Genetic diversity in marine algal virus communities as revealed by sequence analysis of DNA polymerase genes.

Algal-virus-specific PCR primers were used to amplify DNA polymerase gene (pol) fragments (683 to 689 bp) from the virus-sized fraction (0.02 to 0.2 microns) concentrated from inshore and offshore water samples collected from the Gulf of Mexico. Algal-virus-like DNA pol genes were detected in five samples collected from the surface and deep chlorophyll maximum. PCR products from an offshore station were cloned, and the genetic diversity of 33 fragments was examined by restriction fragment length polymorphism and sequence analysis. The five different genotypes or operational taxonomic units (OTUs) that were identified on the basis of restriction fragment length polymorphism banding patterns were present in different relative abundances (9 to 34%). One clone from each OTU was sequenced, and phylogenetic analysis showed that all of the OTUs fell within the family Phycodnaviridae. Four of the OTUs fell within a group of viruses (MpV) which infect the photosynthetic picoplankter Micromonas pusilla. The genetic diversity among these genotypes was as large as that previously found for MpV isolates from different oceans. The remaining genotype formed its own clade between viruses which infect M. pusilla and Chrysochromulina brevifilum. These results imply that marine virus communities contain a diverse assemblage of MpV-like viruses, as well as other unknown members of the Phycodnaviridae.     

Chloroviruses: not your everyday plant virus

Viruses infecting higher plants are among the smallest viruses known and typically have four to ten protein-encoding genes. By contrast, many viruses that infect algae (classified in the virus family Phycodnaviridae) are among the largest viruses found to date and have up to 600 protein-encoding genes. This brief review focuses on one group of plaque-forming phycodnaviruses that infect unicellular chlorella-like green algae. The prototype chlorovirus PBCV-1 has more than 400 protein-encoding genes and 11 tRNA genes. About 40% of the PBCV-1 encoded proteins resemble proteins of known function including many that are completely unexpected for a virus. In many respects, chlorovirus infection resembles bacterial infection by tailed bacteriophages.     

Diverse and dynamic populations of cyanobacterial podoviruses in the Chesapeake Bay unveiled through DNA polymerase gene sequences

Many podoviruses have been isolated which infect marine picocyanobacteria, and they may play a potentially important role in regulating the biomass and population composition of picocyanobacteria. However, little is known about the diversity and population dynamics of autochthonous cyanopodoviruses in marine environments. Using a set of newly designed PCR primers which specifically amplify the DNA pol from cyanopodoviruses, a total of 221 DNA pol sequences were retrieved from eight Chesapeake Bay virioplankton communities collected at different times and locations. All DNA pol sequences clustered with the eight known podoviruses that infect different marine picocyanobacteria, and could be divided into at least 10 different subclusters (I-X). The presence of these cyanopodovirus genotypes based on PCR-amplification of DNA pol gene sequences was supported by the existence of similar DNA pol genotypes with metagenome libraries of Chesapeake Bay virioplankton assemblages. The composition of cyanopodoviruses in the Bay also exhibited distinct winter and summer patterns which were likely related to corresponding seasonal changes in the composition of cyanobacterial populations. Our study suggests that diverse and dynamic populations of cyanopodoviruses are present in the estuarine environment. The PCR method developed in this study provides a specific and sensitive tool to explore the abundance, distribution and phylogenetic diversity of cyanopodoviruses in aquatic environments. Linking the dynamics of host and viral populations in the natural environment is critical to broader characterization of the ecological role of virioplankton within microbial communities.     

PCR amplification of large genomic fragments from human and simian immunodeficiency virus infected cell lines.

Polymerase chain reaction (PCR) has been used to amplify the large fragments from viral genomic DNA of SIV from wild caught, asymptomatic Erythrocebus monkeys from Western Africa (Senegal) and also from HIV-2 infected cell lines. By using consensus primer sequences from highly conserved stretches of gag, pol and env genes, two halves of the viral genome of HIV-2 and SIV (isolated from west African Erythrocebus monkeys) have amplified by PCR. One half spans 5200 bp from within the U3 region of the 5' long terminal repeat (LTR) into pol gene and an overlapping fragment spans 3700 bp from the pol gene into U5 region of 3' LTR. Also fragments ranging from 1-2.3 kb from gag pol and env genes have been successfully amplified. Our data demonstrate that primers used to amplify large segments from viral DNA yield better results if they are derived from a consensus sequence of a highly conserved stretch of the viral genome.     

猴泡沫病毒SFV-1前病毒pol基因克隆及序列分析

采用nested-PCR从猕猴肾组织细胞中获得同源性较高、具有高度保守序列的、长度为465 bp 大小的猴泡沫病毒SFV-1的 pol 基因的cDNA,将其克隆到pMD18-T载体中,并对其进行序列分析,分析结果表明所克隆的基因片断为猴泡沫病毒SFV-1 pol 基因片段,与文献中已发表的来源于灵长类动物SFV-1、SFV-1A、SFV-1B、SFV-2、 SFVmac、SFVkm的 pol 基因核苷酸序列进行比对,显示SFVkm1与上述病毒 pol 基因核苷酸同源性分别为91.06%、90.59%、90.82%、90.21%、89.41%、91.53%.对 pol 基因克隆和序列分析是了解和掌握SFV病毒分子流行病学及其变异趋势的重要手段,7 种不同基因型的泡沫病毒的系统进化树分析显示了SFVkm1与它们之间的相互联系.     

Topoisomerase II from Chlorella virus PBCV-1. Characterization of the smallest known type II topoisomerase

Type II topoisomerases, a family of enzymes that govern topological DNA interconversions, are essential to many cellular processes in eukaryotic organisms. Because no data are available about the functions of these enzymes in the replication of viruses that infect eukaryotic hosts, this led us to express and characterize the first topoisomerase II encoded by one of such viruses. Paramecium bursaria chlorella virus 1 (PBCV-1) infects certain chlorella-like green algae and encodes a 120-kDa protein with a similarity to type II topoisomerases. This protein was expressed in Saccharomyces cerevisiae and was highly active in relaxation of both negatively and positively supercoiled plasmid DNA, catenation of plasmid DNA, and decatenation of kinetoplast DNA networks. Its optimal activity was determined, and the omission of Mg(2+) or its replacement with other divalent cations abolished DNA relaxation. All activities of the recombinant enzyme were ATP dependent. Increasing salt concentrations shifted DNA relaxation from a normally processive mechanism to a distributive mode. Thus, even though the PBCV-1 enzyme is considerably smaller than other eukaryotic topoisomerase II enzymes (whose molecular masses are typically 160-180 kDa), it displays all the catalytic properties expected for a type II topoisomerase.     

Detection of enterotoxigenic Staphylococcus aureus in dried skimmed milk: use of the polymerase chain reaction for amplification and detection of staphylococcal enterotoxin genes entB and entC1 and the thermonuclease gene nuc

The polymerase chain reaction was used to amplify the staphylococcal enterotoxin B and C genes (entB and entC1) and the staphylococcal nuclease gene (nuc). Two sets of primers ("nested primers") were found to be necessary for the detection of low copy numbers of purified DNA in diluent. These allowed detection of ca. 1 fg of purified target DNA, while 100 pg was required before detection of entB, entC1, and nuc with single primer pairs was possible. With nested primers, enterotoxigenic Staphylococcus aureus cells could be detected in artificially contaminated dried skimmed milk samples at levels of ca. 10(5) CFU ml-1 within 8 h. No cross-reaction was observed between the highly homologous entB and entC1 genes. The method showed total specificity for entC1 when tested against a wide variety of other bacteria.     

Detection of enterotoxigenic Staphylococcus aureus in dried skimmed milk: use of the polymerase chain reaction for amplification and detection of staphylococcal enterotoxin genes entB and entC1 and the thermonuclease gene nuc.

The polymerase chain reaction was used to amplify the staphylococcal enterotoxin B and C genes (entB and entC1) and the staphylococcal nuclease gene (nuc). Two sets of primers ("nested primers") were found to be necessary for the detection of low copy numbers of purified DNA in diluent. These allowed detection of ca. 1 fg of purified target DNA, while 100 pg was required before detection of entB, entC1, and nuc with single primer pairs was possible. With nested primers, enterotoxigenic Staphylococcus aureus cells could be detected in artificially contaminated dried skimmed milk samples at levels of ca. 10(5) CFU ml-1 within 8 h. No cross-reaction was observed between the highly homologous entB and entC1 genes. The method showed total specificity for entC1 when tested against a wide variety of other bacteria.     

Paramecium bursaria chlorella virus 1 proteome reveals novel architectural and regulatory features of a giant virus

The 331-kbp chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) genome was resequenced and annotated to correct errors in the original 15-year-old sequence; 40 codons was considered the minimum protein size of an open reading frame. PBCV-1 has 416 predicted protein-encoding sequences and 11 tRNAs. A proteome analysis was also conducted on highly purified PBCV-1 virions using two mass spectrometry-based protocols. The mass spectrometry-derived data were compared to PBCV-1 and its host Chlorella variabilis NC64A predicted proteomes. Combined, these analyses revealed 148 unique virus-encoded proteins associated with the virion (about 35% of the coding capacity of the virus) and 1 host protein. Some of these proteins appear to be structural/architectural, whereas others have enzymatic, chromatin modification, and signal transduction functions. Most (106) of the proteins have no known function or homologs in the existing gene databases except as orthologs with proteins of other chloroviruses, phycodnaviruses, and nuclear-cytoplasmic large DNA viruses. The genes encoding these proteins are dispersed throughout the virus genome, and most are transcribed late or early-late in the infection cycle, which is consistent with virion morphogenesis.     

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.     

Identification of mosquito bloodmeals using mitochondrial cytochrome oxidase subunit I and cytochrome b gene sequences

Abstract.  Primer pairs were designed and protocols developed to selectively amplify segments of vertebrate mitochondrial cytochrome oxidase subunit 1 (COI) and cytochrome b (Cyt b ) mtDNA from the bloodmeals of mosquitoes (Diptera: Culicidae). The protocols use two pairs of nested COI primers and one pair of Cyt b primers to amplify short segments of DNA. Resultant sequences are then compared with sequences in GenBank, using the BLAST function, for putative host identification. Vertebrate DNA was amplified from 88% of our sample of 162 wild-caught, blood-fed mosquitoes from Oregon, U.S.A. and GenBank BLAST searches putatively identified 98% of the amplified sequences, including one amphibian, seven mammalian and 14 avian species. Criteria and caveats for putative identification of bloodmeals are discussed.     

Chlorovirus-mediated membrane depolarization of Chlorella alters secondary active transport of solutes

Paramecium bursaria chlorella virus 1 (PBCV-1) is the prototype of a family of large, double-stranded DNA, plaque-forming viruses that infect certain eukaryotic chlorella-like green algae from the genus Chlorovirus. PBCV-1 infection results in rapid host membrane depolarization and potassium ion release. One interesting feature of certain chloroviruses is that they code for functional potassium ion-selective channel proteins (Kcv) that are considered responsible for the host membrane depolarization and, as a consequence, the efflux of potassium ions. This report examines the relationship between cellular depolarization and solute uptake. Annotation of the virus host Chlorella strain NC64A genome revealed 482 putative transporter-encoding genes; 224 are secondary active transporters. Solute uptake experiments using seven radioactive compounds revealed that virus infection alters the transport of all the solutes. However, the degree of inhibition varied depending on the solute. Experiments with nystatin, a drug known to depolarize cell membranes, produced changes in solute uptake that are similar but not identical to those that occurred during virus infection. Therefore, these studies indicate that chlorovirus infection causes a rapid and sustained depolarization of the host plasma membrane and that this depolarization leads to the inhibition of secondary active transporters that changes solute uptake.     

Sequence analysis of marine virus communities reveals that groups of related algal viruses are widely distributed in nature

Algal-virus-specific PCR primers were used to amplify DNA polymerase (pol) gene fragments from geographically isolated natural virus communities. Natural algal virus communities were obtained from coastal sites in the Pacific Ocean in British Columbia, Canada, and the Southern Ocean near the Antarctic peninsula. Genetic fingerprints of algal virus communities were generated using denaturing gradient gel electrophoresis (DGGE). Sequencing efforts recovered 33 sequences from the gradient gel. Of the 33 sequences examined, 25 encoded a conserved amino acid motif indicating that the sequences were pol gene fragments. Furthermore, the 25 pol sequences were related to pol gene fragments from known algal viruses. In addition, similar virus sequences (>98% sequence identity) were recovered from British Columbia and Antarctica. Results from this study demonstrate that DGGE with degenerate primers can be used to qualitatively fingerprint and assess genetic diversity in specific subsets of natural virus communities and that closely related viruses occur in distant geographic locations. DGGE is a powerful tool for genetically fingerprinting natural virus communities and may be used to examine how specific components of virus communities respond to experimental manipulations.     

Molecular biological detection of anaerobic gut fungi (Neocallimastigales) from landfill sites

Oligonucleotide primers were designed for the 18S rRNA genes of members of the Neocallimastigales and used in a nested PCR protocol to amplify 787-bp fragments of DNA from landfill site samples. The specificities of the primers were confirmed by phylogenetic analysis of the environmental clone sequences, and this method can therefore now be used to investigate the ecology of the obligately anaerobic fungi. To our knowledge, this is the first demonstration of the occurrence of members of the Neocallimastigales outside the mammalian gut, and their distribution across the landfill samples examined here suggests that they are actively involved in cellulose degradation.     

Phycodnavirus potassium ion channel proteins question the virus molecular piracy hypothesis

Phycodnaviruses are large dsDNA, algal-infecting viruses that encode many genes with homologs in prokaryotes and eukaryotes. Among the viral gene products are the smallest proteins known to form functional K(+) channels. To determine if these viral K(+) channels are the product of molecular piracy from their hosts, we compared the sequences of the K(+) channel pore modules from seven phycodnaviruses to the K(+) channels from Chlorella variabilis and Ectocarpus siliculosus, whose genomes have recently been sequenced. C. variabilis is the host for two of the viruses PBCV-1 and NY-2A and E. siliculosus is the host for the virus EsV-1. Systematic phylogenetic analyses consistently indicate that the viral K(+) channels are not related to any lineage of the host channel homologs and that they are more closely related to each other than to their host homologs. A consensus sequence of the viral channels resembles a protein of unknown function from a proteobacterium. However, the bacterial protein lacks the consensus motif of all K(+) channels and it does not form a functional channel in yeast, suggesting that the viral channels did not come from a proteobacterium. Collectively, our results indicate that the viruses did not acquire their K(+) channel-encoding genes from their current algal hosts by gene transfer; thus alternative explanations are required. One possibility is that the viral genes arose from ancient organisms, which served as their hosts before the viruses developed their current host specificity. Alternatively the viral proteins could be the origin of K(+) channels in algae and perhaps even all cellular organisms.     

巢式PCR检测食蟹猴和猕猴中SRV和SFV

目的利用巢式PCR方法检测圈养的食蟹猴（Macaca fascicularis）和猕猴（Macaca mulatta）中猴D型逆转录病毒（Simian Type D Retrovirus SRV）和猴泡沫病毒（Simian foamy virus SFV）。方法针对SRV-env和SFV-pol基因的保守区序列设计特异性外引物,然后再设计特异性内引物。将外引物扩增出的片段克隆到PJET1.2blunt载体中作为阳性对照,运用NCBI中BLAST软件比对测序结果。用巢式PCR方法分别检测食蟹猴和猕猴中SRV和SFV。结果发现食蟹猴中SFV感染率为65.2%,SRV感染率为9.5%,猕猴中SFV感染率为60.5%,SRV感染率为12.8%。结论圈养的食蟹猴和猕猴SFV的感染率均较高,SRV感染率很低。     

Method for discovering novel DNA viruses in blood using viral particle selection and shotgun sequencing

Rapid identification of viruses is needed to monitor the blood supply for emerging threats. Here we present a method that meets these criteria and allows for the shotgun sequencing of novel, uncultured DNA viruses directly from human blood. This method employs selection based on the physical properties of viruses combined with sequence-independent amplification and cloning. We show that both single- and double-stranded DNA viruses can be recovered from blood samples using this approach. In addition, we report the discovery of novel anellovirus sequences in the blood of healthy donors. PCR primers designed to amplify these novel anellovirus sequences were then used to verify the presence of these viruses in the general donor population.     

K-tuple frequency in the human genome and polymerase chain reaction.

The frequency occurrences of K-tuple (overlapping sequences of defined length, K) were computed from the known human genome sequences. The significance of these frequencies for the whole human genome was tested by polymerase chain reaction (PCR). A computer programs based on these results was written to choose primers to amplify DNA target sequences, either of human genes or of human infectious agents. The software also gave nested primer sequences which were used to synthesize non radioactive probes by PCR. We applied these two methods, primer selection and non radioactive probes, to easily and quickly set up very efficient PCR sets to work in the human genome context.     

RNA triphosphatase component of the mRNA capping apparatus of Paramecium bursaria Chlorella virus 1

Paramecium bursaria chlorella virus 1 (PBCV-1) elicits a lytic infection of its unicellular green alga host. The 330-kbp viral genome has been sequenced, yet little is known about how viral mRNAs are synthesized and processed. PBCV-1 encodes its own mRNA guanylyltransferase, which catalyzes the addition of GMP to the 5' diphosphate end of RNA to form a GpppN cap structure. Here we report that PBCV-1 encodes a separate RNA triphosphatase (RTP) that catalyzes the initial step in cap synthesis: hydrolysis of the gamma-phosphate of triphosphate-terminated RNA to generate an RNA diphosphate end. We exploit a yeast-based genetic system to show that Chlorella virus RTP can function as a cap-forming enzyme in vivo. The 193-amino-acid Chlorella virus RTP is the smallest member of a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi and other large eukaryotic DNA viruses (poxviruses, African swine fever virus, and baculoviruses). Chlorella virus RTP is more similar in structure to the yeast RNA triphosphatases than to the enzymes of metazoan DNA viruses. Indeed, PBCV-1 is unique among DNA viruses in that the triphosphatase and guanylyltransferase steps of cap formation are catalyzed by separate viral enzymes instead of a single viral polypeptide with multiple catalytic domains.