Sequence Analysis of a 5.1 kbp Region of the Spodoptera frugiperda Multicapsid Nucleopolyhedrovirus Genome that Comprises a Functional Ecdysteroid UDP-Glucosyltransferase (egt) Gene

The ecdysteroid UDP-glucosyltransferase gene from the Spodoptera frugiperda multicapsid nucleopolyhedrovirus (SfMNPV) was identified using degenerate primers whose sequence were derived from conserved regions of the EGT proteins encoded by other baculoviruses. Analysis of the gene sequence revealed the presence of an open reading frame (ORF) with potential to encode a polypeptide of 525 amino acids. Promoter sequences typical of baculovirus genes were found in the 5 region of this ORF. A polyadenylation signal was identified downstream the translation stop codon. A transient expression assay showed that the product of this ORF was able to conjugate glucose from UDP-glucose with ecdysone confirming that the gene identified was indeed the SfMNPV egt gene. The SfMNPV egt gene and the sequences of other baculovirus egt genes were used to infer a phylogenetic tree. The nucleotide sequence of the entire BamHI fragment that contains the SfMNPV egt gene was determined. Search of the available sequence databases suggested that, besides the egt gene, this region contains 5 ORFs similar to the baculovirus genes gp37 (fusolin), to ptp2 and to ORFs 28, 29, and 30 of Spodoptera exigua multicapsid nucleopolyhedrovirus. Both the phylogenetic analysis of the egt genes and the gene order of the region that flanks the egt gene indicated that SfMNPV is closely related to the baculoviruses that infects S. exigua and Mamestra configurata.     

Combined mitochondrial 16S and 12S rDNA sequences: an effective genetic marker for inter-species phylogenetic analysis of zoonotic trematodes

The present study studied the genetic variation among Schistosoma japonicum isolates from different endemic regions in mainland China and examined the phylogenetic relationships of zoonotic trematodes using the combined mitochondrial 16S and 12S ribosomal DNA sequences. The fragments of 16S and 12S rDNA were amplified from 22 S. japonicum isolates, and sequenced, and the relevant sequences of other nine trematode species belonging to six genera in four families were downloaded from GenBank, and their phylogenetic relationships were re-constructed by unweighted pair-group method with arithmetic averages analyses using the combined 16S and 12S rDNA sequences, with Trichinella spiralis as outgroup. The results showed that the partial sequences of mitochondrial 16S and 12S rDNA of S. japonicum were 757 and 797 bp, respectively, and they were quite conserved among the S. japonicum isolates. Phylogenetic analysis revealed that the combined 16S and 12S rDNA sequences were not able to distinguish S. japonicum isolates in mountainous areas from those in lake/marshland areas in mainland China. However, the combined sequences could distinguish different species of zoonotic trematodes. Therefore, the combined mitochondrial 16S and 12S rDNA sequences provide an effective molecular marker for the inter-species phylogenetic analysis and differential identification of zoonotic trematodes.     

Physical and Partial Genetic Map of <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis> Nucleopolyhedrovirus (SfMNPV) Genome

A Nicaraguan isolate of Spodoptera frugiperda multicapsid nucleopolyhedrovirus (SfMNPV) is undergoing field trials for control of this pest in the Americas. This isolate is composed of multiple genotypes, some of which are deletion mutants. Identification of the genetic changes in deleted genotypes cannot be accomplished without the construction of a detailed physical map. In the present study, combinations of restriction endonuclease analysis and Southern blot analysis was performed. This map was refined by sequencing the termini of cloned restriction fragments. The SfMNPV genome was estimated to be 129.3 kb, 8 kb larger than the previously characterized Sf-2 variant from the United States, due to a deletion between 14.8 and 21.0 m.u. in the physical map described in this study. A total of 27.92 kb were sequenced, which represented 21.5% of the whole genome and included 38 ORFs. Comparison with other sequenced baculoviruses revealed that SfMNPV displayed the highest sequence identity (66%) and gene arrangement (78%) with Spodoptera exigua MNPV, sharing 36 putative ORFs. In addition, the genome organization was similar to that of SeMNPV, with minor differences. Phylogenetic analysis confirmed the close relatedness between SeMNPV and SfMNPV, suggesting they evolved from a common ancestor.     

Genetic variability in the coat protein genes of lettuce big-vein associated virus and Mirafiori lettuce big-vein virus

Summary. Available data suggests that lettuce big-vein disease is caused by the ophiovirus Mirafiori lettuce big-vein virus (MLBVV) but not by the varicosavirus Lettuce big-vein-associated virus (LBVaV), although the latter is frequently associated with the disease. Since the disease occurs worldwide, the putative coat protein (CP) open reading frames of geographically distinct isolates of MLBVV and LBVaV were sequenced. Comparison of both nucleotide and amino acid sequences showed a high level of sequence similarity among LBVaV isolates. Phylogenetic analysis of LBVaV CP nucleotide sequences showed that most of the Spanish isolates clustered in a phylogenetic group whereas English isolates were more similar to the USA isolate. An Australian isolate was closely related to the Dutch isolate. Genetic diversity among MLBVV CP nucleotide sequences was higher ranging from 0.2% to 12%. Phylogenetic analysis of MLBVV CP nucleotide sequences revealed two distinct subgroups. However, this grouping was not correlated with symptom development on lettuce or the geographic origin of the MLBVV isolates. Finally, a quick method based on RFLP analysis of RT-PCR amplicons was developed for assigning MLBVV isolates to the two subgroups.     

Infected cell specific protein and viral DNA synthesis in productive and abortive infections ofSpodoptera frugiperda nuclear polyhedrosis virus

Summary This study examines viral protein and DNA synthesis inSpodoptera frugiperda multicapsid nuclear polyhedrosis virus (SfMNPV) infections ofS. frugiperda andTrichoplusia ni cells. A total of 28 infected cell specific polypeptides (ICSPs) were detected in the productiveS.frugiperda cells. Of these, 14 were identified as structural polypeptides. Based on the change in their rate of synthesis during the replication cycle, these ICSPs were grouped into four classes. Only a 97k and a 29k ICSP were detected in SfMNPV infections ofT.ni cells. Inhibition of host protein synthesis occurred in productive infections only, beginning at 10h postinfection (p.i.) and reaching maximal levels by 20 h p.i. The rate of viral DNA synthesis in the productive cells was maximal between 8 to 16 h postinfection, and only low levels of viral DNA were synthesized inT.ni cells. The data suggest that the productive SfMNPV/S.frugiperda cell infection has a gene expression program similar but not identical to that ofAutographa californica MNPV infections. The SfMNPV/T.ni cell infection is nonpermissive and is restricted at the earliest phase of the viral gene expression program.     

Molecular characterization of Hepatozoon canis in dogs from Colombia

Hepatozoonosis is a tick-borne disease whose transmission to dogs occurs by ingestion of oocysts infected ticks or feeding on preys infested by infected ticks. Until now, there is no previous report of molecular characterization of Hepatozoon sp. in dogs from Colombia. EDTA blood samples were collected from 91 dogs from central-western region of Colombia (Bogotá, Bucaramanga, and Villavicencio cities) and submitted to 18S rRNA Hepatozoon sp. PCR and blood smears confection. Phylogenetic analysis was used to access the identity of Hepatozoon species found in sampled dogs. From 91 sampled dogs, 29 (31.8%) were positive to Hepatozoon sp. (25 dogs were only positive in PCR, 1 was positive only in blood smears, and 3 were positive in both blood smears and PCR). After sequencing, the found Hepatozoon sp. DNA showed 100% of identity with Hepatozoon canis DNA isolates. The phylogenetic tree supported the identity of the found Hepatozoon sp. DNA, showing that the isolates from Colombia were placed in the same clade than other H. canis isolates from Venezuela, Spain, and Taiwan. This is the first molecular detection of H. canis in dogs from Colombia.     

Characterization of Baylisascaris schroederi from Qinling subspecies of giant panda in China by the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA

In the present study, a total of 20 nematode isolates, (including 10 male and 10 female worms) representing Baylisascaris schroederi from 5 Qinling subspecies of giant pandas (Ailuropoda melanoleuca) in Shaanxi Province of China, were characterized and grouped genetically by the first internal transcribed spacer (ITS-1) of nuclear ribosomal DNA (rDNA). The rDNA fragment spanning 3′ end of 18S rDNA, complete ITS-1 rDNA, and 5′ end of 5.8S rDNA were amplified and sequenced. The sequence variability in ITS-1 rDNA was examined within B. schroederi and among parasites in order Ascaridata available in GenBank™, and their phylogenetic relationships were also reconstructed. The sequences of ITS-1 rDNA for all the B. schroederi isolates were 427 bp in length, with no genetic variation detected among these isolates. Phylogenetic analyses based on the ITS-1 rDNA sequences revealed that all the male and female B. schroederi isolates sequenced in the present study were posited into the clade of genus Baylisascaris, sistered to zoonotic nematodes in genus Ascaris, and the ITS-1 rDNA sequence could distinguish different species in order Ascaridata. These results showed that the ITS-1 rDNA provides a suitable molecular marker for the inter-species phylogenetic analysis and differential identification of nematodes in order Ascaridata.     

Molecular characterisation of Raspberry bushy dwarf virus isolates from Sweden and Belarus

The complete coding sequences were determined for RNA-1 and RNA-2 of five raspberry isolates of Raspberry bushy dwarf virus (RBDV) from Belarus (BY1, BY3, BY8, BY22) and Sweden (SE3). The analysed sequences for both RNA-1 and RNA-2 were highly conserved among these isolates. Phylogenetic analyses including available sequences for the CP gene and the MP gene showed that all analysed RBDV isolates from raspberry were closely related. However, there was no strong correlation between the grouping of raspberry isolates in the phylogenetic analyses and their geographical location. In contrast, RBDV isolates showed a host-dependent relationship with isolates from raspberry and grapevine, forming two distinct clades.     

Subgenotype diversity of hepatitis B virus American genotype F in Amerindians from Venezuela and the general population of Colombia

The objective of this study was the evaluation of the genetic diversity found in HBV circulating among Venezuelan Amerindians and the general population in Colombia. Phylogenetic analysis of the S region in 194 isolates showed that genotype F is highly predominant in Colombia and Venezuela. This might be related to the genetic background of the population. F3 is the main subgenotype which circulates in both countries. Phylogenetic analysis of 61 complete genome sequences of HBV American genotypes confirms the presence of two genotypes F and H, and 4 F subgenotypes. In Venezuela, subgenotypes F1, F2, and F3 circulate in East and West Amerindians, while only F3 was found among South Amerindians. Japreira community derived from Yucpa Amerindians around 150 years ago. However, several Japreira HBV sequences were forming a clade that can be classified as subgenotype 2b, differing from Yucpa sequences that belong mainly to subgenotype F3. The apparent absence of correlation between the phylogenetic groupings of HBV isolates with the ethnical origin in aboriginal populations might be suggesting a recent origin of HBV American subgenotypes, or a genetic drift effect.     

Genetic variability and phylogenetic analysis of hosta virus X

Triple gene block 1 (TGB1) and coat protein (CP) sequences of 30 hosta virus X (HVX) isolates from Tennessee (TN), USA, were determined and compared with available sequences in GenBank. The CPs of all known HVX isolates, including those from TN, shared 98.3–100% and 98.2–100% nucleotide and amino acid sequence identity, respectively, whereas TGB1 shared 97.4–100% nucleotide and 97–100% amino acid sequence identity. TGB1 of TN isolates were all longer by one codon from that of a Korean isolate, which is the only sequence publicly available. Phylogenetic analysis of nucleotide and amino acid sequences of TGB1 and CP of all known HVX isolates, separately or combined, revealed a close relationship, suggesting that all of them are derived from a common ancestor. Phylogenetic analysis with the type member of each genus of the family Flexiviridae confirmed that HVX is a member of a distinct species of the genus Potexvirus.     

Molecular characterization of the <Emphasis Type="Italic">env</Emphasis> gene from Brazilian field isolates of Bovine Leukemia Virus

Molecular characterization of Bovine leukemia virus (BLV) isolates from Brazil using the env gene sequences revealed a high conservation of this gene. In most cases the substitutions corresponded to silent transitions. In addition, cystein residues, potential glycosylation sites, neutralization domains and other critical residues involved with the envelope structural domains and viral infectivity were conserved. Most of the substitutions found in the aminoacid sequences of the gp51 protein were localized in the G and H epitopes. Using the SIFT software, it was predicted that they should not alter the protein functions. Phylogenetic analyses showed that partial or complete env gene sequences grouped in three or four phylogenetic clusters, respectively. The sequences from the Brazilian isolates had similar mutation rates as compared to samples from other countries, and belonged to at least two phylogenetic clusters.     

Identification of new isolates of <Emphasis Type="Italic">Turnip mosaic virus</Emphasis> that cluster with less common viral strains

Summary Turnip mosaic virus (TuMV) was found infecting cultivated brassicas and wild and cultivated ornamental Brassicaceae plants in different regions of Spain. Five new TuMV isolates, originating from different host plant species (Brassica cretica, Brassica juncea, Brassica napus, Eruca vesicaria subsp. sativa and Sisymbrium orientale), have been identified. The nucleotide sequences of the coat protein (CP) genes of the five isolates were determined. Phylogenetic analysis of the CP sequences showed that the five isolates grouped into two different clusters. The three isolates from the central region of Spain clustered with a previously reported Pisum sativum isolate from southeastern Spain, whereas the other two isolates from the eastern region clustered with two Italian and two Greek isolates. Both clusters were genetically distinct and belonged to the multi-lineage group OBR. The OBR group contains mainly TuMV isolates from hosts other than Brassica spp. and Raphanus sativus and mostly originating from Mediterranean countries. These new sequences provide further phylogenetic resolution of the OBR group. Although new TuMV isolates have been found in Spain, they were not associated with any serious disease outbreaks.     

Characterization and genetic diversity of sugarcane streak mosaic virus causing mosaic in sugarcane

Sixty-three sugarcane leaf samples were collected from fifty-eight sugarcane varieties, evolved from eleven major sugarcane growing states in India, Australia, South Africa and USA. In RT-PCR, using gene specific primers for sugarcane streak mosaic virus (SCSMV)-CP, 58 of 63 sugarcane samples were found positive to the virus infection and rest of the five samples were negative. Partial CP gene sequences of 42 SCSMV isolates including an isolate from aphid colony (Melanaphis indosacchari) infested on sugarcane variety from this study were characterized after cloning and sequencing for selective isolates represented by at least one isolate from each location. The new sequences identified in the study were named as SCSMV-CB isolates. Fifty two sequences including the 10 database sequences (complete CP cds) deposited earlier from this institute were compared with each other as well as GenBank database sequences of Potyviridae members viz., Rymovirus, Potyvirus, Ipomovirus, Tritimovirus and eight sequences of SCSMV reported from elsewhere. Among the SCSMV-CB isolates sequenced in the study, 85.7–100% (nucleotide) and 89.9–100% (amino acid) sequence identities were observed and with the other data base sequences of SCSMV, the respective identities were 82.2–97.5 and 89.7–98.6%. Grouping of the isolates by the maximum likelihood with molecular clock model, distributed 60 SCSMV sequences including the eight database sequences deposited by other SCSMV working groups from India and USA in 16 different phylogenetic groups. Although the isolates of SCSMV were relatively close to Ipomovirus and Tritimovirus, they were sandwiched between Rymovirus and Ipomovirus. The sequence comparison and phylogenetic studies revealed that the relatedness of SCSMV with the potyviral related genera was comparatively low to consider it as a member of earlier described potyviral genera, hence the genus “Susmovirus” (sugarcane streak mosaic virus) has been proposed, with SCSMV as the sole species to be included. The 52 SCSMV-CB isolates from this institute were distributed in 14 phylogenetic groups and the grouping pattern revealed that the virus isolates could not be grouped based on geographical origin of the host varieties or longevity of the host variety.     

Genomic sequence analysis of the Illinois strain of the <Emphasis Type="Italic">Agrotis ipsilon multiple nucleopolyhedrovirus</Emphasis>

The Agrotis ipsilon multiple nucleopolyhedrovirus (AgipMNPV) is a group II nucleopolyhedrovirus (NPV) from the black cutworm, A. ipsilon, with potential as a biopesticide to control infestations of cutworm larvae. The genome of the Illinois strain of AgipMNPV was completely sequenced. The AgipMNPV genome was 155,122 nt in size and contained 163 open reading frames (ORFs), including 61 ORFs found among all lepidopteran baculoviruses sequenced to date. Phylogenetic inference placed AgipMNPV in a clade with group II NPVs isolated from larvae of Agrotis and Spodoptera species. Though closely related to the Agrotis segetum NPV (AgseNPV), AgipMNPV was found to be missing 15 ORFs present in the AgseNPV genome sequence, including two of the three AgseNPV enhancin genes. Remarkably few polymorphisms were identified in the AgipMNPV sequence even though an uncloned field isolate of this virus was sequenced. A genotype characterized by a 128-bp deletion in the ecdysteroid UDP-glucosyltransferase gene (egt) was identified in the AgipMNPV field isolate and among clonal isolates of AgipMNPV. The deletion in egt was not associated with differences in budded virus or occluded virus production among AgipMNPV clones in cell culture. The nucleotide sequence data reported in this article have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number EU839994.     

Molecular phylogeny of <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> from Central America (Guatemala) and a comparison with South American strains

Molecular phylogenetic analysis was carried out for 21 strains of Trypanosoma cruzi, nine of which were obtained from Guatemala and 12 from South America. Phylogenetic trees were constructed using the nucleotide sequences of two nuclear gene regions, dihydrofolate reductase–thymidylate synthase (DHFR-TS) and trypanothione reductase (TR), and contiguous portions of two mitochondrial genes, cytochrome oxidase subunit II (COII) and reduced nicotinamide adenine dinucleotide dehydrogenase subunit 1 (ND1). Possible genetic exchange between the rather divergent lineages of T. cruzi II from South America was suggested in the trees of the two nuclear genes. T. cruzi I strains obtained from Guatemala and Colombia were identical in all the genes examined, but other T. cruzi I isolates from South America were rather polymorphic in the DHFR-TS and mitochondrial genes. No genetic exchange was identified between T. cruzi I populations from Central and South America in the present study.     

Characterization of Cryptocaryon irritans isolates from marine fishes in Mainland China by ITS ribosomal DNA sequences

Seven isolates of Cryptocaryon irritans from different host species and geographical locations in Mainland China were characterized by the first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA) using two isolates of Ichthyophthirius multifiliis for comparative purposes. The rDNA region including the ITS-1, 5.8S, ITS-2, and flanking 18S and 28S sequences were amplified by polymerase chain reaction and the amplicons were sequenced directly. The ITS-1, 5.8S, and ITS-2 sequences were 129, 160, and 190 bp in length, respectively, for all seven C. irritans isolates, whereas the corresponding sequences for the two I. multifiliis isolates were 142, 153, and 194 bp, respectively. While sequence variation among the seven C. irritans isolates ranged from 0 to 1.6% in both the ITS-1 and ITS-2, and the two I. multifiliis isolates differed by 1.4% in the ITS-1 and 1.0% in the ITS-2; C. irritans differed from I. multifiliis by 57.1–60.9% in the ITS-1 and 79.4–83.0% in the ITS-2, indicating that ITS sequences provide reliable genetic markers for the identification and differentiation of the two species. Phylogenetic analysis using the sequence pairwise-distance data using the neighbor-joining method inferred that the seven C. irritans isolates from Mainland China and two other isolates (T.A and Aus.C) from other countries clustered together to show monophyly, which could be readily distinguished from the other monophyletic group all from other regions. Therefore, ITS sequence data and phylogenetic analysis provided strong support that C. irritans isolates from Mainland China represent a single species. The definition of genetic markers in the ITS rDNA provide opportunities for studying the ecology and population genetic structures of the C. irritans from Mainland China and elsewhere and is also relevant to the diagnosis and control of fish diseases they cause.     

Sequence Divergence of Four Soilborne Sugarbeet-Infecting Viruses

Soilborne viruses are among the most harmful pathogens of sugarbeet (Beta vulgaris L.ssp. vulgaris) but most of them lack information on genetic variability due to paucity of sequence data. Only one isolate of Beet soil borne virus (BSBV; genus Pomovirus), Beet virus Q (BVQ; genus Pomovirus) and Beet soil borne mosaic virus (BSBMV; genus Benyvirus) has been characterised for the coat protein (CP) gene. In this study, the CP gene sequences of three isolates each of BSBV and Beet necrotic yellow vein virus (BNYVV; genus Benyvirus) (France, Germany and USA), two isolates of BVQ (France and Germany), and one isolate of BSBMV (USA) were determined. Phylogenetic analyses including sequences from databanks indicated that the French BNYVV isolate of this study belongs to so-called P-type, the American isolate to A-type and the German isolate to B-type. The CP genes of the three BSBV isolates characterised in this study and the one available from databank were highly identical (98.4–99.0% at nucleotide level; one variable amino acid). The BSBMV isolate studied here differed from the previously characterised isolate for five nucleotides and four amino acids in the CP region. The two BVQ isolates characterised in this study contained three additional nucleotides resulting in an additional amino acid residue (arginine) at CP position 86, as compared to the only isolate available in databank.     

Morphology and genome of Euproctis pseudoconspersa nucleopolyhedrovirus

Euproctis pseudoconspersa NPV (EupsNPV) is pathogenic to the tea tussock (E. pseudoconspersa), one of the major pests of tea bushes in East Asia, and has been used to control the pest. Electron microscope observation showed there were two modes for the virions embedded in each polyhedron, single-nucleocapsid and double-nucleocapsid. The EupsNPV genome contained 141,291 bp and had a G + C content of 40.4%. Of 139 potential ORFs predicted from the sequence, 126 had a homology in other baculoviruses; 13 were unique to EupsNPV. Four homologous repeat sequences (hrs) were present in the EupsNPV genome and the repeat sequences were different between these hrs. Three ORFs were identified to contain two homologues in the EupsNPV genome, including bro, p26 and dbp. Gene parity plots, percent identities of gene homologues and phylogenetic analysis all suggested that EupsNPV is most closely related to EcobNPV in Group II NPV, although its genomic organization was highly distinct.     

18S ribosomal DNA genotypes of Acanthamoeba species isolated from contact lens cases in the Philippines

This study was carried out to document the genotypes of Acanthamoeba present in contact lens cases from 50 randomly selected contact lens wearers living in Quezon City, Metro Manila, Philippines. Acanthamoeba species were isolated from eight (16%) in 50 contact lens cases examined. We analyzed partial 18S ribosomal DNA (Rns) sequences of the eight isolates and found that the sequence differences were sufficient to distinguish the genotypes. After the isolates were genotyped, using the Basic Local Alignment Search Tool program, their phylogenetic positions relative to known Acanthamoeba isolates were determined. The model-based (GTR+Γ+Ι) neighbor-joining, maximum likelihood, and Bayesian inference analyses, as well as the non-model-based maximum parsimony analysis were used. Results showed that of the eight isolates, six were Rns genotype T5 while two were Rns genotype T4. This present study indicates that genotype T5 is also a common contaminant in contact lens storage cases.