A survey of enteric viruses in domestic sewage

In this second study (1979-1981) of the viral content of sewage we have demonstrated the presence of poliovirus types 1, 2, and 3 in Laval and Montreal. Several strains of poliovirus types 2 and 3 were nonvaccinal. This is in contrast with our first study (1977-1978) in which only type 1 poliovirus isolates were nonvaccinal. Coxsackievirus types B-3, B-4, and B-5 and echovirus types 1, 7, and 11 were also isolated from sewage. Interestingly, these isolations coincided with reports of isolation of the same strains during the same period by diagnostic laboratories. Our method based on Vero and BSC-1 cell cultures for virus isolation and immune electron microscopy for identification permitted the recovery not only of several strains of enteroviruses but also of some adenoviruses and reoviruses.     

Agglutination of African Primate and Rodent Erythrocytes by Adenoviruses, Reoviruses, and Enteroviruses

African nonhuman primate and rodent erythrocytes were tested for agglutination by adenoviruses, reoviruses, and enteroviruses. Squirrel erythrocytes were agglutinated by reovirus serotypes and adenovirus types 3, 11, 16, and 21. Adenoviruses also agglutinated brazza monkey erythrocytes to the same titers as those obtained with either rhesus or grey monkey cells. Prototype reovirus types 1 and 2 agglutinated grey monkey erythrocytes to much lower titers than either squirrel or human group O red cells. Among the enteroviruses tested, only echovirus types 7 and 12 agglutinated grey, red-tail, brazza, and rhesus monkey erythrocytes. The specificity of agglutination of squirrel, grey, and brazza monkey erythrocytes by reoviruses, echoviruses, and adenoviruses, respectively, was confirmed by hemagglutination-inhibition tests. The titers obtained were similar to those obtained with erythrocytes usually used in these tests. Erythrocytes of bush babies, potto unstriped grass mice, swamp rat, rusty-nosed rat, bush rat, harsh-furred mice, soft-furred rat, and giant rat were not agglutinated by adenoviruses, reoviruses, or enteroviruses.     

Virus detection in monkeys with diarrhea: the association of adenoviruses with diarrhea and the possible role of rotaviruses.

To explore the role of viruses in the etiology of diarrhea in colony-reared monkeys, direct electron microscopy, the fluorescent virus precipitin test and cell culture inoculation were used to examine the stools of monkeys with and without diarrhea. The animals were predominantly rhesus with a few macaques of other species, and included infants, juveniles and adults. Adenoviruses were isolated from a higher proportion of specimens from rhesus monkeys with diarrhea (73% of specimens from infants and 78% of specimens from juveniles and adults) than from control monkeys without diarrhea (22% of specimens from infants and 26% of specimens from juveniles and adults). SV 20 was the most frequently isolated simian adenovirus type; SV 17 and SV 32 also were recovered. Noncultivable adenoviruses detectable only by electron microscopy were not seen. Although adenovirus excretion was associated with diarrhea, the causal role of adenoviruses was difficult to assess. When serial specimens from animals with chronic or intermittent episodes of diarrhea were examined, sequential infections with different viruses were found to be common. Rotaviruses were detected by electron microscopy and isolated in cell cultures from two infant rhesus monkeys with diarrhea. However, the low detection rate, together with negative serologic data on 40% of infant monkeys with diarrhea, suggested that rotaviruses were not the major cause of gastroenteritis in the monkeys under study.     

Obtaining group-specific fluorescent immunoglobulin for diagnosis of adenovirus infection]

The authors present the results of a comparative study of several schemes of rabbit immunization with mixtures consisting of 4 and 7 (mixture No. 1) nad 2, 4, and 7 (mixture No. 2) adenovirus types for the purpose of obtaining group-specific fluorescent immunoglobulins. Since a direct correlative dependence of the activity of fluorescent immunoglobulins on the antibody level to the adenoviruses of homologous type was revealed in the immune sera a combined scheme of mixture No. 2 administration was elaborated for the immunization of animals.     

在我国患急性腹泻成人中发现一种新轮状病毒

从3例急性成人腹泻患者粪便中,经电镜观察,成人腹泻轮状病毒—酶联免疫吸附试验(ADRV-ELISA)、普通轮状病毒—酶联免疫吸附试验(Rota-ELISA),猪抗C型(组)轮状病毒和鸡抗D型(组)轮状病毒血清分别与本病毒的免疫电镜试验以及RNA电泳等实验结果,发现了一种新轮状病毒,该病毒的形态结构与普通轮状病毒(Rotavirus)、成人腹泻论状病毒(Adult Diarrhoea Rotavirus,简称ADRV)极为相似,但抗原性与普通轮状病毒(A组),成人腹泻轮状病毒(B组),C型轮状病毒(C组),D型轮状病毒(D组)显然无关。基因分析表明,该病毒基因组由11个双链RNA片段组成,但其RNA电泳图型具有独自的特点,与目前公认的A组、B组、C组、D组论状病毒韵RNA电泳图型均不同,免疫电镜证实,该病毒能被人恢复期血清所凝集,表明该病毒可能是腹泻病人的病因。     

Two-day detection of infectious enteric and non-enteric adenoviruses by improved ICC-qPCR

In order to provide a more suitable response to public health concerns, we improved the detection of infectious human adenoviruses in water by optimising the commonly used integrated cell culture–PCR method. Risk evaluation studies seek for rapid detection of infectious adenoviruses, including the enteric types 40 and 41 that are considered as the second most common agents of gastroenteritis in children next to rotaviruses. The here-employed 293A cell line used for infectious status assessment showed its ability to multiply adenoviruses including type 41. Two modifications were moreover applied to the workflow for viral detection. The first occurred at the nucleic acid extraction step performed directly on all infected cells, while the second was the application of real-time quantitative PCR as detection tool. All adaptations led to a 3-day reduction of the response delay and an improved sensitivity especially for the enteric adenoviral types. The infectious status of laboratory strain types 2 and 41 was demonstrated by a more than 2-log₁₀ increase in genome quantity. These conclusions were confirmed and reinforced by the analysis of water samples applying the improved assay. Naturally occurring infectious adenoviruses were detected in wastewater and river water, within 2 days. Types belonging to the species human adenoviruses C and type 31 were observed, but the most frequently identified type was 41 (71 % of identified sequences, n?=?34). This highlights the usefulness of our method for a wide range of types, and especially for the most prevalent and public health-relevant enteric adenoviruses.     

Ultrastructure of cell types within the scolex and cephalic peduncle of the litobothriidean tapeworm,Litobothrium aenigmaticum

Litobothrium aenigmaticum Caira , Jensen , Waeschenbach , & Littlewood 2014 is a cestode species that parasitizes pelagic thresher sharks in Taiwan and the Gulf of California. A previous study using light microscopy suggested the scolex contains four types of unusual tissues, one of which was considered to aid in adhesion of the worm to the mucosa of the spiral intestine. The function of the other tissues was unknown. Also unknown was the function of two laterally paired ducts found extending throughout the length of the worm. The goal of the present study was to use transmission electron microscopy (TEM) to examine the internal anatomy of the scolex and cephalic peduncle of this tapeworm in more detail. Three specimens from Taiwan were examined with TEM and two with light microscopy. Transmission electron microscopy revealed that the laterally paired ducts were excretory, since they were closely associated with numerous protonephridia. The number of cell types, previously referred to as tissues, in the scolex was expanded to 11 based on a combination of features including cell size, nucleus size, ratio of the maximum diameter of the nucleus to maximum diameter of the whole cell, and types of observed organelles, as well as the presence or absence of cytoplasmic electron‐dense and electron‐lucent vesicles. The cytoplasm of all 11 cell types was found to include electron‐dense vesicles, and all types were periodic acid‐Schiff (PAS) positive. This suggests that the electron‐dense vesicles may contain glycoproteins and/or mucoproteins. As all 11 cell types occurred within the region of the scolex that is surrounded by a papilla of host mucosa at the site of tapeworm attachment, we hypothesize that one or more of these cell types may produce a substance that induces this inflammatory reaction. The specific products in the secretory vesicles and how those products are released are still unclear, as are any relationships between or among the cell types in terms of products produced. Further research will be necessary to fully understand the complexity of this organism.     

Seasonal distribution of enteroviruses and adenoviruses in domestic sewage

A seasonal distribution of enteroviruses and adenoviruses in raw sewage effluents of Athens, Greece, was observed over a 15-month surveillance period. All 36 samples tested were positive for both virus groups. Adenovirus concentration levels ranged from 70 to 3200 cytopathic units per litre of sample, whereas the corresponding values for enteroviruses were 90-900 cytopathic units per litre. Peak values for adenoviruses were recorded during the months of April and June 1983, whereas for enteroviruses the peak was recorded in September 1983. All three types of poliovirus were present. Coxsackievirus types B-1, B-2, B-4, and B-5 and echovirus type 7 were also isolated. Adenovirus types 1, 2, 3, 5, 7, and 15 were detected as well.     

A synthetic peptide corresponding to the cleavage region of VP3 from rotavirus SA11 induces neutralizing antibodies.

Antibodies were elicited in rabbits by immunization with the synthetic tetradecapeptide Gln-Asn-Thr-Arg-Asn-Ile-Val-Pro-Val-Ser-Ile-Val-Ser-Arg, corresponding to amino acids 228 to 241 of SA11-VP3. Protein specificity of the antipeptide serum is demonstrated. The antipeptide serum revealed neutralizing activity directed against SA11 in a neutralization assay. Human rotavirus strains Wa, S2, and Hochi and bovine strains NCDV and UK were not neutralized, demonstrating the strain-specific neutralizing activity of the raised antipeptide serum. Upon immune electron microscopy, aggregation of SA11 particles was observed.     

Electron microscopy in the diagnosis of viral gastroenteritis in hospitalised children in the Czech Republic

Our study has been aimed at demonstrating the main role of viruses in the aetiology of acute gastroenteritis in children less than 5 years old and at pointing out the diagnostic potential of electron microscopy in the diagnosis of viral gastroenteritis. A prospective study was conducted to analyse the aetiology of diarrhoeal diseases in children less than 5 years of age admitted to the Department of Infectious Diseases between September 2006 and December 2008. All children were tested by faecal culture, latex agglutination and electron microscopy. A total of 832 children were included in the study. An aetiological agent was detected in 788 children (94.6 %). A bacterial aetiology was found in 22 (2.6 %) children and bacterial–viral co-infection was found in 146 (17.6 %) patients. The most frequent causative agents of gastroenteritis in children were viruses, which were detected in 620 (74.5 %) patients. The main causes of viral gastroenteritis were rotaviruses (detected in 410 children), followed by caliciviruses (42), coronaviruses (28), adenoviruses (19) and astroviruses (14). Dual viral infections were detected in 107 children, with rotavirus–calicivirus co-infection being the most common. Electron microscopy proved to be a more sensitive method in comparison with the latex agglutination test for the diagnosis of rotaviruses and adenoviruses. The major role of viruses in diarrhoeal diseases among children under 5 years of age in the Czech Republic has been confirmed. The diagnostic potential of electron microscopy, particularly in small outbreaks of gastroenteritis, was clearly shown.     

表达轮状病毒G2和G3型vp7基因重组腺病毒的免疫效果

为探索以非复制型腺病毒为表达载体的多价轮状病毒(Rotavirus,RV)基因工程疫苗的可行性,在前期工作的基础上,对表达我国G2和G3型RV流行毒株vp7基因的重组腺病毒的免疫效果进行了研究。分别用表达G2和G3型vp7基因的重组腺病毒rvAdG2VP7、rvAdG3VP7经滴鼻和灌胃两种途径免疫Balb/c小鼠,对免疫后小鼠的血清抗体、黏膜抗体和相关的细胞因子水平进行了检测和比较。结果表明,用表达G2和G3型vp7基因的重组腺病毒经滴鼻和灌胃两种途径免疫小鼠后,均可诱导机体产生较强的RV特异性免疫反应,包括体液免疫、细胞免疫和黏膜免疫,并能产生中和抗体。但免疫反应以Th2类为主,Th1类反应也占有相当的比例。本研究为新型RV基因工程疫苗的深入研究奠定了基础。     

A novel in vivo follicular dendritic cell-dependent iccosome-mediated mechanism for delivery of antigen to antigen-processing cells

Recent scanning electron microscopic studies on isolated follicular dendritic cells (FDC) showed that dendrites of certain FDC were "beaded," i.e., consisting of a series of interconnected immune complex coated bodies (termed "iccosomes," measuring 0.3 to 0.7 micron diameter). In vitro these iccosomes detach from one another with ease. The major objectives herein were to establish whether these structures can be detected in sections and whether iccosomes serve to disseminate antigen in vivo. Beginning at day 1, the time point used for isolating beaded FDC, the popliteal lymph nodes of immune C3H mice were studied with light and transmission electron microscopy for 2 wk (i.e., at days 1, 3, 5, 8, and 14) after hind footpad injection of the histochemically detectable antigen, horseradish peroxidase (HRP). Iccosomes (0.25 to 0.38 micron diameter), contoured by a peroxidase (PO)-positive coat of HRP-anti-HRP complexes, were first detected by transmission electron microscopy at day 1 adjacent to cell bodies of certain FDC. Within their limiting membrane they contained flocculent material that was PO positive. At day 3 by light microscopy, germinal centers were seen enlarged and the antigen-retaining reticulum, composed of antigen-bearing FDC, appeared diffuse. This coincided with the transmission electron microscopic visualization of a dispersed state of iccosomes among the follicular lymphocytes. At that time iccosomes were seen attached to the surface of lymphocytes via PO-positive immune complexes and were surrounded by microvillous processes of these cells. Germinal center lymphocytes and tingible body macrophages both responded to contact with iccosomes by endocytosis. Antigen-containing tingible body macrophage were most conspicuous by light microscopy at day 5, when transmission electron microscopy showed that the majority of germinal center lymphocytes contained endocytosed HRP in secondary lysosome-like granules associated with the Golgi apparatus. The number of dispersed iccosomes was markedly reduced by day 5. In controls injected with HSA, a PO-negative antigen, lymphocytes and tingible body macrophages were PO-negative. The presence of antigen in both cell types was confirmed through the use of a gold-conjugated antigen (goat IgG). Simultaneous immunoperoxidase labeling of the same tissues with anti-Ia showed the gold conjugate containing B cells to be Ia+. Antigen-positive B cells and tingible body macrophages were greatly reduced in numbers by day 14, suggesting the intracellular fragmentation of the antigen.(ABSTRACT TRUNCATED AT 400 WORDS)     

Detection of bovine and porcine adenoviruses for tracing the source of fecal contamination

In this study, a molecular procedure for the detection of adenoviruses of animal origin was developed to evaluate the level of excretion of these viruses by swine and cattle and to design a test to facilitate the tracing of specific sources of environmental viral contamination. Two sets of oligonucleotides were designed, one to detect porcine adenoviruses and the other to detect bovine and ovine adenoviruses. The specificity of the assays was assessed in 31 fecal samples and 12 sewage samples that were collected monthly during a 1-year period. The data also provided information on the environmental prevalence of animal adenoviruses. Porcine adenoviruses were detected in 17 of 24 (70%) pools of swine samples studied, with most isolates being closely related to serotype 3. Bovine adenoviruses were present in 6 of 8 (75%) pools studied, with strains belonging to the genera Mastadenovirus and Atadenovirus and being similar to bovine adenoviruses of types 2, 4, and 7. These sets of primers produced negative results in nested PCR assays when human adenovirus controls and urban-sewage samples were tested. Likewise, the sets of primers previously designed for detection of human adenovirus also produced negative results with animal adenoviruses. These results indicate the importance of further studies to evaluate the usefulness of these tests to trace the source of fecal contamination in water and food and for environmental studies.     

Comparison of three electron microscopy techniques for the detection of human rotaviruses

Rotavirus detection by direct electron microscopy was compared with direct and indirect immune electron microscopy techniques. The latter two approaches permitted the enumeration of 25 and 103 times more rotaviruses respectively, than direct electron microscopy. Also, 70% and 90% of the virus particles were aggregated by direct and indirect immune electron microscopy techniques respectively, thus facilitating their detection.     

Effect of Formamide on the Capsid Morphology of Adenovirus Types 4 and 7

Treatment of adenovirus types 4 and 7 with formamide disrupted the virions, degrading the capsids into predominantly single capsomers. As shown by electron microscopic observation, disruption proceeded in the following sequence: (i) reduction of the electron density of the virions, suggesting release of an internal component; (ii) progressive cleavage of the capsid into two or more segments and the formation (type 7 only) of capsomer “sheets”; (iii) final cleavage of the capsid into single or groups of a few capsomers. The sequence appeared similar for both adenoviruses; for both types, the rate and extent of disruption were dependent on the formamide concentration, but type 7 was more easily disrupted than type 4 by short-term (5 to 10 sec) treatment at the low (10%) concentration. At 30% formamide, the intercapsomer bonds of either type were fully cleaved, and the capsids were completely degraded into predominantly single capsomers. Pretreatment with formaldehyde did not prevent this degradation. Under suitable conditions, virus-derived remnants can be observed among the breakdown products. These remnants have been shorn of capsomers and presumably represent intact internal nucleoprotein.     

Detection of Bovine and Porcine Adenoviruses for Tracing the Source of Fecal Contamination

In this study, a molecular procedure for the detection of adenoviruses of animal origin was developed to evaluate the level of excretion of these viruses by swine and cattle and to design a test to facilitate the tracing of specific sources of environmental viral contamination. Two sets of oligonucleotides were designed, one to detect porcine adenoviruses and the other to detect bovine and ovine adenoviruses. The specificity of the assays was assessed in 31 fecal samples and 12 sewage samples that were collected monthly during a 1-year period. The data also provided information on the environmental prevalence of animal adenoviruses. Porcine adenoviruses were detected in 17 of 24 (70%) pools of swine samples studied, with most isolates being closely related to serotype 3. Bovine adenoviruses were present in 6 of 8 (75%) pools studied, with strains belonging to the genera Mastadenovirus and Atadenovirus and being similar to bovine adenoviruses of types 2, 4, and 7. These sets of primers produced negative results in nested PCR assays when human adenovirus controls and urban-sewage samples were tested. Likewise, the sets of primers previously designed for detection of human adenovirus also produced negative results with animal adenoviruses. These results indicate the importance of further studies to evaluate the usefulness of these tests to trace the source of fecal contamination in water and food and for environmental studies.     

Adenovirus type 11 uses CD46 as a cellular receptor

The 51 human adenovirus serotypes are divided into six species (A to F). Many adenoviruses use the coxsackie-adenovirus receptor (CAR) for attachment to host cells in vitro. Species B adenoviruses do not compete with CAR-binding serotypes for binding to host cells, and it has been suggested that species B adenoviruses use a receptor other than CAR. Species B adenoviruses mainly cause disease in the respiratory tract, the eyes, and in the urinary tract. Here we demonstrate that adenovirus type 11 (Ad11; of species B) binds to Chinese hamster ovary (CHO) cells transfected with CD46 (membrane cofactor protein)-cDNA at least 10 times more strongly than to CHO cells transfected with cDNAs encoding CAR or CD55 (decay accelerating factor). Nonpermissive CHO cells were rendered permissive to Ad11 infection upon transfection with CD46-cDNA. Soluble Ad11 fiber knob but not Ad7 or Ad5 knob inhibited binding of Ad11 virions to CD46-transfected cells, and anti-CD46 antibodies inhibited both binding of and infection by Ad11. From these results we conclude that CD46 is a cellular receptor for Ad11.     

Immune-Complexed Adenovirus Induce AIM2-Mediated Pyroptosis in Human Dendritic Cells

Human adenoviruses (HAdVs) are nonenveloped proteinaceous particles containing a linear double-stranded DNA genome. HAdVs cause a spectrum of pathologies in all populations regardless of health standards. Following repeat exposure to multiple HAdV types, we develop robust and long-lived humoral and cellular immune responses that provide life-long protection from de novo infections and persistent HAdV. How HAdVs, anti-HAdV antibodies and antigen presenting cells (APCs) interact to influence infection is still incompletely understood. In our study, we used physical, pharmacological, biochemical, fluorescence and electron microscopy, molecular and cell biology approaches to dissect the impact of immune-complexed HAdV (IC-HAdV) on human monocyte-derived dendritic cells (MoDCs). We show that IC-HAdV generate stabilized complexes of ~200 nm that are efficiently internalized by, and aggregate in, MoDCs. By comparing IC-HAdV, IC-empty capsid, IC-Ad2ts1 (a HAdV-C2 impaired in endosomal escape due to a mutation that impacts protease encapsidation) and IC-AdL40Q (a HAdV-C5 impaired in endosomal escape due to a mutation in protein VI), we demonstrate that protein VI-dependent endosomal escape is required for the HAdV genome to engage the DNA pattern recognition receptor AIM2 (absent in melanoma 2). AIM2 engagement induces pyroptotic MoDC death via ASC (apoptosis-associated speck protein containing a caspase activation/recruitment domain) aggregation, inflammasome formation, caspase 1 activation, and IL-1β and gasdermin D (GSDMD) cleavage. Our study provides mechanistic insight into how humoral immunity initiates an innate immune response to HAdV-C5 in human professional APCs.     

CD46 is a cellular receptor for all species B adenoviruses except types 3 and 7

The 51 human adenovirus serotypes are divided into six species (A to F). Adenovirus serotypes from all species except species B utilize the coxsackie-adenovirus receptor for attachment to host cells in vitro. Species B adenoviruses primarily cause ocular and respiratory tract infections, but certain serotypes are also associated with renal disease. We have previously demonstrated that adenovirus type 11 (species B) uses CD46 (membrane cofactor protein) as a cellular receptor instead of the coxsackie-adenovirus receptor (A. Segerman et al., J. Virol. 77:9183-9191, 2003). In the present study, we found that transfection with human CD46 cDNA rendered poorly permissive Chinese hamster ovary cells more permissive to infection by all species B adenovirus serotypes except adenovirus types 3 and 7. Moreover, rabbit antiserum against human CD46 blocked or efficiently inhibited all species B serotypes except adenovirus types 3 and 7 from infecting human A549 cells. We also sequenced the gene encoding the fiber protein of adenovirus type 50 (species B) and compared it with the corresponding amino acid sequences from selected serotypes, including all other serotypes of species B. From the results obtained, we conclude that CD46 is a major cellular receptor on A549 cells for all species B adenoviruses except types 3 and 7.     

Comparative study of the morphology and antigenic properties of recombinant analogs of a Marburg virus nucleoprotein]

The full-length gene for Marburg virus (MV) nucleoprotein (NP) was cloned in prokaryotic pQE32 under the control of the T5 promoter and in eukaryotic pTM1 under the control of the promoter for T7 RNA polymerase. Recombinant NP was synthesized in Escherichia coli and in human kidney cell line 293 cotransfected with recombinant vaccinia virus vTF7-3 expressing T7 RNA polymerase. On evidence of electron microscopy with immune detection, recombinant NP formed tubules of two types in E. coli and of a single type in cell line 293. ELISA and immunoblotting with polyclonal and monoclonal antibodies revealed common antigenic determinants in recombinant NP and natural MV NP.