Molecular and Morphological Characterization of Tylenchus Zeae n. Sp. (Nematoda: Tylenchida) from Corn (Zea Mays) in South Carolina.

Specimens of a tylenchid nematode were recovered in 2019 from soil samples collected from a corn field, located in Pickens County, South Carolina, USA. A moderate number of Tylenchus sp. adults (females and males) were recovered. Extracted nematodes were examined morphologically and molecularly for species identification, which indicated that the specimens of the tylenchid adults were a new species, described herein as Tylenchus zeae n. sp. Morphological examination and the morphometric details of the specimens were very close to the original descriptions of Tylenchus sherianus and T. rex. However, females of the new species can be differentiated from these species by body shape and length, shape of excretory duct, distance between anterior end and esophageal intestinal valve, and a few other characteristics given in the diagnosis. Males of the new species can be differentiated from the two closely related species by tail, spicules, and gubernaculum length. Cryo-scanning electron microscopy confirmed head bearing five or six annules; four to six cephalic sensilla represented by small pits at the rounded corners of the labial plate; a small, round oral plate; and a large, pit-like amphidial opening confined to the labial plate and extending three to four annules beyond it. Phylogenetic analysis of 18S rRNA gene sequences placed Tylenchus zeae n. sp. in a clade with Tylenchus arcuatus and several Filenchus spp., and the mitochondrial cytochrome oxidase c subunit 1 (COI) gene region separated the new species from T. arcuatus and other tylenchid species. In the 28S tree, T. zeae n. sp. showed a high level of sequence divergence and was positioned outside of the main Tylenchus-Filenchus clade.

Phytoplasma Infection Blocks Starch Breakdown and Triggers Chloroplast Degradation, Leading to Premature Leaf Senescence, Sucrose Reallocation, and Spatiotemporal Redistribution of Phytohormones.

Witches'-broom (WB, excessive initiation, and outgrowth of axillary buds) is one of the remarkable symptoms in plants caused by phytoplasmas, minute wall-less intracellular bacteria. In healthy plants, axillary bud initiation and outgrowth are regulated by an intricate interplay of nutrients (such as sugars), hormones, and environmental factors. However, how these factors are involved in the induction of WB by phytoplasma is poorly understood. We postulated that the WB symptom is a manifestation of the pathologically induced redistribution of sugar and phytohormones. Employing potato purple top phytoplasma and its alternative host tomato (Solanum lycopersicum), sugar metabolism and transportation, and the spatiotemporal distribution of phytohormones were investigated. A transmission electron microscopy (TEM) analysis revealed that starch breakdown was inhibited, resulting in the degradation of damaged chloroplasts, and in turn, premature leaf senescence. In the infected source leaves, two marker genes encoding asparagine synthetase (Sl-ASN) and trehalose-6-phosphate synthase (Sl-TPS) that induce early leaf senescence were significantly up-regulated. However, the key gibberellin biosynthesis gene that encodes ent-kaurene synthase (Sl-KS) was suppressed. The assessment of sugar content in various infected tissues (mature leaves, stems, roots, and leaf axils) indicated that sucrose transportation through phloem was impeded, leading to sucrose reallocation into the leaf axils. Excessive callose deposition and the resulting reduction in sieve pore size revealed by aniline blue staining and TEM provided additional evidence to support impaired sugar transport. In addition, a spatiotemporal distribution study of cytokinin and auxin using reporter lines detected a cytokinin signal in leaf axils where the axillary buds initiated. However, the auxin responsive signal was rarely present in such leaf axils, but at the tips of the newly elongated buds. These results suggested that redistributed sucrose as well as cytokinin in leaf axils triggered the axillary bud initiation, and auxin played a role in the bud elongation. The expression profiles of genes encoding squamosa promoter-binding proteins (Sl-SBP1), and BRANCHED1 (Sl-BRC1a and Sl-BRC1b) that control axillary bud release, as determined by quantitative reverse transcription (qRT)-PCR, indicated their roles in WB induction. However, their interactions with sugars and cytokinins require further study. Our findings provide a comprehensive insight into the mechanisms by which phytoplasmas induce WB along with leaf chlorosis, little leaf, and stunted growth.

Morphological and molecular characterization of Paratylenchus beltsvillensis n. sp. (Tylenchida: Paratylenchidae) from the rhizosphere of pine tree (Pinus virginiana Mill) in Maryland, USA.

The pin nematode, Paratylechus beltsvillensis n. sp. collected from rhizosphere soil of a Virginia pine tree (Pinus virginiana Mill) growing in Little Paint Branch Park, Beltsville, Prince George's County, Maryland, USA, is described and illustrated along with light and scanning electron photomicrographs. Females, males, and juveniles of this new species were recovered from soil samples using the sugar centrifugal flotation and Baermann funnel extraction methods. Morphologically, females are short, body length ranging from 245 to 267 µm, stylet from 70 to 75 µm long with anchor shaped knobs, vulva located at 70-73% and small vulval flap, spermatheca large, and ovoid filled with sperms. Lateral field with three incisures, of which the outer two are prominent. Tail slender, having a rounded tail terminus. Males without stylet and have a degenerated pharynx, spicules = 17-20 µm and gubernaculum = 5.0-5.5 µm. Both morphological observations and molecular analysis of ITS and partial 28S ribosomal RNA gene sequences indicated that the specimens collected from the soil at Beltsville Park from rhizosphere soil samples from Virginia pine represents a new pin nematode species.

First description of Sarcocystis species infecting Barbary sheep (Ammotragus lervia).

Barbary sheep (Ammotragus lervia) is a North African native wild Caprinae, introduced in the 1970s in new territories such as Spain, the USA, and Mexico. Here, we describe Sarcocystis species in Barbary sheep. Sarcocysts were found in 19 out of 56 adult A. lervia in Southern Spain and characterized morphologically and molecularly. By light microscopy, sarcocysts had thin (< 1 µm) or thick (> 2 µm) walls. By transmission electron microscopy, sarcocysts with thick walls had Type 14 villar protrusions corresponding to S. tenella/S. capracanis of domestic sheep (Ovis aries) or goats (Capra hircus). Sarcocysts with thin walls had Type 7b villar protrusions that corresponded to S. arieticanis/S. hircicanis of domestic sheep or goats. Molecular analyses allowed the identification of only thick-walled Sarcocystis species. Six sarcocysts were assigned to S. tenella (99.2-100% and 95.6-100% sequence similarity within 18S rRNA and COI, respectively) and 19 sarcocysts were assigned to S. capracanis (98.5-99.8% and 97.9-99.0% sequence similarity within 18S rRNA and COI, respectively). Further studies are needed for taxonomic identification of sarcocysts in Barbary sheep because Sarcocystis species in sheep and goats are not cross transmissible despite morphological similarities.

Morphological and Molecular Characterization of Pratylenchus dakotaensis n. sp. (Nematoda: Pratylenchidae), a New Root-Lesion Nematode Species on Soybean in North Dakota, USA.

Root-lesion nematodes (Pratylenchus spp.) of the genus Pratylenchus Filipjev, 1936, are among the most important nematode pests on soybean (Glycine max (L.) Merr.), along with soybean cyst and root-knot nematodes. In May 2015 and 2016, a total of six soil samples were collected from a soybean field in Walcott, Richland County, ND and submitted to the Mycology and Nematology Genetic Diversity and Biology Laboratory (MNGDBL), USDA, ARS, MD for analysis. Later, in 2019, additional nematodes recovered from a greenhouse culture on soybean originally from the same field were submitted for further analysis. Males, females, and juveniles of Pratylenchus sp. were recovered from soil and root samples and were examined morphologically and molecularly. DNA from single nematodes were extracted, and the nucleotides feature of three genomic regions targeting on the D2-D3 region of 28S rDNA and ITS rDNA and mitochondrial cytochrome oxidase subunit I (COX1) gene were characterized. Phylogeny trees were constructed to ascertain the relationships with other Pratylenchus spp., and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to provide a rapid and reliable differentiation from other common Pratylenchus spp. Molecular features indicated that it is a new, unnamed Pratylenchus sp. that is different from morphologically closely related Pratylenchus spp., including P. convallariae, P. pratensis, P. fallax, and P. flakkensis. In conclusion, both morphological and molecular observations indicate that the North Dakota isolate on soybean represents a new root-lesion nematode species which is named and described herein as Pratylenchus dakotaensis n. sp.

Ovipositional Behavior of the Egg Parasitoid Gryon pennsylvanicum (Hymenoptera: Scelionidae) on Two Squash Bug Species Anasa tristis (Hemiptera: Coreidae) and Anasa armigera: Effects of Parasitoid Density, Nutrition, and Host Egg Chorion on Parasitism Rates.

This study examined the ovipositional behavior of Gryon pennsylvanicum Ashmead (Hymenoptera: Scelionidae) on egg masses of two squash bug species Anasa tristis DeGeer and Anasa armigera Say (Hemiptera: Coreidae) by evaluating how parasitoid density and access to nutrition influenced percent parasitism on egg masses of different sizes in laboratory tests. When three parasitoids were exposed to A. tristis egg masses with only three to five eggs, 72.7% of parasitoids became trapped in the eggs and failed to emerge successfully. These results suggest that competition between larvae within the egg may have reduced the fitness of the surviving parasitoid. Continual access to honey water did not significantly influence parasitism rates on A. armigera egg masses and only increased parasitism on A. tristis egg masses with 20-25 eggs. Overall, parasitism rates were higher on A. armigera egg masses than on A. tristis egg masses, and parasitoids were more likely to emerge successfully from A. armigera eggs than from A. tristis eggs. Parasitoids spent the same amount of time probing eggs of the two species, but they spent significantly more time drilling into A. tristis eggs than A. armigera eggs. Measurements taken using transmission electron microscopy determined that the average combined width of the epicuticle and exocuticle of the egg chorion was significantly greater for A. tristis eggs than for A. armigera eggs. This difference may account for the lower rates of parasitism and parasitoid emergence and for the increased time spent drilling into A. tristis eggs compared with A. armigera eggs.

Chromobacterium paludis sp. nov., a novel bacterium isolated from a Chesapeake Bay marsh.

Two isolates of Gram-reaction-negative, motile, violet-pigmented bacteria were isolated from a small pool in marshland near the mouth of the Nanticoke River in Maryland, USA. The isolates IIBBL 257-1T and IIBBL 257-2 had identical 16S rRNA gene sequences as determined by PCR, and highly similar fatty acid and biochemical profiles. The 16S rRNA gene sequences indicated the isolates belonged to the genus Chromobacterium. Genomic sequencing of IIBBL 257-1T revealed a genome of 4.27 Mb, with a G+C content of 63.6 %. Whole genome comparisons with other members of the Chromobacterium using JSpecies and the genome blast distance phylogeny approach indicated that among described species, IIBBL 257-1T was most closely related to C. amazonense and C. phragmitis. Comparison of the IIBBL 257-1T genome with those of type strains of these species resulted in ANIb and dDDH values of ca. 85 and 30 %, respectively, for both. These results demonstrate that IIBBL 257-1T and IIBBL 257-2 represent a new taxon within the genus Chromobacterium. We propose the name Chromobacterium paludis sp. nov. for this taxon; the type strain is IIBBL 257-1T (=NRRL B-65555T=JCM 33770T).

An iflavirus found in stink bugs (Hemiptera: Pentatomidae) of four different species.

An analysis of transcriptomes from the antennae of the three South American stink bugs (Euschistus heros, Chinavia ubica, and Dichelops melacanthus) revealed the presence of picorna-like virus genome-length RNAs with high sequence identity to the genome of Halyomorpha halys virus (HhV), originally discovered in the transcriptome of the brown marmorated stink bug, Halyomorpha halys. Features of the genome, phylogenetic relationships to other viruses, and the appearances of virus-like particles isolated from host stink bugs all confirm that these viruses are iflaviruses and isolates of an undescribed species. Iflavirus RNAs were present at high levels (40%-90% of transcriptome reads) in the stink bug antennal transcriptomes. In whole-insect transcriptomes of H. halys, HhV reads were >500-fold more abundant in adults than in nymphs. We identified from field population a subject of species E. heros infected by this iflavirus. The results of the analysis suggest that these iflaviruses are able to produce large quantities of their RNAs without causing any obvious pathology to their hosts.

Sequencing, genome analysis and prevalence of a cytorhabdovirus discovered in Carica papaya.

The complete genome of a new rhabdovirus infecting papaya (Carica papaya L.) in Ecuador, named papaya virus E, was sequenced and characterized. The negative-sense single-stranded RNA genome consists of 13,469 nucleotides with six canonical open reading frames (ORFs) and two accessory short ORFs predicted between ORFs corresponding to P3 (movement protein) and M (matrix protein). Phylogenetic analyses using amino acid sequences from the nucleocapsid, glycoprotein and polymerase, grouped the virus with members of the genus Cytorhabdovirus, with rice stripe mosaic virus, yerba mate chlorosis-associated virus and Colocasia bobone disease-associated virus as closest relatives. The 3' leader and 5' trailer sequences were 144 and 167 nt long, respectively, containing partially complementary motifs. The motif 3'-AUUCUUUUUG-5', conserved across rhabdoviruses, was identified in all but one intergenic regions; whereas the motif 3'-ACAAAAACACA-5' was found in three intergenic junctions. This is the first complete genome sequence of a cytorhabdovirus infecting papaya. The virus was prevalent in commercial plantings of Los Ríos, the most important papaya producing province of Ecuador. Recently, the genome sequence of bean-associated cytorhabdovirus was reported. The genome is 97% identical to that of papaya virus E, indicating that both should be considered strains of the same virus.

Chromobacterium phragmitis sp. nov., isolated from estuarine marshes.

Thirteen isolates of Gram-stain-negative, motile, violet-pigmented bacteria were isolated from marshes along tidal portions of the Potomac and James rivers in Maryland and Virginia, USA, respectively. 16S rRNA gene sequences and fatty acid analysis revealed a high degree of relatedness among the isolates, and genomic sequencing of two isolates, IIBBL 112-1T and IIBBL 274-1 (from the Potomac and James rivers, respectively), revealed highly similar genomic sequences, with a blast-based average nucleotide identity (ANIb) of ca. 98.7 %. Phylogenetic analysis of 16S rRNA gene sequences suggested that the species most highly related to IIBBL 112-1T were Chromobacterium amazonense, Chromobacterium subtsugae and Chromobacterium sphagni. However, deletion of a 25-nucleotide sequence that may have been horizontally acquired by both IIBBL 112-1T and C. amazonense resulted in a substantially different analysis; in the latter case, the species nearest IIBBL 112-1T were Chromobacterium violaceum, Chromobacterium vaccinii and Chromobacterium piscinae. Whole-genome alignments between either IIBBL 112-1T or IIBBL 274-1 and the type strains of C. vaccinii or C. violaceum resulted in ANIb values in the range of ca. 87 %, while alignment with C. amazonense CBMAI 310T resulted in an ANIb of ca. 83 %. Collectively, these data demonstrate that IIBBL 112-1T and IIBBL 274-1 represent a new taxon within the genus Chromobacterium. We propose the name Chromobacterium phragmitis sp. nov. for this taxon; the type strain is IIBBL 112-1T (=NRRL B-67132T=JCM 31884T).

The complete genome sequence of an alphabaculovirus from Spodoptera exempta, an agricultural pest of major economic significance in Africa.

Spodoptera exempta nucleopolyhedrovirus (SpexNPV) is a viral pathogen of the African armyworm, Spodoptera exempta (Lepidoptera: Noctuidae), a significant agricultural pest of cereal crops in Africa. SpexNPV has been evaluated as a potential insecticide for control of this pest and has served as the subject of research on baculovirus pathology and transmission. Occlusion bodies (OBs) of SpexNPV isolate 244.1 were examined, and the nucleotide sequence of the genome was determined and characterized. SpexNPV-244.1 OBs consisted of irregular polyhedra with a size and appearance typical for alphabaculoviruses. Virions within the polyhedra contained 1-8 nucleocapsids per unit envelope. The SpexNPV-244.1 genome was comprised of a 129,528 bp circular sequence, in which 139 ORFs were annotated. Five homologous regions (hrs) consisting of a variable number of 28-bp imperfect palindromes were identified in the genome. The genome sequence contained the 38 core genes of family Baculoviridae, as well as three ORFs unique to the SpexNPV sequence and one ORF that was apparently acquired by horizontal gene transfer with a betabaculovirus ancestor. Phylogenetic inference with core gene amino acid sequence alignments placed SpexNPV-244.1 in a lineage containing alphabaculoviruses of Spodoptera frugiperda and Spodopotera exigua which in turn is part of a larger group of alphabaculoviruses from the subfamily Noctuinae in the lepidopteran family Noctuidae. Kimura-2-parameter pairwise nucleotide distances indicated that SpexNPV-244.1 represented a different and previously unlisted species in the genus Alphabaculovirus. Gene parity plots indicated that the gene order of SpexNPV-244.l was extensively collinear with that of Spodoptera exigua NPV (SeMNPV). These plots also revealed a group of 17 core genes whose order was conserved in other alpha- and betabaculoviruses.

Varroa destructor feeds primarily on honey bee fat body tissue and not hemolymph.

The parasitic mite Varroa destructor is the greatest single driver of the global honey bee health decline. Better understanding of the association of this parasite and its host is critical to developing sustainable management practices. Our work shows that this parasite is not consuming hemolymph, as has been the accepted view, but damages host bees by consuming fat body, a tissue roughly analogous to the mammalian liver. Both hemolymph and fat body in honey bees were marked with fluorescent biostains. The fluorescence profile in the guts of mites allowed to feed on these bees was very different from that of the hemolymph of the host bee but consistently matched the fluorescence profile unique to the fat body. Via transmission electron microscopy, we observed externally digested fat body tissue in the wounds of parasitized bees. Mites in their reproductive phase were then fed a diet composed of one or both tissues. Mites fed hemolymph showed fitness metrics no different from the starved control. Mites fed fat body survived longer and produced more eggs than those fed hemolymph, suggesting that fat body is integral to their diet when feeding on brood as well. Collectively, these findings strongly suggest that Varroa are exploiting the fat body as their primary source of sustenance: a tissue integral to proper immune function, pesticide detoxification, overwinter survival, and several other essential processes in healthy bees. These findings underscore a need to revisit our understanding of this parasite and its impacts, both direct and indirect, on honey bee health.

The complete genome sequence of a second alphabaculovirus from the true armyworm, Mythimna unipuncta: implications for baculovirus phylogeny and host specificity.

The Mythimna unipuncta nucleopolyhedrovirus isolate KY310 (MyunNPV-KY310) is an alphabaculovirus isolated from a true armyworm (Mythimna unipuncta) population in Kentucky, USA. Occlusion bodies of this virus were examined by electron microscopy and the genome sequence was determined by 454 pyrosequencing. MyunNPV-KY310 occlusion bodies consisted of irregular polyhedra measuring 0.8-1.8 µm in diameter and containing multiple virions, with one to six nucleocapsids per virion. The genome sequence was determined to be 156,647 bp with a nucleotide distribution of 43.9% G+C. 152 ORFs and six homologous repeat (hr) regions were annotated for the sequence, including the 38 core genes of family Baculoviridae and an additional group of 26 conserved alphabaculovirus genes. BLAST queries and phylogenetic inference confirmed that MyunNPV-KY310 is most closely related to the alphabaculovirus Leucania separata nucleopolyhedrovirus isolate AH1, which infects Mythimna separata. In contrast, MyunNPV-KY310 did not exhibit a close relationship with Mythimna unipuncta nucleopolyhedrovirus isolate #7, an alphabaculovirus from the same host species. MyunNPV-KY310 lacks the gp64 envelope glycoprotein, which is a characteristic of group II alphabaculoviruses. However, this virus and five other alphabaculoviruses lacking gp64 are placed outside the group I and group II clades in core gene phylogenies, further demonstrating that viruses of genus Alphabaculovirus do not occur in two monophyletic clades. Potential instances of MyunNPV-KY310 ORFs arising by horizontal transfer were detected. Although there are now genome sequences of four different baculoviruses from M. unipuncta, comparison of their genome sequences provides little insight into the genetic basis for their host specificity.

ICTV Virus Taxonomy Profile: Baculoviridae.

The family Baculoviridae comprises large viruses with circular dsDNA genomes ranging from 80 to 180 kbp. The virions consist of enveloped, rod-shaped nucleocapsids and are embedded in distinctive occlusion bodies measuring 0.15-5 µm. The occlusion bodies consist of a matrix composed of a single viral protein expressed at high levels during infection. Members of this family infect exclusively larvae of the insect orders Lepidoptera, Hymenoptera and Diptera. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Baculoviridae, which is available at www.ictv.global/report/baculoviridae.

Sarcocystis cymruensis: discovery in Western Hemisphere in the Brown rat (Rattus norvegicus) from Grenada, West Indies: redescription, molecular characterization, and transmission to IFN-γ gene knockout mice via sporocysts from experimentally infected domestic cat (Felis catus).

Rodents are intermediate hosts for many species of Sarcocystis. Little is known of Sarcocystis cymruensis that uses the Brown rat (Rattus norvegicus) as intermediate hosts and the domestic cat (Felis catus) as experimental definitive host. Here, we identified and described Sarcocystis cymruensis in naturally infected R. norvegicus from Grenada, West Indies. Rats (n = 167) were trapped in various locations in two parishes (St. George and St. David). Microscopic, thin (< 1 µm) walled, slender sarcocysts were found in 11 of 156 (7.0%) rats skeletal muscles by squash examination. A laboratory-raised cat fed naturally infected rat tissues excreted sporocysts that were infectious for interferon gamma gene knockout (KO) mice, but not to Swiss Webster outbred albino mice. All inoculated mice remained asymptomatic, and microscopic S. cymruensis-like sarcocysts were found in the muscles of KO mice euthanized on day 70, 116, and 189 post inoculation (p.i.). Sarcocysts from infected KO mice were infective for cats at day 116 but not at 70 days p.i. By transmission electron microscopy, the sarcocyst wall was "type 1a." Detailed morphological description of the cyst wall, metrocytes, and bradyzoites is given for the first time. Additionally, molecular data on S. cymruensis are presented also for the first time. Molecular characterization of sarcocysts 18S rDNA and 28S rDNA, ITS-1, and cox1 loci showed the highest similarity with S. rodentifelis and S. muris. In conclusion, the present study described the natural infection of S. cymruensis in Brown rat for the first time in a Caribbean country and provided its molecular characteristics.

The complete genome sequence of a third distinct baculovirus isolated from the true armyworm, Mythimna unipuncta, contains two copies of the lef-7 gene.

A baculovirus isolate from a USDA Forest Service collection was characterized by electron microscopy and analysis of its genome sequence. The isolate, formerly referred to as Pseudoletia (Mythimna) sp. nucleopolyhedrovirus #7 (MyspNPV#7), was determined by barcoding PCR to derive from the host species Mythimna unipuncta (true armyworm) and was renamed Mythimna unipuncta nucleopolyhedrovirus #7 (MyunNPV#7). The occlusion bodies (OBs) and virions exhibited a size and morphology typical for OBs produced by the species of genus Alphabaculovirus, with occlusion-derived virions consisting of 2-5 nucleocapsids within a single envelope. The MyunNPV#7 genome was determined to be 148,482 bp with a 48.58% G+C nucleotide distribution. A total of 159 ORFs of 150 bp or larger were annotated in the genome sequence, including the 38 core genes of family Baculoviridae. The genome contained six homologous repeat regions (hrs) consisting of multiple copies of a 34-bp imperfect palindrome. Phylogenetic inference from concatenated baculovirus core gene amino acid sequence alignments placed MyunNPV#7 with group II alphabaculoviruses isolated from other armyworm and cutworm host species of lepidopteran family Noctuidae. MyunNPV#7 could be distinguished from other viruses in this group on the basis of differences in gene content and order. Pairwise nucleotide distances suggested that MyunNPV#7 represents a distinct species in Alphabaculovirus. The MyunNPV#7 genome was found to contain two copies of the late expression factor-7 (lef-7) gene, a feature not reported for any other baculovirus genome to date. Both copies of lef-7 encoded an F-box domain, which is required for the function of LEF-7 in baculovirus DNA replication.

Atypical fatal sarcocystosis associated with Sarcocystis neurona in a White-nosed coati (Nasua narica molaris).

The protozoan parasite Sarcocystis neurona is an important cause of disease in horses (equine protozoal myeloencephalitis, EPM) and marine mammals. Isolated reports of clinical EPM-like disease have been documented in a zebra, raccoon, domestic cat, domestic dog, ferret, skunk, mink, lynx, red panda and fisher. The predominant disease is encephalomyelitis associated with schizonts in neural tissues. Here, we report highly disseminated sarcocystosis, in many tissues of a captive White-nosed coati (Nasua narica molaris). The 14year old, neutered male coati was euthanized due to progressive weakness, lethargy, and inappetence. Schizonts, including free and intracellular merozoites were detected in many cell types, and differed morphologically from S. neurona schizonts in horses. Only a few sarcocysts were seen in skeletal muscle and the myocardium. Immunohistochemically, the protozoa reacted positively to S. neurona but not to Toxoplasma gondii antibodies. Severe inflammtory disease detected in the stomach, intestine, adrenal and thyroid glands, ciliary body of eye, and urinary bladder associated with schizonts in the coati has not been reported earlier in any host with EPM. Although, a few schizonts were found in the brain, encephalitis was minimal and not the cause of clinical signs. Multilocus PCR-DNA sequencing using DNA derived from the coati lung tissue identified an S. neurona infection using the 18S, 28S and ITS-1 markers, and a novel genotype using primer pairs against antigenic surface proteins (SnSAG3, SnSAG4, SnSAG1-5-6) and microsatellite markers (MS, SN7, SN9). Although the genotype was similar to the widely distributed Type VI strain, it possessed a novel allele at SnSAG5, and a different MS combination of repeats at SN7 and SN9. Whether this severe parasitism was related to the host or the parasite needs further investigation.

The Operophtera brumata Nucleopolyhedrovirus (OpbuNPV) Represents an Early, Divergent Lineage within Genus Alphabaculovirus.

Operophtera brumata nucleopolyhedrovirus (OpbuNPV) infects the larvae of the winter moth, Operophtera brumata. As part of an effort to explore the pesticidal potential of OpbuNPV, an isolate of this virus from Massachusetts (USA)-OpbuNPV-MA-was characterized by electron microscopy of OpbuNPV occlusion bodies (OBs) and by sequencing of the viral genome. The OBs of OpbuNPV-MA consisted of irregular polyhedra and contained virions consisting of a single rod-shaped nucleocapsid within each envelope. Presumptive cypovirus OBs were also detected in sections of the OB preparation. The OpbuNPV-MA genome assembly yielded a circular contig of 119,054 bp and was found to contain little genetic variation, with most polymorphisms occurring at a frequency of < 6%. A total of 130 open reading frames (ORFs) were annotated, including the 38 core genes of Baculoviridae, along with five homologous repeat (hr) regions. The results of BLASTp and phylogenetic analysis with selected ORFs indicated that OpbuNPV-MA is not closely related to other alphabaculoviruses. Phylogenies based on concatenated core gene amino acid sequence alignments placed OpbuNPV-MA on a basal branch lying outside other alphabaculovirus clades. These results indicate that OpbuNPV-MA represents a divergent baculovirus lineage that appeared early during the diversification of genus Alphabaculovirus.