurred, suggesting that the avian influenza virus can infect and spread in aberrant hosts for an extended period of time (5, 14-16, 18, 32).During the past 10 years, H9N2 influenza viruses have become panzootic in Eurasia and have been isolated from outbreaks in poultry worldwide (3, 5, 11, 14-16, 18, 24). A great deal of previous studies demonstrated that H9N2 influenza viruses have become established in terrestrial poultry in different Asian countries (5, 11, 13, 14, 18, 21, 24, 35). In 1994, H9N2 viruses were isolated from diseased chickens in Guangdong province, China, for the first time (4), and later in domestic poultry in other provinces in China (11, 16, 18, 35). Two distinct H9N2 virus lineages represented by A/Chicken/Beijing/1/94 (H9N2) and A/Quail/Hong Kong/G1/98 (H9N2), respectively, have been circulating in terrestrial poultry of southern China (9). Occasionally these viruses expand their host range to other mammals, including pigs and humans (6, 17, 22, 34). Increasing epidemiological and laboratory findings suggest that chickens may play an important role in expanding the host range for avian influenza virus. Our systematic surveillance of influenza viruses in chickens in China showed that H9N2 subtype influenza viruses continued to be prevalent in chickens in mainland China from 1994 to 2008 (18, 19, 36).Eastern China contains one metropolitan city (Shanghai) and five provinces (Jiangsu, Zhejiang, Anhui, Shandong, and Jiangxi), where domestic poultry account for approximately 50% of the total poultry population in China. Since 1996, H9N2 influenza viruses have been isolated regularly from both chickens and other minor poultry species in our surveillance program in the eastern China region, but their genetic diversity and the interrelationships between H9N2 influenza viruses and different types of poultry have not been determined. Therefore, it is imperative to explore the evolution and properties of these viruses. The current report provides insight into the genesis and evolution of H9N2 influenza viruses in eastern China and presents new evidence for the potential crossover between H9N2 and H5N1 influenza viruses in this region.     

Preinfection Human Immunodeficiency Virus (HIV)-Specific Cytotoxic T Lymphocytes Failed To Prevent HIV Type 1 Infection from Strains Genetically Unrelated to Viruses in Long-Term Exposed Partners

Understanding the mechanisms underlying potential altered susceptibility to human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) individuals and the later clinical consequences of breakthrough infection can provide insight into strategies to control HIV-1 with an effective vaccine. From our Seattle ES cohort, we identified one individual (LSC63) who seroconverted after over 2 years of repeated unprotected sexual contact with his HIV-1-infected partner (P63) and other sexual partners of unknown HIV-1 serostatus. The HIV-1 variants infecting LSC63 were genetically unrelated to those sequenced from P63. This may not be surprising, since viral load measurements in P63 were repeatedly below 50 copies/ml, making him an unlikely transmitter. However, broad HIV-1-specific cytotoxic T-lymphocyte (CTL) responses were detected in LSC63 before seroconversion. Compared to those detected after seroconversion, these responses were of lower magnitude and half of them targeted different regions of the viral proteome. Strong HLA-B27-restricted CTLs, which have been associated with disease control, were detected in LSC63 after but not before seroconversion. Furthermore, for the majority of the protein-coding regions of the HIV-1 variants in LSC63 (except gp41, nef, and the 3′ half of pol), the genetic distances between the infecting viruses and the viruses to which he was exposed through P63 (termed the exposed virus) were comparable to the distances between random subtype B HIV-1 sequences and the exposed viruses. These results suggest that broad preinfection immune responses were not able to prevent the acquisition of HIV-1 infection in LSC63, even though the infecting viruses were not particularly distant from the viruses that may have elicited these responses.Understanding the mechanisms of altered susceptibility or control of human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) persons may provide invaluable information aiding the design of HIV-1 vaccines and therapy (9, 14, 15, 33, 45, 57, 58). In a cohort of female commercial sex workers in Nairobi, Kenya, a small proportion of individuals remained seronegative for over 3 years despite the continued practice of unprotected sex (12, 28, 55, 56). Similarly, resistance to HIV-1 infection has been reported in homosexual men who frequently practiced unprotected sex with infected partners (1, 15, 17, 21, 61). Multiple factors have been associated with the resistance to HIV-1 infection in ES individuals (32), including host genetic factors (8, 16, 20, 37-39, 44, 46, 47, 49, 59, 63), such as certain HLA class I and II alleles (41), as well as cellular (1, 15, 26, 55, 56), humoral (25, 29), and innate immune responses (22, 35).Seroconversion in previously HIV-resistant Nairobi female commercial sex workers, despite preexisting HIV-specific cytotoxic T-lymphocyte (CTL) responses, has been reported (27). Similarly, 13 of 125 ES enrollees in our Seattle ES cohort (1, 15, 17) have become late seroconverters (H. Zhu, T. Andrus, Y. Liu, and T. Zhu, unpublished observations). Here, we analyze the virology, genetics, and immune responses of HIV-1 infection in one of the later seroconverting subjects, LSC63, who had developed broad CTL responses before seroconversion.     

Detection of Infectious Adenoviruses in Environmental Waters by Fluorescence-Activated Cell Sorting Assay

Methods for rapid detection and quantification of infectious viruses in the environment are urgently needed for public health protection. A fluorescence-activated cell-sorting (FACS) assay was developed to detect infectious adenoviruses (Ads) based on the expression of viral protein during replication in cells. The assay was first developed using recombinant Ad serotype 5 (rAd5) with the E1A gene replaced by a green fluorescent protein (GFP) gene. Cells infected with rAd5 express GFP, which is captured and quantified by FACS. The results showed that rAd5 can be detected at concentrations of 1 to 10⁴ PFU per assay within 3 days, demonstrating a linear correlation between the viral concentration and the number of GFP-positive cells with an r² value of >0.9. Following the same concept, FACS assays using fluorescently labeled antibodies specific to the E1A and hexon proteins, respectively, were developed. Assays targeting hexon showed greater sensitivity than assays targeting E1A. The results demonstrated that as little as 1 PFU Ads was detected by FACS within 3 days based on hexon protein, with an r² value greater than 0.9 over a 4-log concentration range. Application of this method to environmental samples indicated positive detection of infectious Ads in 50% of primary sewage samples and 33% of secondary treated sewage samples, but none were found in 12 seawater samples. The infectious Ads ranged in quantity between 10 and 165 PFU/100 ml of sewage samples. The results indicate that the FACS assay is a rapid quantification tool for detecting infectious Ads in environmental samples and also represents a considerable advancement for rapid environmental monitoring of infectious viruses.Waterborne viral infection is one of the most important causes of human morbidity in the world. There are hundreds of different types of human viruses present in human sewage, which, if improperly treated, may become the source of contamination in drinking and recreational waters (6, 12, 19). Furthermore, as water scarcity intensifies in the nation, so has consideration of wastewater reuse as a valid and essential alternative for resolving water shortages (31).Currently, routine viral monitoring is not required for drinking or recreational waters, nor is it required for wastewater that is discharged into the environment. This lack of a monitoring effort is due largely to the lack of methods that can rapidly and sensitively detect infectious viruses in environmental samples. In the past 20 years, tremendous progress has been made in detection of viruses in the environment based on molecular technology (32, 33, 35). PCR and quantitative real-time PCR (qPCR) methods have improved both the speed and sensitivity of viral detection compared with detection by the traditional tissue culture method (2, 11, 17, 18). However, they provide little information on viral infectivity, which is crucial for human health risk assessment (22-24, 35). Our previous work using a real-time PCR assay to detect human adenoviruses (Ads) in sewage could not differentiate the infectious viruses in the secondary treated sewage from those killed by chlorination disinfection (15). In this research, we pursued an innovative approach to detecting infectious viruses in water using fluorescence-activated cell sorting (FACS). This method is rapid and sensitive, with an established record in microbiological research (29, 34, 39).FACS is a specialized type of flow cytometry which provides a method for counting and sorting a heterogeneous mixture of biological cells into two or more kinds, one cell at a time, based upon the specific light-scattering and fluorescent characteristics of each cell (4, 25, 34, 38). It is a useful method since it provides fast and quantitative recording of fluorescent signals from individual cells (14, 16, 34, 47). The FACS viral assay is based on the expression of viral protein inside the recipient cell during viral replication (16). Specific antibody labeled with fluorescence is bound to the target viral protein, which results in fluorescence emission from infected cells. Viral particles outside the cell will not be captured, because the size of virus is below the detection limit of flow cytometry. Therefore, detection of cells, which can be captured with fluorescently labeled viral antibody, is a definitive indication of the presence of infectious virus.This research used human Ads as the target for development of the FACS method. The rationale for this choice is as follows. (i) Ads are important human pathogens that may be transmitted by water consumption and water spray (aerosols) (26, 32). The health hazard associated with exposure to Ads has been demonstrated by epidemiological data and clinical research (1, 7, 9, 35, 40, 43). (ii) Ads are among the most prevalent human viruses identified in human sewage and are frequently detected in marine waters and the Great Lakes (17, 32, 33, 35). (iii) Ads are more resistant to UV disinfection than any other bacteria or viruses (3, 5, 10, 24, 41, 42, 44). Thus, they may survive wastewater treatment as increasing numbers of wastewater treatment facilities switch from chlorination to UV to avoid disinfection by-products. (iv) Some serotypes of Ads, including enteric Ad 40 and 41, are fastidious. They are difficult to detect by plaque assay, and a routine assay of infectivity takes 7 to 14 days (8, 20).In this study, recombinant Ad serotype 5 (rAd5) with the E1A gene (the first transcribed gene after infection) replaced by a green fluorescent protein (GFP) gene was first used to test for sensitivity and speed of the assay. Two other viral proteins were then used as targets for development of FACS assays using Ad serotype 2 (Ad2) and Ad41. This study demonstrated the feasibility, sensitivity, and reliability of the assay for detection of infectious Ads in environmental samples.     

Performance Assessment PCR-Based Assays Targeting Bacteroidales Genetic Markers of Bovine Fecal Pollution

There are numerous PCR-based assays available to characterize bovine fecal pollution in ambient waters. The determination of which approaches are most suitable for field applications can be difficult because each assay targets a different gene, in many cases from different microorganisms, leading to variation in assay performance. We describe a performance evaluation of seven end-point PCR and real-time quantitative PCR (qPCR) assays reported to be associated with either ruminant or bovine feces. Each assay was tested against a reference collection of DNA extracts from 247 individual bovine fecal samples representing 11 different populations and 175 fecal DNA extracts from 24 different animal species. Bovine-associated genetic markers were broadly distributed among individual bovine samples ranging from 39 to 93%. Specificity levels of the assays spanned 47.4% to 100%. End-point PCR sensitivity also varied between assays and among different bovine populations. For qPCR assays, the abundance of each host-associated genetic marker was measured within each bovine population and compared to results of a qPCR assay targeting 16S rRNA gene sequences from Bacteroidales. Experiments indicate large discrepancies in the performance of bovine-associated assays across different bovine populations. Variability in assay performance between host populations suggests that the use of bovine microbial source-tracking applications will require a priori characterization at each watershed of interest.The ability to discriminate between bovine and other sources of fecal contamination is necessary for the accurate evaluation of human health risks associated with agricultural runoff and focused water quality management to make waters safe for human use. Many methods have been proposed to identify bovine fecal pollution using a variety of different microbiology and molecular techniques. One of the most widely used approaches utilizes a PCR to amplify a gene target that is specifically found in a host population. Currently, there are numerous PCR-based assays for the detection and/or quantitative assessment of bovine fecal pollution available for microbial source-tracking (MST) applications (1, 5-7, 11, 14, 17, 18, 21, 23). These assays target genes ranging from mitochondrial DNA to ribosomal rRNA to other functional genes involved in microorganism-host interactions.The majority of the reported bovine-associated PCR assays target 16S rRNA genes from the order Bacteroidales. This bacterial group constitutes a large proportion of the normal gut microbiota of most animals, including bovines (28), and contains subpopulations closely associated with other animal hosts such as swine, horse, and human (1, 3, 6, 18, 24). Host-associated PCR-based assays targeting Bacteroidales genetic markers have been used to investigate the sources and levels of fecal pollution at a number of beaches and inland watersheds, with variable levels of success (10, 13, 22, 27). Researchers have postulated that differences in host animal age, health, diet, and geographic location may influence bacterial community structures in the bovine gastrointestinal tract (2, 9, 26). Without a priori knowledge of the potential representational bias introduced by such factors, it may be difficult to use these assays with confidence as indicators of bovine fecal pollution.Assay specificity and sensitivity and the prevalence and abundance of genetic marker determinations are typically estimated from the systematic testing of a collection of reference fecal sources collected from known animal sources. However, the characterization of assay performance has been limited, in most cases, to animal sources originating from a particular geographic region or industry, such as dairy or beef. The determination of assay performance across a range of different host populations is essential as the field moves toward the implementation of PCR-based host-associated fecal pollution assessment approaches.We report a performance study of seven PCR and quantitative PCR (qPCR) assays targeting Bacteroidales genes reported to be associated with either ruminant (e.g., bovine, goat, sheep, deer, and others) or bovine feces. Each assay was tested against a reference collection of DNA extracts from 247 individual bovine fecal samples representing 11 different populations. Assay specificity was determined by testing 175 fecal DNA extracts from 24 different animal species. For qPCR assays, the abundance of each genetic marker was measured within each bovine population and compared to quantities of Bacteroidales 16S rRNA genetic markers. These analyses indicated large discrepancies in assay performance across different bovine populations.     

Ocular Infection of Mice with Influenza A (H7) Viruses: a Site of Primary Replication and Spread to the Respiratory Tract

Avian H7 influenza viruses have been responsible for poultry outbreaks worldwide and have resulted in numerous cases of human infection in recent years. The high rate of conjunctivitis associated with avian H7 subtype virus infections may represent a portal of entry for avian influenza viruses and highlights the need to better understand the apparent ocular tropism observed in humans. To study this, mice were inoculated by the ocular route with viruses of multiple subtypes and degrees of virulence. We found that in contrast to human (H3N2 and H1N1) viruses, H7N7 viruses isolated from The Netherlands in 2003 and H7N3 viruses isolated from British Columbia, Canada, in 2004, two subtypes that were highly virulent for poultry, replicated to a significant titer in the mouse eye. Remarkably, an H7N7 virus, as well as some avian H5N1 viruses, spread systemically following ocular inoculation, including to the brain, resulting in morbidity and mortality of mice. This correlated with efficient replication of highly pathogenic H7 and H5 subtypes in murine corneal epithelial sheets (ex vivo) and primary human corneal epithelial cells (in vitro). Influenza viruses were labeled to identify the virus attachment site in the mouse cornea. Although we found abundant H7 virus attachment to corneal epithelial tissue, this did not account for the differences in virus replication as multiple subtypes were able to attach to these cells. These findings demonstrate that avian influenza viruses within H7 and H5 subtypes are capable of using the eye as a portal of entry.Highly pathogenic avian influenza (HPAI) H5N1 viruses, which have resulted in over 420 documented cases of human infection to date, have generally caused acute, often severe and fatal, respiratory illness (1, 50). While conjunctivitis following infection with H5N1 or human influenza viruses has been rare, most human infections associated with H7 subtype viruses have resulted in ocular and not respiratory disease (1, 9, 37, 38). Infrequent reports of human conjunctivitis infection following exposure to H7 influenza viruses date from 1977, predominantly resulting from laboratory or occupational exposure (21, 40, 48). However, in The Netherlands in 2003, more than 80 human infections with H7N7 influenza virus occurred among poultry farmers and cullers amid widespread outbreaks of HPAI in domestic poultry; the majority of these human infections resulted in conjunctivitis (14, 20). Additionally, conjunctivitis was documented in the two human infections resulting from an H7N3 outbreak in British Columbia, Canada, in 2004, as well as in H7N3- and H7N2-infected individuals in the United Kingdom in 2006 and 2007, respectively (13, 18, 29, 46, 51). The properties that contribute to an apparent ocular tropism of some influenza viruses are currently not well understood (30).Host cell glycoproteins bearing sialic acids (SAs) are the cellular receptors for influenza viruses and can be found on epithelial cells within both the human respiratory tract and ocular tissue (26, 31, 41). Both respiratory and ocular tissues additionally secrete sialylated mucins that function in pathogen defense and protection of the epithelial surface (5, 11, 22). Within the upper respiratory tract, α2-6-linked SAs (the preferred receptor for human influenza viruses) predominate on epithelial cells (26). While α2-3-linked SAs are also present to a lesser degree on respiratory epithelial cells, this linkage is more abundantly expressed on secreted mucins (2). In contrast, α2-3-linked SAs (the preferred receptor for avian influenza viruses) are found on corneal and conjunctival epithelial cells of the human eye (31, 41), while secreted ocular mucins are abundantly composed of α2-6 SAs (5). It has been suggested that avian influenza viruses are more suited to infect the ocular surface due to their general α2-3-linked SA binding preference, but this has not been demonstrated experimentally (30).The mouse model has been used previously to study the role of ocular exposure to respiratory viruses (6, 39). In mice, ocular inoculation with an H3N2 influenza virus resulted in virus replication in nasal turbinates and lung (39), whereas ocular infection with respiratory syncytial virus (RSV) resulted in detectable virus titers in the eye and lung (6). These studies have revealed that respiratory viruses are not limited to the ocular area following inoculation at this site. However, the ability of influenza viruses to replicate specifically within ocular tissue has not been examined.Despite repeated instances of conjunctivitis associated with H7 subtype infections in humans, the reasons for this apparent ocular tropism have not been studied extensively. Here, we present a murine model to study the ability of human and avian influenza viruses to cause disease by the ocular route. We found that highly pathogenic H7 and H5 influenza viruses were capable of causing a systemic and lethal infection in mice following ocular inoculation. These highly pathogenic viruses, unlike human H3N2 and H1N1 viruses, replicated to significant titers in the mouse corneal epithelium and primary human corneal epithelial cells (HCEpiCs). Identification of viruses well suited to infecting the ocular surface is the first step in better understanding the ability of influenza viruses of multiple subtypes to use this tissue as a portal of entry.     

Evaluation of Replication and Cross-Reactive Antibody Responses of H2 Subtype Influenza Viruses in Mice and Ferrets

H2 influenza viruses have not circulated in humans since 1968, and therefore a large segment of the population would likely be susceptible to infection should H2 influenza viruses reemerge. The development of an H2 pandemic influenza virus vaccine candidate should therefore be considered a priority in pandemic influenza preparedness planning. We selected a group of geographically and temporally diverse wild-type H2 influenza viruses and evaluated the kinetics of replication and compared the ability of these viruses to induce a broadly cross-reactive antibody response in mice and ferrets. In both mice and ferrets, A/Japan/305/1957 (H2N2), A/mallard/NY/1978 (H2N2), and A/swine/MO/2006 (H2N3) elicited the broadest cross-reactive antibody responses against heterologous H2 influenza viruses as measured by hemagglutination inhibition and microneutralization assays. These data suggested that these three viruses may be suitable candidates for development as live attenuated H2 pandemic influenza virus vaccines.Influenza pandemics occur when a novel influenza virus enters a population with little preexisting immunity (36). During the pandemics of the last century, novel influenza viruses were introduced either directly from an avian reservoir (34) or were the result of reassortment between contemporaneously circulating human, avian, and swine influenza viruses (5, 29, 36). Due to the lack of preexisting immunity to the novel virus, morbidity and mortality rates are typically higher than in epidemics caused by seasonal influenza viruses (4).Although pandemic preparedness planning has largely focused on the highly pathogenic H5 and H7 avian influenza virus subtypes, the recent emergence of the 2009 pandemic H1N1 viruses underscores the need to consider other influenza virus subtypes as well. Of the 16 hemagglutinin (HA) influenza A virus subtypes that have been identified to date, H1, H2, and H3 have been known to cause influenza pandemics (7, 27), suggesting that these viruses are capable of sustained transmission and can cause disease in humans. While the H1 and H3 subtypes have cocirculated in humans since 1977, H2 influenza viruses have not circulated in humans since 1968 (36) and therefore a large segment of the population would likely be susceptible to infection should H2 influenza viruses reemerge. The 1957 H2 pandemic virus was a reassortant that derived the HA, neuraminidase (NA), and PB1 genes from an avian virus and the remaining gene segments from the circulating H1N1 virus (15, 30). As H2 subtype viruses continue to circulate in avian reservoirs worldwide (12, 17, 18, 22, 33), they remain a potential pandemic threat. The development of an H2 influenza virus vaccine candidate should therefore be considered a priority in future pandemic influenza preparedness planning.Given the low likelihood that a previously selected vaccine virus will exactly match the pandemic virus, the ability to elicit a broadly cross-reactive antibody response to antigenically distinct viruses within a subtype is an important consideration in the selection of a pandemic influenza vaccine candidate. Previous studies have examined the ability of inactivated H2 influenza viruses to provide cross-protection against mouse-adapted variants of reassortant human viruses and an avian H2 influenza virus from 1978 (9, 14). Given the potential for live attenuated influenza virus vaccines to confer a great breadth of heterologous cross-protection (1, 2, 6, 35), we recently conducted a study evaluating cold-adapted A/Ann Arbor/6/1960 (AA CA), an H2 influenza virus used as the backbone of the seasonal live attenuated influenza A virus vaccine currently licensed in the United States (3). However, as H2 influenza virus continues to circulate widely and appear in migratory birds (10, 24, 26), in poultry markets (20), and in swine (21), with evidence of interregional gene transmission (19, 22), a more extensive evaluation of recent isolates may be warranted in the selection of a potential H2 pandemic vaccine candidate.H2 influenza viruses fall into three main lineages: a human lineage, a North American avian lineage, and a Eurasian avian lineage (29). In addition to viruses whose replicative ability in mammals has previously been established (11, 21, 23, 25), we selected a group of geographically and temporally diverse H2 influenza viruses from each lineage. We evaluated the kinetics of replication of each of these viruses in mice and ferrets and compared the abilities of these viruses to induce a broadly cross-reactive antibody response to determine which of these viruses would be suitable for further development as an H2 pandemic influenza vaccine candidate.     

Receptor Specificity of Influenza A H3N2 Viruses Isolated in Mammalian Cells and Embryonated Chicken Eggs

Isolation of human subtype H3N2 influenza viruses in embryonated chicken eggs yields viruses with amino acid substitutions in the hemagglutinin (HA) that often affect binding to sialic acid receptors. We used a glycan array approach to analyze the repertoire of sialylated glycans recognized by viruses from the same clinical specimen isolated in eggs or cell cultures. The binding profiles of whole virions to 85 sialoglycans on the microarray allowed the categorization of cell isolates into two groups. Group 1 cell isolates displayed binding to a restricted set of α2-6 and α2-3 sialoglycans, whereas group 2 cell isolates revealed receptor specificity broader than that of their egg counterparts. Egg isolates from group 1 showed binding specificities similar to those of cell isolates, whereas group 2 egg isolates showed a significantly reduced binding to α2-6- and α2-3-type receptors but retained substantial binding to specific O- and N-linked α2-3 glycans, including α2-3GalNAc and fucosylated α2-3 glycans (including sialyl Lewis x), both of which may be important receptors for H3N2 virus replication in eggs. These results revealed an unexpected diversity in receptor binding specificities among recent H3N2 viruses, with distinct patterns of amino acid substitution in the HA occurring upon isolation and/or propagation in eggs. These findings also suggest that clinical specimens containing viruses with group 1-like receptor binding profiles would be less prone to undergoing receptor binding or antigenic changes upon isolation in eggs. Screening cell isolates for appropriate receptor binding properties might help focus efforts to isolate the most suitable viruses in eggs for production of antigenically well-matched influenza vaccines.Influenza A viruses are generally isolated and propagated in embryonated chicken eggs or in cultures of cells of mammalian origin. Human influenza viruses were previously noted to acquire mutations in the hemagglutinin (HA) gene upon isolation and culture in the allantoic sac of embryonated chicken eggs (herein simply referred to as “eggs”) compared to the sequences of those isolated in mammalian cell substrates (herein referred to as “cells”) (29, 30, 44, 53, 58). These mutations resulted in amino acid substitutions that were found to mediate receptor specificity changes and improved viral replication efficiency in eggs (37). In general, cell-grown viruses are assumed to be more similar than their egg-grown counterparts to the viruses present in respiratory secretions (30, 56). Since their emergence in 1968, influenza A (H3N2) viruses have evolved and adapted to the human host while losing their ability to be efficiently isolated and replicate in eggs, particularly after 1992 (37, 42, 48). The rate of isolation of H3N2 clinical specimens after inoculation into eggs can be up to ∼30 times lower than that in mammalian cell cultures, highlighting the strong selective pressure for the emergence of sequence variants (77).Virtually all influenza vaccines for human use were licensed decades ago by national regulatory authorities, which used a product manufactured from influenza viruses isolated and propagated exclusively in eggs; therefore, cell culture isolates have been unacceptable for this purpose (41, 71). The antigen composition of influenza vaccines requires frequent updates (every 2 years, on average) to closely match their antigenic properties to the most prevalent circulating antigenic drift variant viruses (51). The limited availability of H3N2 viruses isolated in eggs has on one or more occasions delayed vaccine composition updates and may have reduced the efficacy of vaccination against new antigenically drifted viruses (3, 34, 37).Entry of influenza viruses into host cells is mediated by HA, which binds to sialic acid containing glycoconjugates on the surface of epithelial cells in the upper respiratory tract (2, 13). The nature of the linkage between sialic acid and the vicinal sugar (usually galactose) varies in different host species and tissues and may therefore determine whether an influenza virus binds to and infects avian or human cells (40, 46, 59, 62, 72-75). Human influenza viruses preferentially bind to α2-6-linked sialic acids, and avian viruses predominantly bind to α2-3-linked sialic acids (59). Previous studies with chicken embryo chorioallantoic membranes revealed differential lectin binding, suggesting that α2-3-linked but not α2-6-linked sialosides are present on the epithelial cells (28). Human H3N2 viruses isolated in cell culture were reported to bind with a high affinity to α2-6-linked sialosides, while viruses isolated in eggs often had increased specificity for α2-3-linked sialosides (19, 20, 28). The functional classification of avian and mammalian influenza virus receptors is further complicated since in vitro and tissue-binding assays have led to new working hypotheses involving glycan chain length, topology, and the composition of the inner fragments of the carbohydrate chain as additional receptor specificity determinants (9, 17, 65, 66, 82). However, the significance of these in vitro properties remains unknown, since the structures of the natural sialosides on host cells that are used for infectious virus entry are undefined.The techniques most widely used to study the interactions of the influenza virus with host cell receptors employ animal cells in various assay formats (36, 57, 59, 64, 69). To overcome the problems of cell-based techniques, new assays that rely on labeled sialyl-glycoproteins or polymeric sialoglycans have been developed (18). However, these assays are limited by having only a few glycans available in polymeric form and offer low throughput. In contrast, glycan microarrays can assess virus binding to multiple well-defined glycans simultaneously. Previous work with influenza live or β-propiolactone (BPL)-inactivated virions as well as recombinantly produced HAs revealed a good correlation with receptor specificity compared to that achieved by other methods of analysis (4, 11, 57, 58, 65-68).Here we have compared paired isolates derived in eggs or cell cultures from the single clinical specimen to better understand their receptor binding specificity and its implications for vaccine production. We examined the differences in the sequences of the HAs between egg- and cell-grown isolates and analyzed their receptor binding profiles using glycan microarrays. Sequence analysis of the HA and glycan binding results revealed two distinct groups of viruses, with many egg isolates showing unexpectedly reduced levels of binding to α2-3 and α2-6 sialosides compared to the levels for the viruses isolated in mammalian cells. Furthermore, these studies highlighted that specific glycans may be important for H3N2 virus growth in eggs.     

Quantification of Human Polyomaviruses JC Virus and BK Virus by TaqMan Quantitative PCR and Comparison to Other Water Quality Indicators in Water and Fecal Samples

In the United States, total maximum daily load standards for bodies of water that do not meet bacterial water quality standards are set by each state. The presence of human polyomaviruses (HPyVs) can be used as an indicator of human-associated sewage pollution in these waters. We have developed and optimized a TaqMan quantitative PCR (QPCR) assay based on the conserved T antigen to both quantify and simultaneously detect two HPyVs; JC virus and BK virus. The QPCR assay was able to consistently quantify ≥10 gene copies per reaction and is linear over 5 orders of magnitude. HPyVs were consistently detected in human waste samples (57 of 64) and environmental waters with known human fecal contamination (5 of 5) and were not amplified in DNA extracted from 127 animal waste samples from 14 species. HPyV concentrations in sewage decreased 81.2 and 84.2% over 28 days incubation at 25 and 35°C, respectively. HPyVs results were compared to Escherichia coli, fecal coliform, and enterococci concentrations and the presence of three other human-associated microbes: Bacteroidetes, Methanobrevibacter smithii, and adenovirus. HPyVs were the most frequently detected of these in human and contaminated environmental samples and were more human specific than the Bacteroidetes (HF183) or M. smithii. HPyVs and M. smithii more closely mimicked the persistence of adenovirus in sewage than the other microbes. The use of this rapid and quantitative assay in water quality research could help regulatory agencies to identify sources of water pollution for improved remediation of contaminated waters and ultimately protect humans from exposure to pathogens.Maintaining healthy coastal water systems is essential, since poor water quality can have detrimental effects on mangroves, seagrass beds, coral reefs, the fishing and shellfish harvesting industries, and the health of recreational water users (1, 5, 15, 17, 20, 44). Since 1972 in the United States, each state has been required to set total maximum daily loads (TMDLs) for pollutants in water bodies according to section 303(d) of the Clean Water Act (50). The probability that microbial pathogens are present is estimated by enumerating indicator bacteria, which are shed in the feces of humans and most animals. The U.S. Environmental Protection Agency recommends using Escherichia coli and enterococci to assess the quality of freshwater and saline water, respectively (47); however, Florida currently uses fecal coliforms and enterococci as indicators of fecal pollution (42).When bacterial indicators exceed regulatory levels, a plan of action (TMDL implementation) must be developed to reduce pathogens. TMDL plans for “pathogen” reduction are particularly problematic because they rely upon surrogate indicator bacteria, which yield little or no insight as to the source of pollution. High indicator bacteria concentrations can be attributed to many sources, including agricultural runoff, storm water runoff, wildlife, pets, faulty septic systems (onsite wastewater treatment and disposal systems), and a failing central sewer infrastructure (5, 12, 28).To address the issue of source identification, methods have been developed in which the biochemistry or genetics of certain microorganisms are used to indirectly identify probable source(s) of fecal pollution, which is termed microbial source tracking (MST) (48). MST methods based on detection of a source-associated gene (marker) by PCR have proliferated over the past 10 years due to the additional information they can provide to watershed managers on fecal contamination sources (43). Although marker detection by endpoint (binary) PCR can give important insights on the source(s) of fecal contamination, quantitative measurements can provide information about the relative magnitude of contamination from various sources. Moreover, epidemiological studies on the correlation between recreational water use, microbial contamination, and the risk of illness will greatly benefit from the ability to quantify MST markers, rather than simply assessing binary (+/−) detection.Although many bacterial targets have been proposed for MST of human sewage (8, 39, 46a), fewer viral targets have been investigated (19, 24, 33). Polyomavirus is the sole genus in the family Polyomaviridae (22). These viruses have a 5-kbp double-stranded DNA genome surrounded by a 40- to 50-nm icosahedral capsid (38). The JCV and BKV human polyomaviruses (HPyVs) have similarly structured genomes that show ∼75% identity (21). BK virus (BKV) and JC virus (JCV) gained much attention in the late 1970s as the etiological agents of kidney nephritis (i.e., BKV reactivation in the kidneys) and progressive multifocal leukoencephalopathy (i.e., JCV reactivation in brain tissue) in the immunocompromised (16, 34). Serological studies have shown that >70% of adults harbor antibodies to BKV or JCV (27, 30, 44). These viruses are known for producing lifelong, asymptomatic viruria in immunocompetent individuals (37). In 2000 it was first suggested that JCV would be a useful indicator of human sewage in water (11). The obligate host specificity and abundance of BKV and JCV in municipal sewage has led to the successful use of these viruses to indicate human fecal pollution in environmental water samples (12, 29).Due to the health implications of BKV and JCV, several methods have been developed to rapidly detect either BKV or JCV in clinical samples (6, 31, 35, 56). However, from an MST standpoint, it is advantageous to target both BKV and JCV. BKV has been found in feces (54), and both viruses are excreted in the urine (6, 11, 37, 55, 60) either simultaneously or individually. The focus of this research was the modification of the previously developed nested PCR protocol for HPyVs detection (29) to a TaqMan quantitative PCR (QPCR) assay to simultaneously detect and quantify both BKV and JCV. Furthermore, we compared measurements obtained with the newly developed QPCR assay to those of other water quality indicators and MST markers. These indicators included bacterial indicator concentrations (49) and PCR detection of human-associated markers currently used for MST. These included human-associated Bacteroidetes (8), Methanobrevibacter smithii (46a), and adenovirus (36). To assess the potential of HPyVs to mimic the fate of pathogens in water, the persistence of all of the water quality indicators was assessed, and relationships between bacterial indicator organisms and MST markers in both human waste samples as well as contaminated environmental samples were examined.     

Molecular detection and characterization of gastroenteritis viruses occurring naturally in the stream waters of Manaus, central Amazonia, Brazil

To assess the presence of the four main viruses responsible for human acute gastroenteritis in a hydrographic network impacted by a disordered urbanization process, a 1-year study was performed involving water sample collection from streams in the hydrographic basin surrounding the city of Manaus, Amazonas, Brazil. Thirteen surface water sample collection sites, including different areas of human settlement characterized as urban, rural, and primary forest, located in the Tarumã-Açu, São Raimundo, Educandos, and Puraquequara microbasins, were defined with a global positioning system. At least one virus was detected in 59.6% (31/52) of the water samples analyzed, and rotavirus was the most frequent (44.2%), followed by human adenovirus (30.8%), human astrovirus (15.4%), and norovirus (5.8%). The viral contamination observed mainly in the urban streams reflected the presence of a local high-density population and indicated the gastroenteritis burden from pathogenic viruses in the water, principally due to recreational activities such as bathing. The presence of viral genomes in areas where fecal contamination was not demonstrated by bacterial indicators suggests prolonged virus persistence in aquatic environments and emphasizes the enteric virus group as the most reliable for environmental monitoring.Although water is recognized as the most precious natural resource on our planet, human activities disregard this fact by continually polluting freshwater bodies. Increasing worldwide awareness of the poor quality of potable water has occurred mainly due to the significant increase in human morbidity and mortality. More than 2.2 million people die every year from diseases associated with poor quality water and sanitary conditions, mostly in developing countries. The presence of pathogenic enteric microorganisms in aquatic environments reveals how human health can be affected by contamination from sewage discharge into surface waters. It is estimated that nearly a quarter of all hospital beds in the world are occupied by patients presenting complications arising from infections caused by enteric microorganisms (53, 56).Water sanitary quality is usually determined by the concentration of fecal indicator bacteria and occasionally by bacteriophages (8, 17). However, numerous investigations have shown that achieving minimum fecal coliform standards does not predict viral contamination (8, 47). Enteric viruses are highly stable in the environment, maintaining their infectivity even after exposure to treatment processes, and are often the most diluted pathogens in water, thus requiring concentration methods for their detection (2, 8, 42, 53).After replication in the gastrointestinal tract, human enteric pathogenic viruses are excreted in high concentrations in the feces (10⁵ to 10¹¹/g feces) and can enter the environment through the discharge of waste materials from symptomatic or asymptomatic carriers and therefore may be dispersed in environmental waters (2). Difficulties in obtaining viruses from environmental samples have been overcome through the association of virus concentration methods with the use of molecular techniques, such as PCR, which provide rapid, sensitive, and specific detection (2, 15, 16, 32, 35, 46, 48, 54).Although the presence of viruses in water is underestimated, mainly due to the difficulties associated with the detection of such agents in different matrices, enteric viruses have been implicated in waterborne outbreaks in different countries every year (2, 36, 38, 53). Among these, rotaviruses (RV), noroviruses (NoV), human astroviruses (HAstV), and human adenoviruses (HAdV) are recognized as the most important etiologic agents of acute gastroenteritis and have been considered for environmental monitoring (11, 29, 55).Diarrhea, a water-related disease, is a global public health problem and is ranked third among the causes of death affecting children under 5 years old, accounting for 17% of all deaths. It is estimated that 1.5 billion episodes occur each year, mostly in developing countries. It is recognized that a significant proportion of diarrhea cases caused by waterborne transmission in such countries is related to water quality. Levels of diarrhea disease differ between communities due to socioeconomic factors such as water availability and hygienic behavior (9, 45).Despite a significant decrease in diarrhea-related mortality in developed and some developing countries, such as Brazil, diarrhea is still an important cause of morbidity in these countries (37). In the Northern region of Brazil, the city of Manaus reported an increase of 90.5% in the number of diarrhea cases between 1998 and 2000, from 8,878 cases to 16,914 (4).The goal of this study was to assess viral contamination by the four main viruses responsible for acute gastroenteritis (RV, HAdV, HAstV, and NoV) in the hydrographic network that surrounds Manaus. Investigation and determination of the viruses that are dumped into streams from domestic sewage without prior treatment, as occurs in Manaus, could reveal how rapid population growth associated with a disordered urbanization process represents a threat to human health caused by the increased risk of disease transmission.     

Breadth of Neutralizing Antibodies Elicited by Stable,Homogeneous Clade A and Clade C HIV-1 gp140 Envelope Trimers in Guinea Pigs

The native envelope (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) is trimeric, and thus trimeric Env vaccine immunogens are currently being explored in preclinical immunogenicity studies. Key challenges have included the production and purification of biochemically homogeneous and stable trimers and the evaluation of these immunogens utilizing standardized virus panels for neutralization assays. Here we report the binding and neutralizing antibody (NAb) responses elicited by clade A (92UG037.8) and clade C (CZA97.012) Env gp140 trimer immunogens in guinea pigs. These trimers have been selected and engineered for optimal biochemical stability and have defined antigenic properties. Purified gp140 trimers with Ribi adjuvant elicited potent, cross-clade NAb responses against tier 1 viruses as well as detectable but low-titer NAb responses against select tier 2 viruses from clades A, B, and C. In particular, the clade C trimer elicited NAbs that neutralized 27%, 20%, and 47% of tier 2 viruses from clades A, B, and C, respectively. Heterologous DNA prime, protein boost as well as DNA prime, recombinant adenovirus boost regimens expressing these antigens, however, did not result in an increased magnitude or breadth of NAb responses in this system. These data demonstrate the immunogenicity of stable, homogeneous clade A and clade C gp140 trimers and exemplify the utility of standardized tier 1 and tier 2 virus panels for assessing the NAb responses of candidate HIV-1 Env immunogens.The development and evaluation of novel HIV-1 Env immunogens are critical priorities of the HIV-1 vaccine field (2, 10, 25). The major antigenic target for neutralizing antibodies (NAbs) is the trimeric Env glycoprotein on the virion surface (4, 18, 30). Monomeric gp120 immunogens have not elicited broadly reactive NAbs in animal models (5, 13, 28, 29) or humans (16, 31), and thus several groups have focused on generating trimer immunogens that better mimic the native Env spike found on virions (3, 7, 14, 15, 20, 22, 27). It has, however, proven difficult to produce stable and conformationally homogeneous Env trimers. Strategies to modify Env immunogens have therefore been explored, including the removal of the cleavage site between gp120 and gp41 (3, 7, 23, 39, 40), the incorporation of an intramolecular disulfide bond to stabilize cleaved gp120 and gp41 moieties (6), and the addition of trimerization motifs such as the T4 bacteriophage fibritin “fold-on” (Fd) domain (8, 17, 39).Preclinical evaluation of candidate Env immunogens is critical for concept testing and for the prioritization of vaccine candidates. Luciferase-based virus neutralization assays with TZM.bl cells (21, 24) have been developed as high-throughput assays that can be standardized (26). However, the optimal use of this assay requires the generation of standardized virus panels derived from multiple clades that reflect both easy-to-neutralize (tier 1) and primary isolate (tier 2) viruses (21, 24). A tiered approach for the evaluation of novel Env immunogens has been proposed, in which tier 1 viruses represent homologous vaccine strains and a small number of heterologous neutralization-sensitive viruses while tier 2 viruses provide a greater measure of neutralization breadth for the purpose of comparing immunogens (24).We screened a large panel of primary HIV-1 isolates for Env stability and identified two viruses, CZA97.012 (clade C) (32) and 92UG037.8 (clade A) (17), that yielded biochemically homogeneous and stable Env trimers with well defined and uniform antigenic properties (17). The addition of the T4 bacteriophage fibritin “fold-on” (Fd) trimerization domain further increased their yield and purity (17). In the present study, we assessed the immunogenicity of these stable clade A and clade C gp140 trimers in guinea pigs. Both trimers elicited high-titer binding antibody responses and cross-clade neutralization of select tier 1 viruses as well as low-titer but detectable NAb responses against select tier 2 viruses from clades A, B, and C. These data demonstrate the immunogenicity of these stable gp140 trimers and highlight the utility of standardized virus panels in the evaluation of novel HIV-1 Env immunogens.     

Genetic and Phenotypic Characterization of GII-4 Noroviruses That Circulated during 1987 to 2008

The predominance and continual emergence of new variants in GII-4 noroviruses (NVs) in recent years have raised questions about the role of host immunity and histo-blood group antigens (HBGAs) in NV evolution. To address these questions, we performed a genetic and phenotypic characterization of GII-4 variants circulating in the past decade (1998 to 2008). Ninety-three GII-4 sequences were analyzed, and of them, 16 strains representing 6 genetic clusters were selected for further characterization. The HBGA binding properties were determined by both saliva- and oligosaccharide-binding assays using P particles as a model of NV capsid. The antigenic properties were also examined by enzyme immunoassay (EIA), Western blot analysis, and receptor blocking assay, using P-particle-specific antibodies from immunized mice and GII-4 virus-infected patients. Our results showed that 15 of the 16 GII-4 viruses bound to saliva of all A, B, and O secretors. Oligosaccharide binding assays yielded largely consistent results, although the binding affinities to some oligosaccharides varied among some strains. The only nonbinder had a mutation in the binding site. While antigenic variations were detected among the 16 strains, significant cross-blocking on the HBGA binding was also noted. Sequence alignment revealed high conservation of HBGA binding interfaces with some variations in adjacent regions. Taken together, our data suggested that the ability of GII-4 to recognize different secretor HBGAs persisted over the past decade, which may explain the predominance of GII-4 over other genotypes. Our data also indicated that both the host immunity and HBGAs play a role in NV evolution. While host immunity may continue driving NV for antigenic change, the functional selection by the HBGAs tends to lock the architecture of the capsid/HBGA interfaces and allows only limited variations outside the HBGA binding sites. A potential outcome of such counterselection between theses two factors in NV evolution is discussed.Noroviruses (NVs) have been recognized as the most important cause of nonbacterial acute gastroenteritis in both developed and developing countries, affecting people of all ages (13, 35, 39, 44, 48, 56). They are single-stranded positive-sense RNA viruses belonging to the family Caliciviridae. NVs are highly contagious, spreading by a fecal/oral pathway through person-to-person contact and by contaminated food and/or water and usually causing large outbreaks within closed communities in a variety of settings, such as hospitals, nursing homes, schools, childcare centers, restaurants, cruise ships, and the military (11, 63). Human NVs have been difficult to study due to diverse members and the lack of an efficient cell culture and animal model for human NVs. The cloning of the NV genomes (33, 36, 73) and subsequent expression of the viral capsid proteins in baculovirus and other expression systems (3, 31, 32) have greatly advanced the research of NVs, including host-virus interaction, immunology, diagnosis, molecular virology, and epidemiology (16, 17, 19, 20, 25, 28-30, 46, 51, 59, 73).Several lines of evidence indicate that NVs recognize human histo-blood group antigens (HBGAs) as a ligand or receptor in a strain-specific manner (63, 64). HBGAs are complex carbohydrates presenting on red blood cells and on the epithelia of digestive, respiratory, and genitourinary tracts. They also exist in biologic fluid, such as milk and saliva. NVs are highly diverse in recognizing the human HBGAs, and a number of HBGA-binding patterns involving the ABO, secretor, and Lewis families of human HBGAs have been described (19, 20, 23, 24, 26, 28, 43, 45, 55). The association of HBGA binding with clinical infection and illness has been demonstrated by volunteer challenge studies and outbreak investigations (25, 27, 42, 62, 66), although exceptions also have been reported (41, 50, 53). Further study has mapped the HBGA binding site in the protruding (P) domain of the viral capsid protein (60). Using the P domain as a model, the atomic structures of the HBGA binding interfaces have been resolved by X-ray crystallography (5, 7, 9). The interfaces are comprised of several amino acids located on the top of the P dimer, within the outermost surface of the viral capsid. Extensive hydrogen bond networks between the P dimer and the HBGAs were elucidated and further confirmed by mutagenesis analyses (61, 68, 69). Despite significant differences in genetics and HBGA binding patterns, the sequences of the HBGA-binding interfaces are highly conserved within, but not between, the two major human-related genogroups (GI and GII) of NVs, suggesting that HBGAs are important factors in NV evolution (9, 69).The NV capsid is composed of a single major structural protein, the capsid protein (VP1), which can be divided into two major domains: the shell (S) and the protruding (P) domains (52). Expression of the full-length VP1 by a eukaryotic system forms empty virus-like particles (VLPs) that have been used as a surrogate for NVs for many years, e.g., in diagnostic tests. Recent studies showed that expression of the P domain alone results in the formation of a subviral particle, the P particle (54, 60). Owing to its easy production in an Escherichia coli system and the same HBGA-binding properties and antigenicity as its parental VLP, the P particle has been used as a research tool of NV-HBGA interaction in a number of studies (54, 59, 60, 67, 68, 69). This report took advantage of the convenient P particle model to study the phenotypic HBGA-binding properties and antigenicity of GII-4 NVs that have circulated in the past decade.NVs are grouped into five genogroups (GI to GV), of which GI and GII are involved in the majority of acute viral gastroenteritis cases in humans. Strains within each genogroup can be further divided into genotypes, and up to 30 genotypes of GI and GII NVs have been described (75). NVs can be detected throughout the year, with peaks during the fall and winter seasons. Strains representing multiple genotypes can be found cocirculating in the same geographical area during a season. However, a single genotype of NVs, GII-4 (genogroup II genotype 4), has been the predominant cause of major acute gastroenteritis epidemics in many countries since the mid-1990s, and the number of GII-4 epidemics has increased in recent years (49). Overall, the GII-4 genotype is estimated to be responsible for 60 to 80% of all NV-associated outbreaks worldwide (43).Molecular surveillance has found that the GII-4 viruses are continuously changing, with new variants emerging every 2 or 3 years (1, 2, 57, 71, 72). One hypothesis suggests that the GII-4 viruses might be under selection pressure of the herd immunity, similar to the epochal evolution model used to describe the evolution of influenza (flu) viruses (56). New antigenic variants of GII-4 derived by genetic shift (replacement) accompanied by changes of HBGA binding specificities have been reported (43). However, the HBGA-binding interfaces of NVs have been found to be highly conserved among NVs within each of the two major genogroups, supporting HBGAs as an important factor in NV evolution (69). In fact, it has been shown that the major HBGA-binding pattern of GII-4 viruses to the H3, Le^b, and Le^y antigens has remained unchanged from 1974 to 1997 (4, 23, 24).The objective of this study was to elucidate the roles of HBGAs and host immunity in NV evolution using GII-4 viruses as a model. Since most of the studies on the epochal evolution of GII-4 were based on genetic analysis and focused on GII-4 variants identified in the past decade, we performed a study on the GII-4 variants in the same period by both genetic and phenotypic characterizations. Phylogenetic analysis revealed 6 genetic clusters of GII-4 viruses similar to those reported before. Characterization of HBGA-binding patterns of the GII-4 viruses revealed a consensus phenotype of binding to all A, B, and O secretor HBGAs, with some variations in affinity to these antigens. We also discussed the role of both host immunity and HBGAs in NV evolution. While the host immunity may drive NVs for change, as a functional selection factor, the HBGAs may restrict variation. This counterselection mechanism may help in understanding the epochal evolution hypothesis. The principles found through the study of GII-4 NVs can also be applied to other genotypes, which may eventually lead to a refined functional classification of all NVs.