Two novel alphaherpesviruses associated with fatal disseminated infections in Atlantic bottlenose dolphins.

Two immature female bottlenose dolphins (Tursiops truncatus) were found stranded on the Atlantic coast of the USA. Necropsy and histopathologic examination of both dolphins demonstrated acute necrotizing lesions in multiple organ systems. Commonly seen in these lesions were cells with enlarged nuclei that contained single 4 to 6 microm diameter homogeneous eosinophilic inclusion bodies that were often surrounded by a clear halo. Ultrastructural examination revealed that intranuclear inclusions contained 90 to 110 nm diameter viral particles with electron-dense cores and hexagonal profiles. Viral particles were also present in the cytoplasm, and these were surrounded by variably electron-dense envelopes. Enveloped virions were 140 nm in diameter. Polymerase chain reactions targeting the DNA polymerase and terminase genes of herpesviruses were carried out on unfixed tissues of both animals, and analysis of the DNA products indicated the presence of two novel alphaherpesviruses. The gross, histologic, ultrastructural, and molecular genetic findings indicate disseminated herpesviral infections, and support the conclusion that the alphaherpesviruses caused the deaths of the two dolphins. This is the first report of disseminated herpesviral infection in cetaceans.

An Epstein-Barr-related herpesvirus from marmoset lymphomas.

Epstein-Barr virus (EBV) is implicated in the development of human B cell lymphomas and carcinomas. Although related oncogenic herpesviruses were believed to be endemic only in Old World primate species, we now find these viruses to be endemic in New World primates. We have isolated a transforming, EBV-related virus from spontaneous B cell lymphomas of common marmosets (Callithrix jacchus). Sequencing of two-thirds of the genome reveals considerable divergence from the genomes of EBV and Old World primate EBV-related viruses, including differences in genes important for virus-induced cell growth transformation and pathogenesis. DNA related to the C. jacchus herpesvirus is frequently detected in squirrel monkey peripheral blood lymphocytes, indicating that persistent infection with EBV-related viruses is prevalent in both New World primate families. Understanding how these more divergent EBV-related viruses achieve similar biologic outcomes in their natural host is likely to provide important insights into EBV infection, B cell growth transformation, and oncogenesis.

Common metastatic carcinoma of California sea lions (Zalophus californianus): evidence of genital origin and association with novel gammaherpesvirus.

Tissues from 10 adult California sea lions (Zalophus californianus, seven females and three males) that had metastatic carcinoma in sublumbar area lymph nodes were examined histologically. A distinctive epithelial proliferative lesion interpreted as intraepithelial neoplasia was found in genital tracts of all ten animals; in vagina (5/7), cervix (7/7), uterus (3/7), penis (3/3) and prepuce (3/3). Intraepithelial neoplasia closely resembled metastatic carcinomas and was directly contiguous with invasive carcinoma in one animal. Rare eosinophilic intranuclear inclusion bodies were found in penile and preputial intraepithelial neoplasia (one animal), cervical intraepithelial neoplasia (one animal), invasive cervical carcinoma (one animal) and metastatic carcinoma (two animals). Electron microscopic examination of tissues from two sea lions (one with intraepithelial neoplasia and one with metastatic carcinoma) demonstrated viral particles consistent with a herpesvirus. An immunohistochemical stain for the latent membrane protein of Epstein-Barr virus was positive in intraepithelial neoplasia in one sea lion. Herpesvirus DNA sequences were detected by consensus primer polymerase chain reaction (PCR) in metastatic carcinomas from all four sea lions from which unfixed tumor samples were available. Results of sequencing were consistent with a novel gammaherpesvirus in the genus Rhadinovirus. DNA extracted from the four metastatic carcinomas also was tested for papillomavirus by Southern blot and PCR with consensus papillomavirus primers; all samples were negative by both methods. These findings support the genital origin of the sea lion carcinoma and implicate a novel gammaherpesvirus as a possible cause.

Fatal lymphoproliferative disease associated with a novel gammaherpesvirus in a captive population of common marmosets.

BACKGROUND AND PURPOSE: Callitrichids (marmosets and tamarins) are extremely susceptible to experimental tumor induction by herpesviruses native to other primate species. A colony of common marmosets developed a syndrome of weight loss, inappetence, diarrhea, and in several animals, palpable abdominal masses. METHODS: Marmosets in the colony were subjected to histologic examination and serologic testing for Epstein-Barr virus (EBV). The DNA from tumors that developed in the marmosets was subjected to consensus primer polymerase chain reaction (PCR) analysis designed to amplify conserved regions of herpesvirus genomes. RESULTS: The mesenteric lymph nodes and intestinal mucosa were consistently infiltrated by principally B lymphocytes, which often obliterated the normal architecture. Of 84 clinically normal marmosets, 52 were seropositive for EBV. The tumor DNA contained previously unreported herpesvirus sequences closely related to but distinct from those of EBV, Herpesvirus papio, and these lymphocryptovirus, a novel gammaherpesvirus. Results of PCR analysis of circulating lymphocytes from EBV-positive, clinically normal marmosets were negative for EBV antibodies and were positive for marmoset lymphocryptovirus; PCR analysis of circulating lymphocytes from EBV-negative marmosets yielded negative results for EBV and this novel marmoset lymphocryptovirus. CONCLUSION: This novel gammaherpesvirus possibly associated with tumor development may have important management implications for captive callitrichids.

Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium that is one of the top three causes of opportunistic human infections. A major factor in its prominence as a pathogen is its intrinsic resistance to antibiotics and disinfectants. Here we report the complete sequence of P. aeruginosa strain PAO1. At 6.3 million base pairs, this is the largest bacterial genome sequenced, and the sequence provides insights into the basis of the versatility and intrinsic drug resistance of P. aeruginosa. Consistent with its larger genome size and environmental adaptability, P. aeruginosa contains the highest proportion of regulatory genes observed for a bacterial genome and a large number of genes involved in the catabolism, transport and efflux of organic compounds as well as four potential chemotaxis systems. We propose that the size and complexity of the P. aeruginosa genome reflect an evolutionary adaptation permitting it to thrive in diverse environments and resist the effects of a variety of antimicrobial substances.

Aminoglycoside-resistance mechanisms for cystic fibrosis Pseudomonas aeruginosa isolates are unchanged by long-term, intermittent, inhaled tobramycin treatment.

Aminoglycoside-resistance mechanisms were characterized in Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients during a recent clinical trial of inhaled tobramycin. Impermeability, in which bacteria have reduced susceptibility to all aminoglycosides, was the predominant mode of resistance in isolates obtained both before and after 6 months of cyclic treatment with tobramycin or placebo administered by aerosol. Enzymatic resistance mechanisms were found in fewer than 10% of resistant isolates. P. aeruginosa from individual patients could be grouped on the basis of genetic relatedness. When enzymatic resistance was involved, all isolates in a group had elevated tobramycin MICs. When impermeability occurred, MICs of a genotypic group varied from susceptible to resistant. These findings suggest that impermeability resistance occurs in only a fraction of the P. aeruginosa population in lungs of persons with CF and that this form of resistance arises by a process involving multiple small changes in MIC.

Activities of tobramycin and six other antibiotics against Pseudomonas aeruginosa isolates from patients with cystic fibrosis.

The in vitro activity of tobramycin was compared with those of six other antimicrobial agents against 1,240 Pseudomonas aeruginosa isolates collected from 508 patients with cystic fibrosis during pretreatment visits as part of the phase III clinical trials of tobramycin solution for inhalation. The tobramycin MIC at which 50% of isolates are inhibited (MIC(50)) and MIC(90) were 1 and 8 microg/ml, respectively. Tobramycin was the most active drug tested and also showed good activity against isolates resistant to multiple antibiotics. The isolates were less frequently resistant to tobramycin (5.4%) than to ceftazidime (11.1%), aztreonam (11.9%), amikacin (13.1%), ticarcillin (16.7%), gentamicin (19.3%), or ciprofloxacin (20.7%). For all antibiotics tested, nonmucoid isolates were more resistant than mucoid isolates. Of 56 isolates for which the tobramycin MIC was > or = 16 microg/ml and that were investigated for resistance mechanisms, only 7 (12.5%) were shown to possess known aminoglycoside-modifying enzymes; the remaining were presumably resistant by an incompletely understood mechanism often referred to as "impermeability."

Association of herpesvirus with fibropapillomatosis of the green turtle Chelonia mydas and the loggerhead turtle Caretta caretta in Florida.

Sea turtle fibropapillomatosis (FP) is a disease marked by proliferation of benign but debilitating cutaneous fibropapillomas and occasional visceral fibromas. Transmission experiments have implicated a chloroform-sensitive transforming agent present in filtered cell-free tumor homogenates in the etiology of FP. In this study, consensus primer PCR methodology was used to test the association of a chelonian herpesvirus with fibropapillomatosis. Fibropapilloma and skin samples were obtained from 17 green and 2 loggerhead turtles affected with FP stranded along the Florida coastline. Ninety-three cutaneous and visceral tumors from the 19 turtles, and 33 skin samples from 16 of the turtles, were tested. All turtles affected with FP had herpesvirus associated with their tumors as detected by PCR. Ninety-six percent (89/93) of the tumors, but only 9% (3/33) of the skin samples, from affected turtles contained detectable herpesvirus. The skin samples that contained herpesvirus were all within 2 cm of a fibropapilloma. Also, 1 of 11 scar tissue samples from sites where fibropapillomas had been removed 2 to 51 wk earlier from 5 green turtles contained detectable herpesvirus. None of 18 normal skin samples from 2 green and 2 loggerhead turtles stranded without FP contained herpesvirus. The data indicated that herpesvirus was detectable only within or close to tumors. To determine if the same virus infected both turtle species, partial nucleotide sequences of the herpesvirus DNA polymerase gene were determined from 6 loggerhead and 2 green turtle samples. The sequences predicted that herpesvirus of loggerhead turtles differed from those of green turtles by only 1 of 60 amino acids in the sequence examined, indicating that a chelonian herpesvirus exhibiting minor intratypic variation was the only herpesvirus present in tumors of both green and loggerhead turtles. The FP-associated herpesvirus resisted cultivation on chelonian cell lines which support the replication of other chelonian herpesviruses. These results lead to the conclusion that a chelonian herpesvirus is regularly associated with fibropapillomatosis and is not merely an incidental finding in affected turtles.

Effect of chronic intermittent administration of inhaled tobramycin on respiratory microbial flora in patients with cystic fibrosis.

Pseudomonas aeruginosa endobronchial infection causes significant morbidity and mortality among cystic fibrosis patients. Microbiology results from two multicenter, double-blind, placebo-controlled trials of inhaled tobramycin in cystic fibrosis were monitored for longitudinal changes in sputum microbial flora, antibiotic susceptibility, and selection of P. aeruginosa isolates with decreased tobramycin susceptibility. Clinical response was examined to determine whether current susceptibility standards are applicable to aerosolized administration. Treatment with inhaled tobramycin did not increase isolation of Burkholderia cepacia, Stenotrophomonas maltophilia, or Alcaligenes xylosoxidans; however, isolation of Candida albicans and Aspergillus species did increase. Although P. aeruginosa tobramycin susceptibility decreased in the tobramycin group compared with that in the placebo group, there was no evidence of selection for the most resistant isolates to become most prevalent. The definition of resistance for parenteral administration does not apply to inhaled tobramycin: too few patients had P. aeruginosa with a tobramycin MIC >/=16 microgram/mL to define a new break point on the basis of clinical response.

Focal segmental glomerulosclerosis: a need for caution in live-related renal transplantation.

Focal segmental glomerulosclerosis (FSGS) has increasingly been recognized to occur in a familial pattern. We have observed the development of biopsy-confirmed FSGS and subsequent end-stage renal disease (ESRD) in one live related kidney donor and ESRD without biopsy in another. Both donors had family members with ESRD secondary to FSGS. Both donors were apparently healthy by routine physical examination, urinalysis, and serum creatinine at the time of evaluation as live related donors. We believe these cases emphasize the need for great caution when evaluating siblings as potential live related donors.

Novel endotheliotropic herpesviruses fatal for Asian and African elephants.

A highly fatal hemorrhagic disease has been identified in 10 young Asian and African elephants at North American zoos. In the affected animals there was ultrastructural evidence for herpesvirus-like particles in endothelial cells of the heart, liver, and tongue. Consensus primer polymerase chain reaction combined with sequencing yielded molecular evidence that confirmed the presence of two novel but related herpesviruses associated with the disease, one in Asian elephants and another in African elephants. Otherwise healthy African elephants with external herpetic lesions yielded herpesvirus sequences identical to that found in Asian elephants with endothelial disease. This finding suggests that the Asian elephant deaths were caused by cross-species infection with a herpesvirus that is naturally latent in, but normally not lethal to, African elephants. A reciprocal relationship may exist for the African elephant disease.

Detection and analysis of diverse herpesviral species by consensus primer PCR.

A consensus primer PCR method which amplifies a region of herpesviral DNA-directed DNA polymerase (EC 2.7.7.7) and which uses degenerate primers in a nested format was developed. Primers were designed to target sequences coding for highly conserved amino acid motifs covering a region of approximately 800 bp. The assay was applied to 22 species of herpesviruses (8 human and 14 animal viruses), with PCR products obtained for 21 of 22 viruses. In the process, 14 previously unreported amino acid-coding sequences from herpesviral DNA polymerases were obtained, including regions of human herpesviruses 7 and 8. The 50 to 60 amino acid-coding sequences recovered in the present study were determined to be unique to each viral species studied, with very little sequence variation between strains of a single species when studied. Template dilution studies in the presence of human carrier DNA demonstrated that six human herpesviruses (herpesviruses 1, 2, 3, 4, 5, and 6B) could be detected at levels at or below 100 genome equivalents per 100 ng of carrier DNA. These data suggest that consensus primer PCR targeted to herpesviral DNA polymerase may prove to be useful in the detection and identification of known herpesviruses in clinical samples and the initial characterization of new herpesviral genomes.

Plaque-associated expression of human herpesvirus 6 in multiple sclerosis.

Representational difference analysis was used to search for pathogens in multiple sclerosis brains. We detected a 341-nucleotide fragment that was 99.4% identical to the major DNA binding protein gene of human herpesvirus 6 (HHV-6). Examination of 86 brain specimens by PCR demonstrated that HHV-6 was present in > 70% of MS cases and controls and is thus a commensal virus of the human brain. By DNA sequencing, 36/37 viruses from MS cases and controls were typed as HHV-6 variant B group 2. Other herpesviruses, retroviruses, and measles virus were detected infrequently or not at all. HHV-6 expression was examined by immunocytochemistry with monoclonal antibodies against HHV-6 virion protein 101K and DNA binding protein p41. Nuclear staining of oligodendrocytes was observed in MS cases but not in controls, and in MS cases it was observed around plaques more frequently than in uninvolved white matter. MS cases showed prominent cytoplasmic staining of neurons in gray matter adjacent to plaques, although neurons expressing HHV-6 were also found in certain controls. Since destruction of oligodendrocytes is a hallmark of MS, these studies suggest an association of HHV-6 with the etiology or pathogenesis of MS.

The Drosophila homeotic mutation Nasobemia (AntpNs) and its revertants: an analysis of mutational reversion.

The homeotic gene Antennapedia (Antp) controls determination of many different cell types in the thorax and abdomen of Drosophila melanogaster. The spontaneous mutant allele Nasobemia (AntpNs) and its revertants have been widely used to infer normal Antp gene function but have not themselves been thoroughly characterized. Our analysis reveals that AntpNs consists of an internal 25-kb partial duplication of the Antp gene as well as a complex insertion of > 40 kb of new DNA including two roo transposons. The duplication gives the mutant gene three Antp promoters, and transcripts from each of these are correctly processed to yield functional ANTP proteins. At least two of the promoters are ectopically active in the eye-antenna imaginal discs, leading to homeotic transformation of the adult head. A molecular and genetic description of several AntpNs revertants shows them to be diverse in structure and activity, including a restoration of the wild type, rearrangements separating two of the AntpNs promoters from the coding sequences, and protein nulls and hypomorphs affecting expression from all three of the promoters. Finally, one revertant has a suppressing lesion in the osa locus far away from Antp. These features explain the unusual homozygous viable nature of AntpNs, suggest a mechanism by which its homeotic transformation occurs, and exemplify the diversity of ways in which mutational reversion can take place.

Positive and negative control of the Antennapedia promoter P2.

To understand the nature of the regulatory signals impinging on the second promoter of the Antennapedia gene (Antp P2), analysis of its expression in mutants and in inhibitory drug injected embryos has been carried out. The maternally-active gene osk is identified as one of two general repressors of P2 which prevent Antp transcription until division cycle 14. Products of the zygotically-active segmentation genes ftz, hb, Kr, gt and kni then act as activators or repressors of Antp P2 in a combinatorial fashion. The timing of these events, and their positive versus negative nature, is critical for generating the expression patterns normal for Antp.

Alternative RNA splicing that is spatially regulated: generation of transcripts from the Antennapedia gene of Drosophila melanogaster with different protein-coding regions.

We have shown previously that transcription of the Drosophila homeotic gene Antennapedia results in four major RNA species which differ in long 5'- and 3'-untranslated sequences. The protein-coding portion of these transcripts, however, is located in exons common to all. Using RNase protection assays and further cDNA clone isolation, we have now detected two alternative splicing events between exons of this region. These result in four RNA variations which, if translated, would encode a family of Antennapedia proteins. By analyzing transcripts from various developmental stages and isolated tissues, we show that alternative splicing is under strict temporal and spatial regulation. For example, while similar patterns of splicing were found for all wild-type thoracic imaginal disks examined, these differed distinctly from the patterns observed in neural tissues. Our results suggest that individual RNAs may be associated with different biological roles, and provide molecular evidence that the Antennapedia gene is involved in multiple functions.

Function and misfunction of the two promoters of the Drosophila Antennapedia gene.

In the Antennapedia (Antp) gene of Drosophila melanogaster, structurally distinct RNAs arise from different transcription initiation sites. When the two sites are separated by a chromosome inversion, transcripts are produced from each fragment of the split Antp locus, and these RNAs initiate at the same nucleotide as in wild-type animals. Thus, the initiation sites are regulated by independent promoters. We show by in situ hybridization that transcripts from each promoter accumulate in spatially distinct patterns in a subset of wild-type imaginal discs. Importantly, these patterns are generally maintained in the inversion mutant. We conclude that the promoters possess independent and dissimilar regulatory elements for spatial activation. Finally, we have looked at transcription in seven different dominant Antp mutants, all of which show a transformation of head tissue to thoracic tissue. In each mutant, the second promoter is improperly activated in the eye-antennal imaginal disc. Because all but one of these mutations have inversion breakpoints distantly upstream of the activated promoter, they probably act via long-range euchromatic position effects. Our studies define how the dual promoters and chromatin structure of the Antp gene contribute to the generation of a complex pattern of transcription.

Multiple transcripts from the Antennapedia gene of Drosophila melanogaster.

The structures of four major transcripts from the homeotic gene Antennapedia of Drosophila melanogaster were determined. These transcripts constitute two RNA classes, each class initiating from a unique promoter but sharing 3' exons. Within the shared sequences is a major open reading frame encoding a 378-amino-acid protein as well as alternative polyadenylation sites. Although the RNA classes differ in their 5' sequences, both leaders contain many AUGs upstream of the major open reading frame. For the two RNA classes, neither gross tissue nor temporal specificity was observed. However, the second poly(A) site is preferred in neural tissue. The structural diversity of the RNAs is discussed in relation to biological functions of the Antennapedia locus.

An inversion that disrupts the Antennapedia gene causes abnormal structure and localization of RNAs.

Mutations in Drosophila homeotic genes lead to the developmental replacement of one normal body part by another. We have examined the mechanism by which a dominant allele of the Antennapedia (Antp) gene causes the antennae to be replaced by legs. Normal Antp gene activity is required for thoracic but not for head development. We demonstrate that the Antp73b inversion mutation results in Antp transcription in the head. Antp transcripts found in this abnormal location are of two types: normal-sized Antp RNAs and fusion RNAs joining Antp exons (including the protein-coding region) to 5' exons from another gene. This foreign gene is normally expressed in the head, suggesting the cause for abnormal activation of Antp. The result is a change of cell identity from head to thorax.

A homologous protein-coding sequence in Drosophila homeotic genes and its conservation in other metazoans.

The homeotic genes of the bithorax complex (BX-C) and the Antennapedia complex (ANT-C) of Drosophila appear to specify the developmental fate of segments or parts of segments of the fly. We have previously reported weak DNA sequence homology between 3' portions of the Antennapedia and fushi tarazu genes of the ANT-C and the Ultrabithorax gene of the BX-C. Here we show that this DNA homology (the homeo box) is due to a conserved protein-coding sequence present in these three pattern-formation genes. Thus the functional homology between these developmental controlling genes is reflected in a structural homology in their gene products. The homeo box sequence is also present in a few copies in the genomes of some other invertebrates, and is even conserved in vertebrate genomes, including the human genome. Apparently at least a part of these developmental switch genes from Drosophila is highly conserved during evolution, and might perform an analogous function in many metazoans .