The fecal virome of pigs on a high-density farm

Swine are an important source of proteins worldwide but are subject to frequent viral outbreaks and numerous infections capable of infecting humans. Modern farming conditions may also increase viral transmission and potential zoonotic spread. We describe here the metagenomics-derived virome in the feces of 24 healthy and 12 diarrheic piglets on a high-density farm. An average of 4.2 different mammalian viruses were shed by healthy piglets, reflecting a high level of asymptomatic infections. Diarrheic pigs shed an average of 5.4 different mammalian viruses. Ninety-nine percent of the viral sequences were related to the RNA virus families Picornaviridae, Astroviridae, Coronaviridae, and Caliciviridae, while 1% were related to the small DNA virus families Circoviridae, and Parvoviridae. Porcine RNA viruses identified, in order of decreasing number of sequence reads, consisted of kobuviruses, astroviruses, enteroviruses, sapoviruses, sapeloviruses, coronaviruses, bocaviruses, and teschoviruses. The near-full genomes of multiple novel species of porcine astroviruses and bocaviruses were generated and phylogenetically analyzed. Multiple small circular DNA genomes encoding replicase proteins plus two highly divergent members of the Picornavirales order were also characterized. The possible origin of these viral genomes from pig-infecting protozoans and nematodes, based on closest sequence similarities, is discussed. In summary, an unbiased survey of viruses in the feces of intensely farmed animals revealed frequent coinfections with a highly diverse set of viruses providing favorable conditions for viral recombination. Viral surveys of animals can readily document the circulation of known and new viruses, facilitating the detection of emerging viruses and prospective evaluation of their pathogenic and zoonotic potentials.     

Rapid identification of known and new RNA viruses from animal tissues

Viral surveillance programs or diagnostic labs occasionally obtain infectious samples that fail to be typed by available cell culture, serological, or nucleic acid tests. Five such samples, originating from insect pools, skunk brain, human feces and sewer effluent, collected between 1955 and 1980, resulted in pathology when inoculated into suckling mice. In this study, sequence-independent amplification of partially purified viral nucleic acids and small scale shotgun sequencing was used on mouse brain and muscle tissues. A single viral agent was identified in each sample. For each virus, between 16% to 57% of the viral genome was acquired by sequencing only 42-108 plasmid inserts. Viruses derived from human feces or sewer effluent belonged to the Picornaviridae family and showed between 80% to 91% amino acid identities to known picornaviruses. The complete polyprotein sequence of one virus showed strong similarity to a simian picornavirus sequence in the provisional Sapelovirus genus. Insects and skunk derived viral sequences exhibited amino acid identities ranging from 25% to 98% to the segmented genomes of viruses within the Reoviridae family. Two isolates were highly divergent: one is potentially a new species within the orthoreovirus genus, and the other is a new species within the orbivirus genus. We demonstrate that a simple, inexpensive, and rapid metagenomics approach is effective for identifying known and highly divergent new viruses in homogenized tissues of acutely infected mice.     

2009年云南省边境地区健康儿童肠道病毒测序定型和ECHO7、ECHO13病毒的基因特征分析

了解2009年缅甸入境健康儿童和云南省口岸地区健康儿童肠道病毒(enterovirus,EV)带毒情况并对埃柯病毒7型(ECHO7)和埃柯病毒13型(ECHO13)的基因特征进行了描述。采集9个边境口岸小于15岁的健康儿童粪便标本271份,进行病毒分离和基因测序定型。271份便标本中共检测到EV30株(带毒率为11.1%),其中脊髓灰质炎病毒(poliovirus,PV)6株(阳性率2.8%),均为疫苗株,未发现脊灰野病毒;检测到非脊灰病毒(NPV)24株(阳性率8.9%)。经VP1区核苷酸序列测定,24株NPV全部为人类肠道病毒B组(HEV-B,6个血清型),其中13株为埃柯病毒7型(echovirus 7,ECHO7,占54.17%),5株为ECHO13(占20.83%)。未分离到HEV-A组,HEV-C组和HEV-D组病毒。2009年缅甸入境健康儿童和云南省口岸地区健康儿童中肠道病毒携带率较高,且均为HEV-B组病毒,其中主要型别为ECHO7和ECHO13,这两种病毒存在基因多样性特点(即存在不同的基因型)。     

Co-circulation and evolution of polioviruses and species C enteroviruses in a district of Madagascar

Between October 2001 and April 2002, five cases of acute flaccid paralysis (AFP) associated with type 2 vaccine-derived polioviruses (VDPVs) were reported in the southern province of the Republic of Madagascar. To determine viral factors that favor the emergence of these pathogenic VDPVs, we analyzed in detail their genomic and phenotypic characteristics and compared them with co-circulating enteroviruses. These VDPVs appeared to belong to two independent recombinant lineages with sequences from the type 2 strain of the oral poliovaccine (OPV) in the 5'-half of the genome and sequences derived from unidentified species C enteroviruses (HEV-C) in the 3'-half. VDPV strains showed characteristics similar to those of wild neurovirulent viruses including neurovirulence in poliovirus-receptor transgenic mice. We looked for other VDPVs and for circulating enteroviruses in 316 stools collected from healthy children living in the small area where most of the AFP cases occurred. We found vaccine PVs, two VDPVs similar to those found in AFP cases, some echoviruses, and above all, many serotypes of coxsackie A viruses belonging to HEV-C, with substantial genetic diversity. Several coxsackie viruses A17 and A13 carried nucleotide sequences closely related to the 2C and the 3D(pol) coding regions of the VDPVs, respectively. There was also evidence of multiple genetic recombination events among the HEV-C resulting in numerous recombinant genotypes. This indicates that co-circulation of HEV-C and OPV strains is associated with evolution by recombination, resulting in unexpectedly extensive viral diversity in small human populations in some tropical regions. This probably contributed to the emergence of recombinant VDPVs. These findings give further insight into viral ecosystems and the evolutionary processes that shape viral biodiversity.     

Acute diarrhea in west african children: diverse enteric viruses and a novel parvovirus genus

Parvoviruses cause a variety of mild to severe symptoms or asymptomatic infections in humans and animals. During a viral metagenomic analysis of feces from children with acute diarrhea in Burkina Faso, we identified in decreasing prevalence nucleic acids from anelloviruses, dependoviruses, sapoviruses, enteroviruses, bocaviruses, noroviruses, adenoviruses, parechoviruses, rotaviruses, cosavirus, astroviruses, and hepatitis B virus. Sequences from a highly divergent parvovirus, provisionally called bufavirus, were also detected whose NS1 and VP1 proteins showed <39% and <31% identities to those of previously known parvoviruses. Four percent of the fecal samples were PCR positive for this new parvovirus, including a related bufavirus species showing only 72% identity in VP1. The high degree of genetic divergence of these related genomes from those of other parvoviruses indicates the presence of a proposed new Parvoviridae genus containing at least two species. Studies of the tropism and pathogenicity of these novel parvoviruses will be facilitated by the availability of their genome sequences.     

Molecular identification and characterization of a new type of bovine enterovirus

Bovine enteroviruses belong to the family Picornaviridae. Little is known about their pathogenic potential; however, they cause asymptomatic infections in cattle and are excreted in feces. In the present study, viruses isolated from environmental samples were sequenced. According to phylogenetic analyses and standard picornavirus nomenclature, these isolates constitute a new type of bovine enterovirus serogroup A.     

Prevalence and genetic diversity of waterborne pathogenic viruses in surface waters of tropical urban catchments

Aims: To study the virological quality of surface water from highly urbanized tropical water catchment areas and to determine predominant enteric viral genotypes in surface water. Methods and Results: A wide range of human pathogenic viruses in urban surface waters was screened by nested PCR assays after concentration by ultrafiltration. Among the 84 water samples collected, at least one virus was detected in 70 (83·3%) of these samples. Noroviruses were determined to be the most prevalent enteric viruses detected in urban surface water samples, followed by astroviruses, enteroviruses, adenoviruses and hepatitis A viruses. The molecular characterization of environmental viral isolates suggested co‐circulation of multiple genotypes of both noroviruses GI and GII, astroviruses and enteroviruses in urban surface waters. Conclusions: Human enteric viruses with great genetic diversity were detected in surface waters, indicating the presence of human origin of faecal contamination in highly urbanized water catchment areas. Significance and Impact of the Study: The present study identifies and characterizes potential viral hazards of source waters for drinking water supply and recreational activities. This will enable scientific decisions to be made regarding the selection and prioritization of human pathogenic viruses to be included in the future risk assessment and treatment evaluation for water and wastewater.     

Genome Sequencing and Phylogenetic Analysis of 39 Human Parainfluenza Virus Type 1 Strains Isolated from 1997–2010

Thirty-nine human parainfluenza type 1 (HPIV-1) genomes were sequenced from samples collected in Milwaukee, Wisconsin from 1997–2010. Following sequencing, phylogenetic analyses of these sequences plus any publicly available HPIV-1 sequences (from GenBank) were performed. Phylogenetic analysis of the whole genomes, as well as individual genes, revealed that the current HPIV-1 viruses group into three different clades. Previous evolutionary studies of HPIV-1 in Milwaukee revealed that there were two genotypes of HPIV-1 co-circulating in 1991 (previously described as HPIV-1 genotypes C and D). The current study reveals that there are still two different HPIV-1 viruses co-circulating in Milwaukee; however, both groups of HPIV-1 viruses are derived from genotype C indicating that genotype D may no longer be in circulation in Milwaukee. Analyses of genetic diversity indicate that while most of the genome is under purifying selection some regions of the genome are more tolerant of mutation. In the 40 HPIV-1 genomes sequenced in this study, the nucleotide sequence of the L gene is the most conserved while the sequence of the P gene is the most variable. Over the entire protein coding region of the genome, 81 variable amino acid residues were observed and as with nucleotide diversity, the P protein seemed to be the most tolerant of mutation (and contains the greatest proportion of non-synonymous to synonymous substitutions) while the M protein appears to be the least tolerant of amino acid substitution.     

Epidemics and Frequent Recombination within Species in Outbreaks of Human Enterovirus B-Associated Hand,Foot and Mouth Disease in Shandong China in 2010 and 2011

The epidemiology and molecular characteristics of human enterovirus B (HEV-B) associated with hand, foot and mouth disease (HFMD) outbreaks in China are not well known. In the present study, we tested 201 HEV isolates from 233 clinical specimens from patients with severe HFMD during 2010–2011 in Linyi, Shandong, China. Of the 201 isolates, 189 were fully typed and 18 corresponded to HEV-B species (six serotypes CVA9, CVB1, CVB4, Echo 6, Echo 25 and Echo 30) using sensitive semi-nested polymerase chain reaction analysis of VP1 gene sequences. Phylogenetic analysis based on the VP1 region showed that eight E30SD belonged to a novel sub-genogroup D2; E25SD belonged to a novel sub-genogroup D6; E6SD belonged to sub-lineage C6 and five CVB1SD belonged to subgroup 4C; and B4SD belonged sub-lineage D2. The full viral genomes of the CVB1SD, E6SD, E25SD and E30SD isolates were sequenced. Analysis of phylogenetic and similarity plots indicated that E25SD recombined with E25-HN-2, E30FDJS03 and E4AUS250 at noncontiguous P2A–P3D regions, while E30SD, E30FDJ03, E25-HN-2 and E9 DM had shared sequences in discrete regions of P2 and P3. Both E6SD and B1SD shared sequences with E1-HN, B4/GX/10, B5-HN, and A9-Alberta in contiguous regions of most of P2 and P3. Genetic algorithm recombination detection analysis further confirmed the existence of multiple potential recombination points. In conclusion, analysis of the complete genomes of E25SD, E30SD, CVB1SD and E6SD isolated from HFMD patients revealed that they formed novel subgenogroup. Given the prevalence and recombination of these viruses in outbreaks of HFMD, persistent surveillance of HFMD-associated HEV-B pathogens is required to predict potential emerging viruses and related disease outbreaks.     

Human oral viruses are personal,persistent and gender-consistent

Viruses are the most abundant members of the human oral microbiome, yet relatively little is known about their biodiversity in humans. To improve our understanding of the DNA viruses that inhabit the human oral cavity, we examined saliva from a cohort of eight unrelated subjects over a 60-day period. Each subject was examined at 11 time points to characterize longitudinal differences in human oral viruses. Our primary goals were to determine whether oral viruses were specific to individuals and whether viral genotypes persisted over time. We found a subset of homologous viral genotypes across all subjects and time points studied, suggesting that certain genotypes may be ubiquitous among healthy human subjects. We also found significant associations between viral genotypes and individual subjects, indicating that viruses are a highly personalized feature of the healthy human oral microbiome. Many of these oral viruses were not transient members of the oral ecosystem, as demonstrated by the persistence of certain viruses throughout the entire 60-day study period. As has previously been demonstrated for bacteria and fungi, membership in the oral viral community was significantly associated with the sex of each subject. Similar characteristics of personalized, sex-specific microflora could not be identified for oral bacterial communities based on 16S rRNA. Our findings that many viruses are stable and individual-specific members of the oral ecosystem suggest that viruses have an important role in the human oral ecosystem.     

Relationship between group G chromosomes, especially chromosome 21, and the sensitivity of human cells to Coxsackie B viruses]

The R-method of differential chromosome staining by length was applied to comparative karyological studies on the culture of J 96 human cells susceptible to enteroviruses, and on the J 41 cell line derived from this culture and possessing high specific resistance to Coxsackie B viruses. Karyotype of the J 41 cell line was shown to be deprived of chromosome G21 (P less than 0.0001). The number of other chromosomes varied from cell to cell, but they are constantly present in the majority of cells of both the J 96 and J 41 cell lines. A conclusion is drawn that chromosome G21 incorporates gene(s) which controls the human cells susceptibility to Coxsackie B viruses.     

Bovine enteroviruses as indicators of fecal contamination

Surface waters frequently have been contaminated with human enteric viruses, and it is likely that animal enteric viruses have contaminated surface waters also. Bovine enteroviruses (BEV), found in cattle worldwide, usually cause asymptomatic infections and are excreted in the feces of infected animals in large numbers. In this study, the prevalence and genotype of BEV in a closed herd of cattle were evaluated and compared with BEV found in animals in the immediate environment and in environmental specimens. BEV was found in feces from 76% of cattle, 38% of white-tailed deer, and one of three Canada geese sharing the same pastures, as well as the water obtained from animal watering tanks, from the pasture, from streams running from the pasture to an adjacent river, and from the river, which emptied into the Chesapeake Bay. Furthermore, BEV was found in oysters collected from that river downstream from the farm. These findings suggest that BEV could be used as an indicator of fecal pollution originating from animals (cattle and/or deer). Partial sequence analysis of the viral genomes indicates that different viral variants coexist in the same area. The possibility of identifying the viral strains found in the animals and in the contaminated areas by sequencing the RNA genome, could provide a tool to find the origin of the contamination and should be useful for epidemiological and viral molecular evolution studies.     

Bovine Enteroviruses as Indicators of Fecal Contamination

Surface waters frequently have been contaminated with human enteric viruses, and it is likely that animal enteric viruses have contaminated surface waters also. Bovine enteroviruses (BEV), found in cattle worldwide, usually cause asymptomatic infections and are excreted in the feces of infected animals in large numbers. In this study, the prevalence and genotype of BEV in a closed herd of cattle were evaluated and compared with BEV found in animals in the immediate environment and in environmental specimens. BEV was found in feces from 76% of cattle, 38% of white-tailed deer, and one of three Canada geese sharing the same pastures, as well as the water obtained from animal watering tanks, from the pasture, from streams running from the pasture to an adjacent river, and from the river, which emptied into the Chesapeake Bay. Furthermore, BEV was found in oysters collected from that river downstream from the farm. These findings suggest that BEV could be used as an indicator of fecal pollution originating from animals (cattle and/or deer). Partial sequence analysis of the viral genomes indicates that different viral variants coexist in the same area. The possibility of identifying the viral strains found in the animals and in the contaminated areas by sequencing the RNA genome, could provide a tool to find the origin of the contamination and should be useful for epidemiological and viral molecular evolution studies.     

Characterization of the Non-Polio Enterovirus Infections Associated with Acute Flaccid Paralysis in South-Western India

Non-polio enteroviruses (NPEVs) have been reported frequently in association with acute flaccid paralysis (AFP) cases during Polio Surveillance Programs (PSPs) worldwide. However, there is limited understanding on the attributes of their infections. This study reports characteristics of NPEVs isolated from AFP cases, investigated during PSPs held in 2009–2010, in Karnataka and Kerala states of south-western India having varied climatic conditions. NPEV cell culture isolates derived from stool specimens that were collected from 422 of 2186 AFP cases (<1–14 years age) and 17 of 41 asymptomatic contacts; and details of all AFP cases/contacts were obtained from National Polio Laboratory, Bangalore. The distribution of NPEV infections among AFP cases and circulation pattern of NPEV strains were determined by statistical analysis of the data. Genotyping of all NPEV isolates was carried out by partial VP1 gene sequencing and phylogenetic analysis. NPEV positive AFP cases were significantly higher in children aged <2 years; with residual paralysis; in summer months; and in regions with relatively hot climate. Genotyping of NPEVs identified predominance of human enteroviruses (HEV)-B species [81.9%—Echoviruses (E): 57.3%; coxsackieviruses (CV) B: 15%; numbered EVs: 8.9%; CVA9: 0.7%] and low levels of HEV-A [14.5%—CVA: 6%; numbered EVs: 8.5%] and HEV-C [3.6%—CVA: 2.6%; numbered EVs: 1%] species, encompassing 63 genotypes. EV76 (6.3%) and each of E3, CVB3 and E9 (4.97%) were found frequently during 2009 while E11 (6.7%), CVB1 (6.1%), E7 (5.1%) and E20 (5.1%) were detected commonly in 2010. A marked proportion of AFP cases from children aged <2 years; presenting with fever; and from north and south interior parts of Karnataka state was detected with E/numbered EVs than that found with CVA/CVB. This study highlights the extensive genetic diversity and diverse circulation patterns of NPEV strains in AFP cases from different populations and climatic conditions.     

Characteristics of stork feces‐derived H5N1 viruses that are preferentially transmitted to primary human airway epithelial cells

Avian influenza viruses are a possible threat to human health as they may cause an influenza pandemic. Asian open‐bill storks are migratory birds that brought H5N1 viruses into Thailand during the 2004–2005 epidemic. However, to date, there are no reports of direct transmission of stork‐derived H5N1 viruses to Thais. Therefore, we questioned whether or not H5N1 viruses secreted in the feces of infected storks could directly infect cells derived from the human respiratory tract. To answer this question, we used primary NHBE cells as a model. We found that H5N1 viruses from two of the three cloacal swabs rapidly replicated and caused severe structural damage to the infected NHBE cells within the early phase of infection. Viruses from the remaining swab replicated poorly and caused no damage to the infected cells. The rapid‐replicating viruses were able to replicate efficiently even in the presence of a high level of type I IFN production and stimulated a high level of IL‐6 production but not the immunosuppressive cytokine, IL‐10. The genotypic study revealed that the major genotypes of the two rapid‐replicating viruses present in stork feces were the best‐fit genotypes for replication in the primary NHBE cells. In contrast, the major NA‐based genotype found in the cloacal swab containing slow‐replicating viruses could not survive in the primary NHBE cells. Altogether, the data suggested that those stork‐derived H5N1 viruses that preferentially replicated in human airway epithelial cells may exist in nature, and may not require additional mutations in order to defeat the species barrier.     

Non-Human Primates Harbor Diverse Mammalian and Avian Astroviruses Including Those Associated with Human Infections

Astroviruses (AstVs) are positive sense, single-stranded RNA viruses transmitted to a wide range of hosts via the fecal-oral route. The number of AstV-infected animal hosts has rapidly expanded in recent years with many more likely to be discovered because of the advances in viral surveillance and next generation sequencing. Yet no study to date has identified human AstV genotypes in animals, although diverse AstV genotypes similar to animal-origin viruses have been found in children with diarrhea and in one instance of encephalitis. Here we provide important new evidence that non-human primates (NHP) can harbor a wide variety of mammalian and avian AstV genotypes, including those only associated with human infection. Serological analyses confirmed that >25% of the NHP tested had antibodies to human AstVs. Further, we identified a recombinant AstV with parental relationships to known human AstVs. Phylogenetic analysis suggests AstVs in NHP are on average evolutionarily much closer to AstVs from other animals than are AstVs from bats, a frequently proposed reservoir. Our studies not only demonstrate that human astroviruses can be detected in NHP but also suggest that NHP are unique in their ability to support diverse AstV genotypes, further challenging the paradigm that astrovirus infection is species-specific.     

Comprehensive discovery of CRISPR-targeted terminally redundant sequences in the human gut metagenome: Viruses,plasmids, and more

Viruses are the most numerous biological entity, existing in all environments and infecting all cellular organisms. Compared with cellular life, the evolution and origin of viruses are poorly understood; viruses are enormously diverse, and most lack sequence similarity to cellular genes. To uncover viral sequences without relying on either reference viral sequences from databases or marker genes that characterize specific viral taxa, we developed an analysis pipeline for virus inference based on clustered regularly interspaced short palindromic repeats (CRISPR). CRISPR is a prokaryotic nucleic acid restriction system that stores the memory of previous exposure. Our protocol can infer CRISPR-targeted sequences, including viruses, plasmids, and previously uncharacterized elements, and predict their hosts using unassembled short-read metagenomic sequencing data. By analyzing human gut metagenomic data, we extracted 11,391 terminally redundant CRISPR-targeted sequences, which are likely complete circular genomes. The sequences included 2,154 tailed-phage genomes, together with 257 complete crAssphage genomes, 11 genomes larger than 200 kilobases, 766 genomes of Microviridae species, 56 genomes of Inoviridae species, and 95 previously uncharacterized circular small genomes that have no reliably predicted protein-coding gene. We predicted the host(s) of approximately 70% of the discovered genomes at the taxonomic level of phylum by linking protospacers to taxonomically assigned CRISPR direct repeats. These results demonstrate that our protocol is efficient for de novo inference of CRISPR-targeted sequences and their host prediction.     

Genomic diversity and evolution of the lyssaviruses

Lyssaviruses are RNA viruses with single-strand, negative-sense genomes responsible for rabies-like diseases in mammals. To date, genomic and evolutionary studies have most often utilized partial genome sequences, particularly of the nucleoprotein and glycoprotein genes, with little consideration of genome-scale evolution. Herein, we report the first genomic and evolutionary analysis using complete genome sequences of all recognised lyssavirus genotypes, including 14 new complete genomes of field isolates from 6 genotypes and one genotype that is completely sequenced for the first time. In doing so we significantly increase the extent of genome sequence data available for these important viruses. Our analysis of these genome sequence data reveals that all lyssaviruses have the same genomic organization. A phylogenetic analysis reveals strong geographical structuring, with the greatest genetic diversity in Africa, and an independent origin for the two known genotypes that infect European bats. We also suggest that multiple genotypes may exist within the diversity of viruses currently classified as 'Lagos Bat'. In sum, we show that rigorous phylogenetic techniques based on full length genome sequence provide the best discriminatory power for genotype classification within the lyssaviruses.