Chlorella viruses isolated in China.

Plaque-forming viruses of the unicellular, eucaryotic, exsymbiotic, Chlorella-like green algae strain NC64A, which are common in the United States, were also present in fresh water collected in the People's Republic of China. Seven of the Chinese viruses were examined in detail and compared with the Chlorella viruses previously isolated in the United States. Like the American viruses, the Chinese viruses were large polyhedra and sensitive to chloroform. They contained numerous structural proteins and large double-stranded DNA genomes of at least 300 kilobase pairs. Each of the DNAs from the Chinese viruses contained 5-methyldeoxycytosine, which varied from 12.6 to 46.7% of the deoxycytosine, and N6-methyldeoxyadenosine, which varied from 2.2 to 28.3% of the deoxyadenosine. Four of the Chinese virus DNAs hybridized extensively with DNA from the American virus PBCV-1, and three hybridized poorly.     

Chlorella viruses isolated in China

Plaque-forming viruses of the unicellular, eucaryotic, exsymbiotic, Chlorella-like green algae strain NC64A, which are common in the United States, were also present in fresh water collected in the People's Republic of China. Seven of the Chinese viruses were examined in detail and compared with the Chlorella viruses previously isolated in the United States. Like the American viruses, the Chinese viruses were large polyhedra and sensitive to chloroform. They contained numerous structural proteins and large double-stranded DNA genomes of at least 300 kilobase pairs. Each of the DNAs from the Chinese viruses contained 5-methyldeoxycytosine, which varied from 12.6 to 46.7% of the deoxycytosine, and N6-methyldeoxyadenosine, which varied from 2.2 to 28.3% of the deoxyadenosine. Four of the Chinese virus DNAs hybridized extensively with DNA from the American virus PBCV-1, and three hybridized poorly.     

Restriction endonuclease activity induced by NC-1A virus infection of a Chlorella-like green alga.

A type II restriction endonuclease, CviBI, was isolated from a eukaryotic, Chlorella-like green alga infected with the dsDNA containing virus NC-1A. The enzyme recognizes the sequence GANTC and cleaves DNA between the G and A. Methylation of deoxyadenosine in the GANTC sequence probably inhibits enzyme activity. In vitro CviBI cleaves host nuclear DNA but not viral DNA. A survey of 18 other viruses which infect the same Chlorella sp. revealed that infection with 5 of these viruses also induced a restriction endonuclease which cleaves DNA into the same size fragments as CviBI.     

NUCLEOTIDE SEQUENCE OF THE GENES FOR RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE LARGE SUBUNIT AND RIBOSOMAL PROTEIN S14 FROM A CHLORELLA-LIKE GREEN ALGA1

Two chloroplast genes were sequenced from an exsymbiotic strain of a eukaryotic, Chlorella-like green alga. The genes for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) and the ribosomal protein S14 (rps14) were oriented in the same direction and were separated by 402 bp. The rbcLs of the exsymbiont and a free living Chlorella ellipsoidea were compared with other reported rbcL sequences. The rbcL gene of the exsymbiont is closely related to that of free-living Chlorella ellipsoidea. This is the first published report of an rps 14 gene sequence from an alga.     

Isolation and characterization of Chlorella viruses from freshwater sources in Korea

We isolated 23 Chlorella viruses from 9 Korean cities. The viruses were initially amplified in the Chlorella strain NC64A. Pure isolates were obtained by repeated plaque isolations. A SDS-PAGE analysis revealed similar but distinct protein patterns, both among the group of purified viruses and in comparison with the prototype Chlorella virus PBCV-1. Digestions of the 330- to 350-kb genomic DNAs with 10 restriction enzymes revealed different restriction fragment patterns among the isolates. One isolate, SS-1, was resistant to digestion with HindIII, PvuII, AluI, and HaeIII, indicating methylation at the AGCT or GC sequences. Some isolates reacted with antiserum against PBCV-1. The others that did not react to this PBCV-1 antibody reacted to the antibody that was raised against purified HS-2 virion. The tRNA-coding regions of 8 Chlorella viruses were cloned and sequenced. These viruses contained 14-16 tRNA genes within a 1.2- to 2-kb region, except for the SS-1 isolate, which had a 1039-bp spacer in a cluster of 11 tRNA genes. The SS-1 spacer contained an open-reading frame (ORF) of 294 amino acids. This ORF had a 51% amino acid sequence similarity to the PBCV-1 ORF A478L. A Southern blot analysis suggested that it was a novel gene that lacked a homologue in PBCV-1.     

中国小球藻病毒及其分子生物学性质

以小球藻NC64A株系为寄主,从17个省市采集的上百个水样中分离到了11个病毒分离物（BJ－1、BJ－2、BJ－3、BJ－4、FJ－1、FJ－2、NJ－1、HCJ－1、CDT－1、SCB—1、SCC－1）。这些病毒具有许多相同的性质,如均为球形多面体,基因组为300kb的dsDNA。但它们的DNA限制性酶切图谱,碱基组成和蛋白组分等均有差异。FJ－1的主要外壳蛋白的分子量小于54000,其它10个分离物则与PBCV-1一样,主要外壳蛋白的分子量为54000。Westernblot分析的结果显示,除FJ－1外其它病毒分离物与PBCV—1的抗血清有较强的免疫交叉反应。说明这些病毒与PBCV－1的同源性较高。在11个病毒分离物中,FJ－1在蛋白组分、碱基组成等方面与其它分离物和PBCV—1差别较大。     

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.     

Association of Paramecium bursaria Chlorella viruses with Paramecium bursaria cells: ultrastructural studies

Paramecium bursaria Chlorella viruses were observed by applying transmission electron microscopy in the native symbiotic system Paramecium bursaria (Ciliophora, Oligohymenophorea) and the green algae Chlorella (Chlorellaceae, Trebouxiophyceae). Virus particles were abundant and localized in the ciliary pits of the cortex and in the buccal cavity of P. bursaria. This was shown for two types of the symbiotic systems associated with two types of Chlorella viruses - Pbi or NC64A. A novel quantitative stereological approach was applied to test whether virus particles were distributed randomly on the Paramecium surface or preferentially occupied certain zones. The ability of the virus to form an association with the ciliate was investigated experimentally; virus particles were mixed with P. bursaria or with symbiont-free species P. caudatum. Our results confirmed that in the freshwater ecosystems two types of P. bursaria -Chlorella symbiotic systems exist, those without Chlorella viruses and those associated with a large amount of the viruses. The fate of Chlorella virus particles at the Paramecium surface was determined based on obtained statistical data and taking into account ciliate feeding currents and cortical reorganization during cell division. A life cycle of the viruses in the complete symbiotic system is proposed.     

Cross-protection of newly emerging HPAI H5 viruses by neutralizing human monoclonal antibodies: A viable alternative to oseltamivir

Newly emerging highly pathogenic avian influenza (HPAI) H5N2, H5N3, H5N5, H5N6, H5N8 and H5N9 viruses have been spreading in poultry and wild birds. The H5N6 viruses have also caused 10 human infections with 4 fatal cases in China. Here, we assessed the cross-neutralization and cross-protection of human and mouse monoclonal antibodies against 2 viruses: a HPAI H5N8 virus, A/chicken/Netherlands/14015526/2014 (NE14) and a HPAI H5N6 virus, A/Sichuan/26221/2014 (SC14). The former was isolated from an infected chicken in Netherlands in 2014 and the latter was isolated from an infected human patient in Sichuan, China. We show that antibodies FLA5.10, FLD21.140, 100F4 and 65C6, but not AVFluIgG01, AVFluIgG03, S139/1 and the VRC01 control, potently cross-neutralize the H5N8 NE14 and H5N6 SC14 viruses. Furthermore, we show that a single injection of >1 mg/kg of antibody 100F4 at 4 hours before, or 20 mg/kg antibody 100F4 at 72 hours after, a lethal dose of H5N8 NE14 enables mice to withstand the infection. Finally, we show that a single injection of 0.5 or 1 mg/kg antibody 100F4 prophylactically or 10 mg/kg 100F4 therapeutically outperforms a 5-day course of 10 mg/kg/day oseltamivir treatment against lethal H5N8 NE14 or H5N6 SC14 infection in mice. Our results suggest that further preclinical evaluation of human monoclonal antibodies against newly emerging H5 viruses is warranted.     

Planktonic viruses,heterotrophic bacteria,and nanoflagellates in fresh and coastal marine waters of the Kara Sea Basin (the Arctic)

In August–September 2009, the concentration of dissolved organic matter and quantitative distribution of virioplankton, bacterioplankton, and heterotrophic nanoflagellates were studied in the coastal waters of the Kara Sea, the fresh waters of the islands and the coasts of the sea, and the estuaries of the Ob’ and Yenisei rivers. A high positive correlation was observed between the abundances of viruses and bacteria. The frequency of visibly infected bacteria in marine waters ranged from 0.6 to 4.3% (an average of 1.6%); in the fresh waters of islands and coastline and in estuaries, it ranged from 0.3 to 3.9% (an average of 1.5%) and from 0.5 to 1.6% (an average of 1.1%) respectively. In most surveyed water bodies, the role of viruses in bacterioplankton mortality was considerably higher than that of heterotrophic flagellates.     

Nuclear envelope disruption involving host caspases plays a role in the parvovirus replication cycle

Parvoviruses are small, nonenveloped, single-stranded DNA viruses which replicate in the nucleus of the host cell. We have previously found that early during infection the parvovirus minute virus of mice (MVM) causes small, transient disruptions of the nuclear envelope (NE). We have now investigated the mechanism used by MVM to disrupt the NE. Here we show that the viral phospholipase A2, the only known enzymatic domain on the parvovirus capsid, is not involved in causing NE disruption. Instead, the virus utilizes host cell caspases, which are proteases involved in causing NE breakdown during apoptosis, to facilitate these nuclear membrane disruptions. Studies with pharmacological inhibitors indicate that caspase-3 in particular is involved. A caspase-3 inhibitor prevents nuclear lamin cleavage and NE disruption in MVM-infected mouse fibroblast cells and reduces nuclear entry of MVM capsids and viral gene expression. Caspase-3 is, however, not activated above basal levels in MVM-infected cells, and other aspects of apoptosis are not triggered during early MVM infection. Instead, basally active caspase-3 is relocalized to the nuclei of infected cells. We propose that NE disruption involving caspases plays a role in (i) parvovirus entry into the nucleus and (ii) alteration of the compartmentalization of host proteins in a way that is favorable for the virus.     

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.     

Virus-bacterium interactions in water and sediment of West African inland aquatic systems

The ecology of virioplankton in tropical aquatic ecosystems is poorly documented, and in particular, there are no references concerning African continental waters in the literature. In this study, we examined virus-bacterium interactions in the pelagic and benthic zones of seven contrasting shallow inland waters in Senegal, including one hypersaline lake. SYBR Gold-stained samples revealed that in the surface layers of the sites, the numbers of viruses were in the same range as the numbers of viruses reported previously for productive temperate systems. Despite high bacterial production rates, the percentages of visibly infected cells (as determined by transmission electron microscopy) were similar to the lowest percentages (range, 0.3 to 1.1%; mean, 0.5%) found previously at pelagic freshwater or marine sites, presumably because of the local environmental and climatic conditions. Since the percentages of lysogenic bacteria were consistently less than 8% for pelagic and benthic samples, lysogeny did not appear to be a dominant strategy for virus propagation at these sites. In the benthic samples, viruses were highly concentrated, but paradoxically, no bacteria were visibly infected. This suggests that sediment provides good conditions for virus preservation but ironically is an unfavorable environment for proliferation. In addition, given the comparable size distributions of viruses in the water and sediment samples, our results support the paradigm that aquatic viruses are ubiquitous and may have moved between the two compartments of the shallow systems examined. Overall, this study provides additional information about the relevance of viruses in tropical areas and indicates that the intensity of virus-bacterium interactions in benthic habitats may lower than the intensity in the adjacent bodies of water.     

Virus-Bacterium Interactions in Water and Sediment of West African Inland Aquatic Systems

The ecology of virioplankton in tropical aquatic ecosystems is poorly documented, and in particular, there are no references concerning African continental waters in the literature. In this study, we examined virus-bacterium interactions in the pelagic and benthic zones of seven contrasting shallow inland waters in Senegal, including one hypersaline lake. SYBR Gold-stained samples revealed that in the surface layers of the sites, the numbers of viruses were in the same range as the numbers of viruses reported previously for productive temperate systems. Despite high bacterial production rates, the percentages of visibly infected cells (as determined by transmission electron microscopy) were similar to the lowest percentages (range, 0.3 to 1.1%; mean, 0.5%) found previously at pelagic freshwater or marine sites, presumably because of the local environmental and climatic conditions. Since the percentages of lysogenic bacteria were consistently less than 8% for pelagic and benthic samples, lysogeny did not appear to be a dominant strategy for virus propagation at these sites. In the benthic samples, viruses were highly concentrated, but paradoxically, no bacteria were visibly infected. This suggests that sediment provides good conditions for virus preservation but ironically is an unfavorable environment for proliferation. In addition, given the comparable size distributions of viruses in the water and sediment samples, our results support the paradigm that aquatic viruses are ubiquitous and may have moved between the two compartments of the shallow systems examined. Overall, this study provides additional information about the relevance of viruses in tropical areas and indicates that the intensity of virus-bacterium interactions in benthic habitats may lower than the intensity in the adjacent bodies of water.     

Characterization of target cells for MCF viruses in AKR mice

The recombinant (MCF) class of murine leukemia virus appears to play an important role in lymphomagenesis in AKR and other mice. Although much effort has been extended in characterizing MCF viruses, relatively little is known about the cells they infect. I examined what cells were targets in AKR mice for both lymphomagenic and nonlymphomagenic MCF viruses. Lymphomagenic MCF viruses of thymic origin (AKR-247 and C58L1) were found to infect and replicate selectively in immature lymphocytes only present in thymic cortex, whereas nonlymphomagenic MCF viruses of splenic origin (C58v-1-C77 and C58v-2-C45) selectively infected and replicated in cells that appeared to B lymphocytes. Virus-binding studies suggested that neither T- nor B-lymphocyte tropisms were determined by selective attachment of virus to the respective cells. These findings demonstrate that in contrast with ecotropic viruses, which can infect many types of cells in the mouse, specific cellular tropisms can exist for MCF viruses, and that MCF infection, and therefore oncogenicity, is closely linked to cellular differentiation.     

Preliminary studies on sap-transmissible viruses of red clover (Trifolium pratense L.) in England and Wales

Red clover plants, collected from nine widely separated permanent pastures in England and Wales, were tested for sap-transmissible viruses. Viruses were identified by the symptoms they caused in test plants, by electron microscopy, and by serological tests. Of the 265 plants tested 14% were infected. Only pea mosaic virus was common and widespread; it was found in 8% of the plants, and in seven of the fields. Other viruses isolated were arabis mosaic, bean yellow mosaic, red clover mottle, and red clover vein mosaic; only red clover mottle virus produced diagnostic symptoms in red clover. No viruses were detected in seedlings grown from seed from eighty-nine commercial seed crops. Attempts to transmit red clover mottle virus by the Collembolan Sminthurus viridis L., which is common on red clover, failed.     

Complement fixation reaction in the typing of influenza viruses isolated in Rio de Janeiro]

The author studied by the complement fixation test the influenza virus strains isolated in Rio de Janeiro during the 1973 epidemic. He prepared immunesera in hamsters by the inoculation of the allantoic fluid from infected chick embryos with each of the 7 isolated strains and the standard strains. The soluble antigens were prepared with the allantoic fluid of infected chick embryos. The tests were identically positive with the A2/Hong Kong/68 and A2/England/72 antigens and negative with the B/Mass/66. The tests were type specific and the behaviour of the A2/Hong Kong/68 and the A2/England/72 and the 7 strains of the isolated viruses was almost the same. They fixed 3 or 4 units of complement. The variants PR8, FM1 and Asia fixed only 2 units of complement.     

Detection of animal and human enteric viruses in water from the Assomption River and its tributaries

Animal enteroviruses, reoviruses, and human enteric viruses were detected in water samples (20 L) from a major river system, the Assomption River in the province of Quebec. Animal enteroviruses, probably of porcine origin (this region is a major producer of pork), were isolated on porcine cell cultures and were found in 29 to 60% of water samples from the different sites on the river and in 19 to 48% of the water samples from the tributaries. The average concentration of these animal enteroviruses in water from the Assomption River was 2 to 7 mpniu/L (most probable number of infectious units per litre), and that from the tributaries varied from 3 to 24 mpniu/L. Reoviruses were detected in infected cell cultures by an enzyme-linked immunosorbent assay. Their origin is probably avian (broiler chicken farms) or human (untreated domestic waste waters) and they were detected in 19 to 52% of the water samples from the Assomption River at an average concentration of 3 to 12 mpniu/L. In water samples from the tributaries, 5 to 71% of the samples were positive at an average concentration of 5 to 24 mpniu/L. Human enteric viruses were detected in MA-104 cells by an immunoperoxidase assay using human immune serum globulin. They were detected in 13 to 72% of water samples from the Assomption River and 14 to 71% of the water samples from the tributaries. The average concentration of these human enteric viruses in Assomption River water varied from 1 to 12 and from 2 to 145 mpniu/L in water samples from the tributaries.(ABSTRACT TRUNCATED AT 250 WORDS)     

Degradation of urea by bacteria and algae in mass algal cultures.

Studies were conducted to determine the role of bacteria and algae in the degradation of urea in industrial waste waters. The microflora accompanying algae in continuous cultures on waste waters from the nitrogen fertilizers industry was found to include bacteria capable of intensive degradation of urea. Urea hydrolyzing bacteria are with time eliminated from the algal culture. This elimination can be attributed to algal metabolites which inhibit the development of sensitive bacteria. The Chlorella vulgaris strain used in the treatment of wast waters from the nitrogen fertilizers industry did not hydrolyse urea.