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.     

Bat Airway Epithelial Cells: A Novel Tool for the Study of Zoonotic Viruses

Bats have been increasingly recognized as reservoir of important zoonotic viruses. However, until now many attempts to isolate bat-borne viruses in cell culture have been unsuccessful. Further, experimental studies on reservoir host species have been limited by the difficulty of rearing these species. The epithelium of the respiratory tract plays a central role during airborne transmission, as it is the first tissue encountered by viral particles. Although several cell lines from bats were established recently, no well-characterized, selectively cultured airway epithelial cells were available so far. Here, primary cells and immortalized cell lines from bats of the two important suborders Yangochiroptera and Yinpterochiroptera, Carollia perspicillata (Seba''s short-tailed bat) and Eidolon helvum (Straw-colored fruit bat), were successfully cultured under standardized conditions from both fresh and frozen organ specimens by cell outgrowth of organ explants and by the use of serum-free primary cell culture medium. Cells were immortalized to generate permanent cell lines. Cells were characterized for their epithelial properties such as expression of cytokeratin and tight junctions proteins and permissiveness for viral infection with Rift-Valley fever virus and vesicular stomatitis virus Indiana. These cells can serve as suitable models for the study of bat-borne viruses and complement cell culture models for virus infection in human airway epithelial cells.     

Metagenomics: A New Direction in Ecology

Biology Bulletin - The prospects for application of metagenomic technologies in environmental studies are discussed. The advantages in investigating the taxonomic composition of aquatic and...     

Enhanced Passive Bat Rabies Surveillance in Indigenous Bat Species from Germany - A Retrospective Study

In Germany, rabies in bats is a notifiable zoonotic disease, which is caused by European bat lyssaviruses type 1 and 2 (EBLV-1 and 2), and the recently discovered new lyssavirus species Bokeloh bat lyssavirus (BBLV). As the understanding of bat rabies in insectivorous bat species is limited, in addition to routine bat rabies diagnosis, an enhanced passive surveillance study, i.e. the retrospective investigation of dead bats that had not been tested for rabies, was initiated in 1998 to study the distribution, abundance and epidemiology of lyssavirus infections in bats from Germany. A total number of 5478 individuals representing 21 bat species within two families were included in this study. The Noctule bat (Nyctalus noctula) and the Common pipistrelle (Pipistrellus pipistrellus) represented the most specimens submitted. Of all investigated bats, 1.17% tested positive for lyssaviruses using the fluorescent antibody test (FAT). The vast majority of positive cases was identified as EBLV-1, predominately associated with the Serotine bat (Eptesicus serotinus). However, rabies cases in other species, i.e. Nathusius'' pipistrelle bat (Pipistrellus nathusii), P. pipistrellus and Brown long-eared bat (Plecotus auritus) were also characterized as EBLV-1. In contrast, EBLV-2 was isolated from three Daubenton''s bats (Myotis daubentonii). These three cases contribute significantly to the understanding of EBLV-2 infections in Germany as only one case had been reported prior to this study. This enhanced passive surveillance indicated that besides known reservoir species, further bat species are affected by lyssavirus infections. Given the increasing diversity of lyssaviruses and bats as reservoir host species worldwide, lyssavirus positive specimens, i.e. both bat and virus need to be confirmed by molecular techniques.     

Isolation and Identification of a Natural Reassortant Mammalian Orthoreovirus from Least Horseshoe Bat in China

Background

Mammalian orthoreoviruses (MRVs) have a wide geographic distribution and can infect virtually all mammals. Infections in humans may be either symptomatic or asymptomatic. This study describes the isolation and identification of a natural reassortant MRV from least horseshoe bats (Rhinolophus pusillu) in China, referred to as RpMRV-YN2012.

Methods and Results

The RpMRV-YN2012 was obtained from urine samples of Rhinolophus pusillus by cell culture. Negative-staining electron microscopy revealed that RpMRV-YN2012 was a non-enveloped icosahedral virus with ∼75 nm in diameter. Polyacrylamide gel electrophoresis (PAGE) migration patterns of the genome segments showed that RpMRV-YN2012 contained 10 segments in a 3:3:4 arrangement. The whole genome sequence of RpMRV2012 was determined. The consensus terminal sequences of all segments of 5’-GCUAh…yUCAUC-3’ (h = A, U or C; y = C or U) were similar to the MRV species within the genus Orthoreovirus. Its evolution and evidence of genetic reassortment were analyzed by sequence comparison and phylogenetic analysis. The results showed that RpMRV-YN2012 is a novel serotype 2 MRV that may have originated from reassortment among bat, human, and/or pig MRV strains which associated with diarrhea, acute gastroenteritis and necrotizing encephalopathy in animals and humans.

Conclusions

RpMRV-YN2012 is a novel bat reassortant MRV, which may have resulted from a reassortment involving MRVs known to infect humans and animals. It is necessary to identify whether RpMRV-YN2012 is associated with diarrhea, acute gastroenteritis and necrotizing encephalopathy in clinical patients. In addition, we should carefully monitor its evolution and virulence in real time.     

Large Tailed Spindle Viruses of Archaea: a New Way of Doing Viral Business

Viruses of Archaea continue to surprise us. Archaeal viruses have revealed new morphologies, protein folds, and gene content. This is especially true for large spindle viruses, which infect only Archaea. We present a comparison of particle morphologies, major coat protein structures, and gene content among the five characterized large spindle viruses to elucidate defining characteristics. Structural similarities and a core set of genes support the grouping of the large spindle viruses into a new superfamily.     

Identification and Genome Characterization of the First Sicinivirus Isolate from Chickens in Mainland China by Using Viral Metagenomics

Unlike traditional virus isolation and sequencing approaches, sequence-independent amplification based viral metagenomics technique allows one to discover unexpected or novel viruses efficiently while bypassing culturing step. Here we report the discovery of the first Sicinivirus isolate (designated as strain JSY) of picornaviruses from commercial layer chickens in mainland China by using a viral metagenomics technique. This Sicinivirus isolate, which contains a whole genome of 9,797 nucleotides (nt) excluding the poly(A) tail, possesses one of the largest picornavirus genome so far reported, but only shares 88.83% and 82.78% of amino acid sequence identity to that of ChPV1 100C ({"type":"entrez-nucleotide","attrs":{"text":"KF979332","term_id":"672942599","term_text":"KF979332"}}KF979332) and Sicinivirus 1 strain UCC001 ({"type":"entrez-nucleotide","attrs":{"text":"NC_023861","term_id":"602259859","term_text":"NC_023861"}}NC_023861), respectively. The complete 939 nt 5′UTR of the isolate strain contains at least twelve stem-loop domains (A–L), representing the highest set of loops reported within Sicinivirus genus. The conserved ''barbell-like'' structure was also present in the 272 nt 3′UTR of the isolate as that in the 3′ UTR of Sicinivirus 1 strain UCC001. The 8,586 nt large open reading frame encodes a 2,862 amino acids polyprotein precursor. Moreover, Sicinivirus infection might be widely present in commercial chicken farms in Yancheng region of the Jiangsu Province as evidenced by all the tested stool samples from three different farms being positive (17/17) for Sicinivirus detection. This is the first report on identification of Sicinivirus in commercial layer chickens with a severe clinical disease in mainland China, however, further studies are needed to evaluate the pathogenic potential of this picornavirus in chickens.     

Adaptation of Pandemic H2N2 Influenza A Viruses in Humans

The 1957 A/H2N2 influenza virus caused an estimated 2 million fatalities during the pandemic. Since viruses of the H2 subtype continue to infect avian species and pigs, the threat of reintroduction into humans remains. To determine factors involved in the zoonotic origin of the 1957 pandemic, we performed analyses on genetic sequences of 175 newly sequenced human and avian H2N2 virus isolates and all publicly available influenza virus genomes.     

Oxidative Activation of K-Cl Cotransport by Diamide in Erythrocytes from Humans with Red Cell Disorders,and from Several Other Mammalian Species

Red blood cells (RBCs) from different mammalian species were investigated for the presence of diamide-induced oxidative activation of K-Cl cotransport reported to be present in sheep but absent in human RBCs. K efflux was measured in RBCs from human with hemoglobin (Hb) A or S, glucose-phosphate dehydrogenase (G6PDH) and a cytoskeletal deficiency, and from rat, mouse and rabbit. RBCs were incubated with diamide (0–1.0 mm) in K-free Cl or NO₃ media of variable osmolalities (200–450 mOsM). Cl-dependent K efflux or K-Cl cotransport (estimated as the difference between K efflux rate constants in Cl and NO₃) was activated by diamide in a sigmoidal fashion. Relative maximum K-Cl cotransport followed the sequence: human HbA (1) < rabbit (1.8) < sheep (6.9) < human HbS (9.5) ∼ rat (9.7). Relative diamide concentrations for half maximal activation of K-Cl cotransport followed the sequence: sheep (1.9) > human Hb A (1) > rabbit (0.75) > human HbS and rat (0.67). Cell swelling in 200 mOsM doubled K-Cl cotransport in diamide, both in human HbA and S cells but reduced that in rat RBCs. In contrast, cell shrinkage at 450 mOsM obliterated K-Cl cotransport in human HbA and S but not in rat RBCs. Human RBCs with G6PDH and a cytoskeleton deficiency behaved like HbA RBCs. In mouse RBCs, diamide-activated K-Cl cotransport was 30% higher in isotonic than in hypotonic medium. In human HbA and S, and in low or high K sheep RBCs fractionated by Percoll density gradient, diamide increased the activity of K-Cl cotransport, an effect inversely correlated with cell density. Analysis of pooled data reveals that K-Cl cotransport accounted for about 80% of all K flux in Cl. There was a statistically significant correlation between K-Cl cotransport and K efflux in Cl (P < 0.00001) and in NO₃ (P < 0.00001). In conclusion, a diamide-activated K-Cl cotransport was present in human RBCs and in all other mammalian RBCs tested, with a large inter-, and for human and sheep, intraspecies variability for its maximum activity. Received: 5 June 1996/Revised: 4 October 1996     

Effects of Renal Denervation on Renal Artery Function in Humans: Preliminary Study

Aim

To study the effects of RD on renal artery wall function non-invasively using magnetic resonance.

Methods and Results

32 patients undergoing RD were included. A 3.0 Tesla magnetic resonance of the renal arteries was performed before RD and after 6-month. We quantified the vessel sharpness of both renal arteries using a quantitative analysis tool (Soap-Bubble®). In 17 patients we assessed the maximal and minimal cross-sectional area of both arteries, peak velocity, mean flow, and renal artery distensibility. In a subset of patients wall shear stress was assessed with computational flow dynamics. Neither renal artery sharpness nor renal artery distensibility differed significantly. A significant increase in minimal and maximal areas (by 25.3%, p = 0.008, and 24.6%, p = 0.007, respectively), peak velocity (by 16.9%, p = 0.021), and mean flow (by 22.4%, p = 0.007) was observed after RD. Wall shear stress significantly decreased (by 25%, p = 0.029). These effects were observed in blood pressure responders and non-responders.

Conclusions

RD is not associated with adverse effects at renal artery level, and leads to an increase in cross-sectional areas, velocity and flow and a decrease in wall shear stress.     

Inborn Errors of RNA Lariat Metabolism in Humans with Brainstem Viral Infection

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Immunohistochemical Localization of Phosphodiesterase 2A in Multiple Mammalian Species

Phosphodiesterases (PDEs) comprise a family of enzymes that regulate the levels of cyclic nucleotides, key second messengers that mediate a diverse array of functions. PDE2A is an evolutionarily conserved cGMP-stimulated cAMP and cGMP PDE. In the present study, the regional and cellular distribution of PDE2A in tissues of rats, mice, cynomolgus monkeys, dogs, and humans was evaluated by immunohistochemistry. A polyclonal antibody directed to the C-terminal portion of PDE2A specifically detected PDE2A by Western blotting and by immunohistochemistry. The pattern of PDE2A immunoreactivity (ir) was consistent across all species. Western blot analysis demonstrated that PDE2A was most abundant in the brain relative to peripheral tissues. PDE2A ir was heterogeneously distributed within brain and was selectively expressed in particular peripheral tissues. In the brain, prominent immunoreactivity was apparent in components of the limbic system, including the isocortex, hippocampus, amygdala, habenula, basal ganglia, and interpeduncular nucleus. Cytoplasmic PDE2A staining was prominent in several peripheral tissues, including the adrenal zona glomerulosa, neurons of enteric ganglia, endothelial cells in all organs, lymphocytes of spleen and lymph nodes, and pituitary. These studies suggest that PDE2A is evolutionarily conserved across mammalian species and support the hypothesis that the enzyme plays a fundamental role in signal transduction. (J Histochem Cytochem 57:933–949, 2009)     

我国大陆细蜉属Caenis 修订及一新种描述(蜉蝣目:细蜉科)

我国大陆细蜉属Caenis共知5种,其中短铗细蜉Caenis parviforcipis(Zhou et al.)n．comb．由短尾蜉属Brachycercus转移而来;中华细蜉Caenis sinensis Gui et al．稚虫形态在本文中首次描述;另外从标本来看,已知的黑铗细蜉Caenis nigroforceps Zhou et al．实际为近岸细蜉Caenis rivulorum Eaton的新异名;本文描述1新种：花斑细蜉Caenis melanoleuca,sp．nov．;第5种黑点细蜉Caenis nigropunctata Klapalek也根据标本得到了确认。文中还给出了以上5种成虫和4种稚虫的检索表。所有标本都保存在南京师范大学生命科学学院。     

A New Technique for the Study of Viruses in the Electron Microscope

A method is described for the application of immunochemical stains to virus-infected cells in preparation for examination in the electron microscope. Specific antibodies to viral particles and to purified viral antigens have been rendered visible in the electron microscope by conjugation with the ironcontaining protein, ferritin. Examination of vaccinia-infected and influenza-infected cells treated with their specific ferritin-labelled antiserum has revealed the disposition of mature virus and viral precursors during various stages of the infection. Virus particles maturing at the cell surface and within the cytoplasm were specifically tagged and, in the case of influenza virus, the soluble, nucleoprotein viral-precursor was identified in distinct portions of the nucleus.     

甜瓜属一新物种(双二倍体)合成及定性

采用胚胎拯救方法,首次成功实现了栽培黄瓜(CucumissativusL.,2n=14)与同属野生种C.hystrixChakr.(2n=24)间可重复的种间杂交.杂交F1植株形态一致.其中多分枝、密棕茸毛(尤其是在花瓣和雌蕊上)、桔黄色花冠及卵圆形果实这些特征与亲本C.hystrix相似,而第一雌花节位则与亲本黄瓜(C.sativus)相似.其它性状如株径、节长、叶和花的形状和大小等都介于双亲之间而呈中间型.将杂种F1植株自交并与两亲本进行回交,结果表明F1杂种的雄蕊和雌蕊都是不育的.这可能是由于杂种染色体数目为奇数(2n=19,其中7条来自黄瓜,12条来自野生黄瓜),缺乏同源性而导致减数分裂不正常.利用体细胞无性系突变方法,对杂种的染色体数进行了加倍.流动细胞计量仪测定表明,加倍的F1植株(双二倍体)占再生植株的7.3%,形态一致,其DNA含量为2.35pg,而F1(二倍体)的含量为1.17pg.新合成的双二倍体植株能释放花粉,并且能形成含种子的果实.对生长和发育、营养价值及抗性等方面初步研究表明,这一新物种可望成为一新型作物种,在未来农业中占有一席之地.通过不同杂交可形成两种类型的果实一种为腌渍类型,该株系每株可着生30多个大约10cm长的果实,可一次性采收;另一种为耐弱光类型,果形细长,基本上无种子,适合于部分遮荫的环境,如温室栽培.营养分析表明,新物种果实的蛋白质含量为0.78%,矿物质0.35%,均分别高于普通黄瓜含量0.62%和0.27%.对根结线虫筛选试验结果表明,C.hystrix具有高度抗性,其抗性通过正反交可部分转移到F1代和双二倍体中.