Partial Genome Sequences Research Articles

Molecular characterization and prevalence of two capulaviruses: Alfalfa leaf curl virus from France and Euphorbia caput-medusae latent virus from South Africa

Little is known about the prevalence, diversity, evolutionary processes, genomic structures and population dynamics of viruses in the divergent geminivirus lineage known as the capulaviruses. We determined and analyzed full genome sequences of 13 Euphorbia caput-medusae latent virus (EcmLV) and 26 Alfalfa leaf curl virus (ALCV) isolates, and partial genome sequences of 23 EcmLV and 37 ALCV isolates. While EcmLV was asymptomatic in uncultivated southern African Euphorbia caput-medusae, severe alfalfa disease symptoms were associated with ALCV in southern France. The prevalence of both viruses exceeded 10% in their respective hosts. Besides using patterns of detectable negative selection to identify ORFs that are probably functionally expressed, we show that ALCV and EcmLV both display evidence of inter-species recombination and biologically functional genomic secondary structures. Finally, we show that whereas the EcmLV populations likely experience restricted geographical dispersion, ALCV is probably freely moving across the French Mediterranean region.

Genome sequencing in plants and the genus Panax L.

Advances in genome sequencing technologies have created a new genomic era of life sciences research worldwide in which a number of modern and sophisticated techniques and tools have been developed and employed. Many countries have invested in plant genome sequencing as part of a sustainable development strategy. Each year, the number of plant genomes and transcriptomes sequenced has increased. The results obtained offer opportunities for fundamental and applied research, provide valuable data for identification of genes or molecular markers linked to traits that are important for selection, cultivation, and/or production. In Vietnam, partial or complete genome sequencing of crops has been recently conducted, primarily as part of international collaborative projects. The genus Panax L. (Araliaceae family) is comprised of several species of commercial value with narrow distributions such as P. bipinnatifidus Seem., P. stipuleanatus H.T.Tsai & K.M.Feng, and Panax vietnamensis Ha et Grushv. Despite their very important roles in traditional medicine, understanding of their genetic characteristics is still limited. Molecular studies on the genus have, so far, only evaluated limited markers for phylogenetic analysis. Therefore, genome sequencing of these important herbal plants is needed to understand their genetic characteristics, their evolutionary history and the genes and biochemical pathways contributing to medicinally important metabolites. This review summarizes all related genome sequencing technologies including the most recent advances in the last decade and their applications in genome and transcriptome sequencing of plants in general and in the genus Panax L. in particular.

Hyperexpansion of RNA Bacteriophage Diversity.

Bacteriophage modulation of microbial populations impacts critical processes in ocean, soil, and animal ecosystems. However, the role of bacteriophages with RNA genomes (RNA bacteriophages) in these processes is poorly understood, in part because of the limited number of known RNA bacteriophage species. Here, we identify partial genome sequences of 122 RNA bacteriophage phylotypes that are highly divergent from each other and from previously described RNA bacteriophages. These novel RNA bacteriophage sequences were present in samples collected from a range of ecological niches worldwide, including invertebrates and extreme microbial sediment, demonstrating that they are more widely distributed than previously recognized. Genomic analyses of these novel bacteriophages yielded multiple novel genome organizations. Furthermore, one RNA bacteriophage was detected in the transcriptome of a pure culture of Streptomyces avermitilis, suggesting for the first time that the known tropism of RNA bacteriophages may include gram-positive bacteria. Finally, reverse transcription PCR (RT-PCR)-based screening for two specific RNA bacteriophages in stool samples from a longitudinal cohort of macaques suggested that they are generally acutely present rather than persistent.

Comparative molecular epidemiology of two closely related coronaviruses, bovine coronavirus (BCoV) and human coronavirus OC43 (HCoV-OC43), reveals a different evolutionary pattern

Bovine coronaviruses (BCoVs) are widespread around the world and cause enteric or respiratory infections among cattle. The current study includes 13 samples from BCoVs collected in Normandy during an 11-year period (from 2003 to 2014), 16 French HCoV-OC43s, and 113 BCoVs or BCoVs-like sequence data derived from partial or complete genome sequences available on GenBank. According to a genotyping method developed previously for HCoV-OC43, BCoVs and BCoVs-like are distributed on three main sub-clusters named C1, C2, and C3. Sub-cluster C1 includes BCoVs and BCoVs-like from America and Asia. Sub-cluster C2 includes BCoVs from Europe. Sub-cluster C3 includes prototype, vaccine, or attenuated BCoV strains. The phylogenetic analyses revealed the monophyletic status of the BCoVs from France reported here for the first time. Moreover, BCoV exhibits a relative genetic stability when compared to HCoV-OC43 we previously described from the same region. The numerous recombination detected between HCoV-OC43 were much less frequent for BCoV. The analysis points thus to the influence of different evolutive constraints in these two close groups.

Association of NetF-positive type A Clostridium perfringens with necrotizing enteritis in neonatal foals

A role for type A Clostridium perfringens in necrotizing enterocolitis of neonatal foals has long been suspected but incompletely characterized. The supernatant of an isolate made from a foal that died from this disease was highly cytotoxic for an equine ovarian (EO) cell line. Partial genome sequencing of an isolate revealed three novel putative toxin genes encoding proteins related to the pore-forming Leukocidin/Hemolysin Superfamily; these were designated netE, netF, and netG. netE and netF were located on one large conjugative plasmid, and netG was located with a cpe enterotoxin gene on a second large conjugative plasmid. Mutation and complementation showed that only netF was associated with the cytotoxicity. There was a highly significant association (p<0.0002) between the presence of netF with type A strains isolated from cases of foal necrotizing enterocolitis. netE and netF were found in all cytotoxic isolates, as was cpe, but netG was less consistently present. Pulsed-field gel electrophoresis showed that netF-positive isolates belonged to a clonal lineage that included canine-origin netF strains; some canine and equine netF-positive isolates were genetically indistinguishable. Equine antisera to recombinant Net proteins showed that only antiserum to rNetF had high supernatant cytotoxin neutralizing activity. The genomes of two C. perfringens strains recovered from a case of foal necrotizing enteritis (JP55) and canine haemorrhagic gastroenteritis (JP838), respectively, were completely sequenced using Single Molecule, Real-Time (SMRT) technology-PacBio and Illumina Hiseq2000. The JP55 and JP838 genomes include a single 3.34 Mb and 3.53 Mb circular chromosome, respectively, and both genomes additionally consist of five circular plasmids. Comparison of these two C. perfringens chromosomes with three fully sequenced reference chromosome sequences identified regions (~69 kb) shared between the two isolates, including regions forming a mosaic of plasmid-integrated segments, suggesting that these elements were acquired early in a clonal lineage of netF-positive C. perfringens strains. In addition, plasmid annotation revealed that both netF-positive C. perfringens strains, JP55 and JP838, harbour three tcp-conjugative plasmids in common, including a NetF/NetE toxins-encoding plasmid, a CPE/CPB2 toxins-encoding plasmid and a putative bacteriocin-encoding plasmid. We also found that JP55 and JP838 strains share unique virulence genes on conserved

GenSeed-HMM: A Tool for Progressive Assembly Using Profile HMMs as Seeds and its Application in Alpavirinae Viral Discovery from Metagenomic Data.

This work reports the development of GenSeed-HMM, a program that implements seed-driven progressive assembly, an approach to reconstruct specific sequences from unassembled data, starting from short nucleotide or protein seed sequences or profile Hidden Markov Models (HMM). The program can use any one of a number of sequence assemblers. Assembly is performed in multiple steps and relatively few reads are used in each cycle, consequently the program demands low computational resources. As a proof-of-concept and to demonstrate the power of HMM-driven progressive assemblies, GenSeed-HMM was applied to metagenomic datasets in the search for diverse ssDNA bacteriophages from the recently described Alpavirinae subfamily. Profile HMMs were built using Alpavirinae-specific regions from multiple sequence alignments (MSA) using either the viral protein 1 (VP1; major capsid protein) or VP4 (genome replication initiation protein). These profile HMMs were used by GenSeed-HMM (running Newbler assembler) as seeds to reconstruct viral genomes from sequencing datasets of human fecal samples. All contigs obtained were annotated and taxonomically classified using similarity searches and phylogenetic analyses. The most specific profile HMM seed enabled the reconstruction of 45 partial or complete Alpavirinae genomic sequences. A comparison with conventional (global) assembly of the same original dataset, using Newbler in a standalone execution, revealed that GenSeed-HMM outperformed global genomic assembly in several metrics employed. This approach is capable of detecting organisms that have not been used in the construction of the profile HMM, which opens up the possibility of diagnosing novel viruses, without previous specific information, constituting a de novo diagnosis. Additional applications include, but are not limited to, the specific assembly of extrachromosomal elements such as plastid and mitochondrial genomes from metagenomic data. Profile HMM seeds can also be used to reconstruct specific protein coding genes for gene diversity studies, and to determine all possible gene variants present in a metagenomic sample. Such surveys could be useful to detect the emergence of drug-resistance variants in sensitive environments such as hospitals and animal production facilities, where antibiotics are regularly used. Finally, GenSeed-HMM can be used as an adjunct for gap closure on assembly finishing projects, by using multiple contig ends as anchored seeds.

Identification of an HIV-1 BG Intersubtype Recombinant Form (CRF73_BG), Partially Related to CRF14_BG, Which Is Circulating in Portugal and Spain.

HIV-1 exhibits a characteristically high genetic diversity, with the M group, responsible for the pandemic, being classified into nine subtypes, 72 circulating recombinant forms (CRFs) and numerous unique recombinant forms (URFs). Here we characterize the near full-length genome sequence of an HIV-1 BG intersubtype recombinant virus (X3208) collected in Galicia (Northwest Spain) which exhibits a mosaic structure coincident with that of a previously characterized BG recombinant virus (9601_01), collected in Germany and epidemiologically linked to Portugal, and different from currently defined CRFs. Similar recombination patterns were found in partial genome sequences from three other BG recombinant viruses, one newly derived, from a virus collected in Spain, and two retrieved from databases, collected in France and Portugal, respectively. Breakpoint coincidence and clustering in phylogenetic trees of these epidemiologically-unlinked viruses allow to define a new HIV-1 CRF (CRF73_BG). CRF73_BG shares one breakpoint in the envelope with CRF14_BG, which circulates in Portugal and Spain, and groups with it in a subtype B envelope fragment, but the greatest part of its genome does not appear to derive from CRF14_BG, although both CRFs share as parental strain the subtype G variant circulating in the Iberian Peninsula. Phylogenetic clustering of partial pol and env segments from viruses collected in Portugal and Spain with X3208 and 9691_01 indicates that CRF73_BG is circulating in both countries, with proportions of around 2–3% Portuguese database HIV-1 isolates clustering with CRF73_BG. The fact that an HIV-1 recombinant virus characterized ten years ago as a URF has been shown to represent a CRF suggests that the number of HIV-1 CRFs may be much greater than currently known.

Two Complete Genome Sequences of Phasey Bean Mild Yellows Virus, a Novel Member of the Luteoviridae from Australia.

We present here the complete genome sequences of a novel polerovirus from Trifolium subterraneum (subterranean clover) and Cicer arietinum (chickpea) and compare these to a partial viral genome sequence obtained from Macroptilium lathyroides (phasey bean). We propose the name phasey bean mild yellows virus for this novel polerovirus.

Molecular detection and characterization of noroviruses in river water in Thailand.

Norovirus (NoV) generally exists as a mixture of multiple genotype variants in nature. However, there has been no published report monitoring NoV in natural settings in Thailand. To obtain information on mixed presence of the NoV RNA genome, we conducted viral genome analysis of 15 water specimens collected from five sites in a river near Bangkok between August 2013 and August 2014. The number of viral RNA copies per specimen declined progressively from the most upstream to the most downstream site. Following direct nucleotide sequencing of the PCR products, we obtained three partial genome sequences of the NoV GI strain and 13 partial genome sequences of the NoV GII strains. Phylogenetic analysis indicated the presence of four GII.4 variant groups pro-circulated after the Den Haag_2006b, New Orleans_2009 and Sydney_2012 outbreaks. On the other hand, only GI.4 was observed from the specimens collected on April, 2014. These results indicated that multiple genogroups and genotypes of noroviruses are present and are circulating in the natural environment in Thailand as in other countries. Our study provides comprehensive information on the occurrence of new variants. Our study is the first paper that multiple genogroups and genotypes of norovirus exist, and are circulating in the river water near Bangkok, Thailand. Phylogenetic analysis indicated the presence of four GII.4 variant groups pro-circulated after the Den Haag_2006b, New Orleans_2009 and Sydney_2012 that caused outbreaks in the world. Continued research will be essential for understanding the natural history of NoV and the control of future outbreaks.

Cucumis melo endornavirus: Genome organization, host range and co-divergence with the host

A high molecular weight dsRNA was isolated from a Cucumis melo L. plant (referred to as ‘CL01’) of an unknown cultivar and completely sequenced. Sequence analyses showed that dsRNA is associated with an endornavirus for which a name Cucumis melo endornavirus (CmEV) is proposed. The genome of CmEV-CL01 consists of 15,078 nt, contains a single, 4939 codons-long ORF and terminates with a stretch of 10 cytosine residues. Comparisons of the putative CmEV-encoded polyprotein with available references in protein databases revealed a unique genome organization characterized by the presence of the following domains: viral helicase Superfamily 1 (Hel-1), three glucosyltransferases (doublet of putative capsular polysaccharide synthesis proteins and a putative C_28_Glycosyltransferase), and an RNA-dependent RNA polymerase (RdRp). The presence of three glycome-related domains of different origin makes the genome organization of CmEV unique among endornaviruses. Phylogenetic analyses of viral RdRp domains showed that CmEV belongs to a specific lineage within the family Endornaviridae made exclusively of plant-infecting endornaviruses. An RT-PCR based survey demonstrated high incidence of CmEV among melon germplasm accession (>87% of tested samples). Analyses of partial genome sequences of CmEV isolates from 26 different melon genotypes suggest fine-tuned virus adaptation and co-divergence with the host. Finally, results of the present study revealed that CmEV is present in plants belonging to three different genera in the family Cucurbitaceae. Such diverse host range is unreported for known endornaviruses and suggests a long history of CmEV association with cucurbits predating their speciation.

Sequence variation in Brassica AP2 and analysis of interaction of AP2-miR172 regulatory module

Floral organ identity in Arabidopsis thaliana is regulated via interaction of APEATALA2 (AP2) transcripts with miR172. In complex genomes of Brassicas, the impact of polyploidization on sequence and expression variation in AP2 and aspects of its interaction with Brassica miR172 is poorly understood. Herein we report sequence variation and expression pattern of BrassicaAP2 homologs and demonstrate interaction of AP2 transcripts with cognate miR172. Four genomic and 3 cDNA variants of AP2 were isolated from B. rapa and B. juncea. This included a splice variant (cBju2_AP2) from B. juncea which retained first intron leading to premature termination of predicted protein. Phylogenetic analysis depicted two major clades and allowed assignment of BrassicaAP2 sequences to LF and MF1 subgenomes even from uncharacterized B. juncea genome. Expression analysis of two LF specific AP2 homologs (cBju1_AP2 and cBju2_AP2) revealed near identical expression pattern. However, cBju1_AP2 exhibited significantly higher expression levels compared to cBju2_AP2 implying regulatory diversification. Higher expression levels in reproductive stages implicated Brassica AP2 in flower development. Analysis of 19 BrassicaAP2 partial genomic sequences isolated from 6 Brassica species showed a conserved miR172 binding site, while considerable polymorphism was observed in flanking regions. In-silico analysis of hybridization energies between miR172 (miR172b, miR172d, miR172e) and AP2 variants revealed differing total free energy of binding suggesting complex interaction patterns in Brassicas. The predicted miR172 guided cleavage of AP2 transcript (cBju1_AP2) from B. juncea was experimentally validated through tobacco transient assays. Conservation of miR172 binding site in all BrassicaAP2 sequences is indicative of them being true targets of miR172 mediated PTGS albeit with varying efficiency. This suggests that in spite of polyploidy induced polymorphisms in AP2 transcript, miR172 binding site has remained conserved to preserve dynamics of interaction.

Assessing the Role of Seabirds in the Ecology of Influenza A Viruses.

Wild waterbirds, specifically waterfowl, gulls, and shorebirds, are recognized as the primordial reservoir of influenza A viruses (IAVs). However, the role of seabirds, an abundant, diverse, and globally distributed group of birds, in the perpetuation and transmission of IAVs is less clear. Here we summarize published and publicly available data for influenza viruses in seabirds, which for the purposes of this study are defined as birds that exhibit a largely or exclusively pelagic lifestyle and exclude waterfowl, gulls, and shorebirds, and we review this collective dataset to assess the role of seabirds in the influenza A ecology. Since 1961, more than 40,000 samples have been collected worldwide from the seabirds considered here and screened, using a variety of techniques, for evidence of active or past IAV infection. From these data, the overall prevalence of active infection has been estimated to be very low; however, serological data provide evidence that some seabird species are more frequently exposed to IAVs. Sequence data for viruses from seabirds are limited, except for murres (common murre, Uria aalge, and thick-billed murre, Uria lomvia; family Alcidae) for which there are full or partial genome sequences available for more than 80 viruses. Characterization of these viruses suggests that murres are infected with Group 1 hemagglutinin subtype viruses more frequently as compared to Group 2 and also indicates that these northern, circumpolar birds are frequently infected by intercontinental reassortant viruses. Greater temporal and spatial sampling and characterization of additional viruses are required to better understand the role of seabirds in global IAV dynamics.

The rearranged mitochondrial genome of Leptopilina boulardi (Hymenoptera: Figitidae), a parasitoid wasp of Drosophila.

The partial mitochondrial genome sequence of Leptopilina boulardi (Hymenoptera: Figitidae) was characterized. Illumina sequencing was used yielding 35,999,679 reads, from which 102,482 were utilized in the assembly. The length of the sequenced region of this partial mitochondrial genome is 15,417 bp, consisting of 13 protein-coding, two rRNA, and 21tRNA genes (the trnaM failed to be sequenced) and a partial A+T-rich region. All protein-coding genes start with ATN codons. Eleven protein-coding genes presented TAA stop codons, whereas ND6 and COII that presented TA, and T nucleotides, respectively. The gene pattern revealed extensive rearrangements compared to the typical pattern generally observed in insects. These rearrangements involve two protein-coding and two ribosomal genes, along with the 16 tRNA genes. This gene order is different from the pattern described for Ibalia leucospoides (Ibaliidae, Cynipoidea), suggesting that this particular gene order can be variable among Cynipoidea superfamily members. A maximum likelihood phylogenetic analysis of the main groups of Apocrita was performed using amino acid sequence of 13 protein-coding genes, showing monophyly for the Cynipoidea superfamily within the Hymenoptera phylogeny.

Identification of 15 novel polymorphic microsatellite loci in pearl oyster (Pinctada fucata).

The pearl oyster Pinctada fucata is a commercially important marine shellfish. As a result, genetic improvement and selective-breeding program have been conducted for this species. Polymorphic microsatellites are effective molecular markers to investigate molecular marker-assisted selection and genetic variance. In this study, microsatellite DNAs were screened and characterized based on the partial genome sequence of P. fucata. We identified 111 microsatellite DNA motifs through mining the published draft genome sequence of P. fucata. Forty-two loci were screened with 8 P. fucata individuals, and 15 were found to be polymorphic and were therefore further evaluated using 40 wild individuals from the Daya Bay, Shenzhen City, Guangdong Province, China. The number of alleles per locus ranged from 3 to 8, with an average of 5.2667 for the 15 polymorphic loci. Observed and expected heterozygosities ranged from 0.1154 to 0.6216 (0.3321 on average) and 0.4950 to 0.8491 (0.6768 on average), respectively. Of the 15 polymorphic loci, 12 loci deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.0033). Polymorphism information content ranged from 0.44 to 0.83 with a mean value of 0.63. The results suggest that the markers isolated in this study can be used for research on molecular marker-assisted selection and genetic variance of P. fucata.

Current situation of tomato yellow leaf curl disease (TYLCD) in Antalya, Turkey

Gul-Seker M., Ekinci H., Ozturk C., Elibuyuk I.O. (2015): Current situation of tomato yellow leaf curl disease (TYLCD) in Antalya, Turkey. Plant Protect. Sci., 51: 208–213. We studied the distribution of virus/viruses causing tomato yellow leaf curl disease (TYLCD) in Antalya and to charac terise the partial genome sequences of the selected isolates. Tomato yellow leaf curl virus (TYLCV) was only detected by the triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) and polymerase chain reaction (PCR). 24 and 80 greenhouses were found to be TYLCV-infected in the spring and autumn cultivations in 2011, respectively. The rate of TYLCV infection was found to be 29% in the spring cultivation and 43.7% in the autumn cultivation. The partial nucleotide sequences of the isolates were also determined.

Whole genome analysis of Japanese bovine toroviruses reveals natural recombination between porcine and bovine toroviruses

Bovine toroviruses (BToVs), belong to the subfamily Toroviridae within the family Coronaviridae, and are pathogens, causing enteric disease in cattle. In Japan, BToVs are distributed throughout the country and cause gastrointestinal infection of calves and cows. In the present study, complete genome sequences of two Japanese BToVs and partial genome sequences of two Japanese BToVs and one porcine torovirus (PToV) from distant regions in Japan were determined and genetic analyses were performed. Pairwise nucleotide comparison and phylogenetic analyses revealed that Japanese BToVs shared high identity with each other and showed high similarities with BToV Breda1 strain in S, M, and HE coding regions. Japanese BToVs showed high similarities with porcine toroviruses in ORF1a, ORF1b, and N coding regions and the 5′ and 3′ untranslated regions, suggestive of a natural recombination event. Recombination analyses mapped the putative recombinant breakpoints to the 3′ ends of the ORF1b and HE regions. These findings suggest that the interspecies recombinant nature of Japanese BToVs resulted in a closer relationship between BToV Breda1 and PToVs.

First report of Cymbidium mosaic virus and Odontoglossum ring spot virus in Argentina

Oncidium Sharry Baby orchids from nursery greenhouses in Buenos Aires, Argentina with viral symptoms were processed for transmission electron microscope observations. Two types of viral particles, morphologically different, were observed. The aim of this work is identification of viral particles infecting the orchids. Serological analysis and partial genome sequence confirmed the presence of Cymbidium mosaic virus (CymMV) and Odontoglossum ring spot virus (ORSV). To our knowledge this is the first report of ORSV and CymMV in Argentina.

Chikungunya Virus Sequences Across the First Epidemic in Nicaragua, 2014-2015.

Chikungunya is caused by the mosquito-borne arthrogenic alphavirus, chikungunya virus (CHIKV). Chikungunya was introduced into the Americas in late 2013 and Nicaragua in mid-2014. Here, we sequenced five imported and 30 autochthonous Nicaraguan CHIKV from cases identified in the first epidemic in the country between August 2014 and April 2015. One full-length and two partial genomic sequences were obtained by deep sequencing; Sanger methodology yielded 33 E1 sequences from five imported and 28 autochthonous cases. Phylogenetic analysis indicates that Nicaraguan CHIKV all belonged to the Asian genotype, Caribbean clade. Moreover, E1 gene sequences revealed accumulation of mutations in later months of the epidemic, including four silent mutations in 11 autochthonous cases and three non-synonymous mutations in three autochthonous cases. No mutations contributing to increased transmissibility by Aedes albopictus were identified in the E1 gene. This represents the most comprehensive set of CHIKV sequences available from the Americas to date.

Molecular evidence of spotted fever group rickettsiae and Anaplasmataceae from ticks and stray dogs in Bangladesh.

Emerging tick-borne diseases (TBDs) are important foci for human and animal health worldwide. However, these diseases are sometimes over looked, especially in countries with limited resources to perform molecular-based surveys. The aim of this study was to detect and characterize spotted fever group (SFG) rickettsiae and Anaplasmataceae in Bangladesh, which are important tick-borne pathogens for humans and animals worldwide. A total of 50 canine blood samples, 15 ticks collected from dogs, and 154 ticks collected from cattle were screened for the presence of SFG rickettsiae and Anaplasmataceae using molecular-based methods such as PCR and real-time PCR. The sequence analysis of the amplified products detected two different genotypes of SFG rickettsiae in ticks from cattle. The genotype detected in Rhipicephalus microplus was closely related to Rickettsia monacensis, while the genotype detected in Haemaphysalis bispinosa was closely related to Rickettsia sp. found in Korea and Japan. Anaplasma bovis was detected in canine blood and ticks (Rhipicephalus sanguineus and H. bispinosa). Unexpectedly, the partial genome sequence of Wolbachia sp., presumably associated with the nematode Dirofilaria immitis, was identified in canine blood. The present study provides the first molecular evidence of SFG rickettsiae and A. bovis in Bangladesh, indicating the possible emergence of previously unrecognized TBDs in this country.

HERPESVIRUSES INCLUDING NOVEL GAMMAHERPESVIRUSES ARE WIDESPREAD AMONG PHOCID SEAL SPECIES IN CANADA.

Little is known about herpesviruses in Canadian pinnipeds. We measured prevalence of antibodies to herpesviruses in the sera from Canadian phocid seals by an indirect enzyme-linked immunosorbent assay. Wild harbor seals (Phoca vitulina) and captive harbor seals were positive for antibodies to Phocid herpesvirus 1 (PhoHV-1) at prevalences of 91% and 100%, respectively. Sera from wild hooded seals (Cystophora cristata), harp seals (Pagophilus groenlandica), and grey seals (Halichoerus grypus) were positive for antibodies to PhoHV-1 antigenically related herpesvirus antigens at 73%, 79%, and 96%, respectively. We isolated new herpesviruses in cell culture from two hunter-harvested ringed seals (Pusa hispida) in poor body condition from Ulukhaktok, Northwest Territories, Canada; one lethargic hooded seal from the St. Lawrence Estuary, Québec, Canada; and one captive, asymptomatic harp seal from the Magdalen Islands, Québec. Partial sequencing of the herpesvirus DNA polymerase gene revealed that all four virus isolates were closely related to PhoHV-2, a member of the Gammaherpesvirinae subfamily, with nucleotide similarity ranging between 92.8% and 95.3%. The new seal herpesviruses were genetically related to other known pinniped herpesviruses, such as PhoHV-1, Otariid herpesvirus 3, Hawaiian monk (Monachus schauinslandi) seal herpesvirus, and Phocid herpesvirus 5 with 47-48%, 55%, 77%, and 70-77% nucleotide similarities, respectively. The harp seal herpesvirus and both ringed seal herpesviruses were almost identical to each other, whereas the hooded seal herpesvirus was genetically different from the three others (92.8% nucleotide similarity), indicating detection of at least two novel seal herpesviruses. These findings are the first isolation, partial genome sequencing, and identification of seal gammaherpesviruses in three species of Canadian phocid seals; two species of which were suspected of exposure to one or more antigenically related herpesviruses based on serologic analyses.