High prevalence of Nova hantavirus infection in the European mole (Talpa europaea) in France.

SUMMARY: Recent discovery of genetically distinct hantaviruses in shrews and moles (order Soricomorpha, family Soricidae and Talpidae) has challenged the conventional view that rodents serve as the principal reservoir hosts. Nova virus (NVAV), previously identified in archival liver tissue of a single European mole (Talpa europaea) from Hungary, represents one of the most highly divergent hantaviruses identified to date. To ascertain the spatial distribution and genetic diversity of NVAV, we employed RT-PCR to analyse lungs from 94 moles, captured in two locations in France, during October 2012 to March 2013. NVAV was detected in more than 60% of moles at each location, suggesting efficient enzootic virus transmission and confirming that this mole species serves as the reservoir host. Although the pathogenic potential of NVAV is unknown, the widespread geographical distribution of the European mole might pose a hantavirus exposure risk for humans.

Modulation of adhesion molecules and monocyte chemoattractant protein by tumor necrosis factor-alpha and salicylic acid in primary human coronary artery endothelial cells.

Kawasaki disease (KD) is an illness characterized by vascular inflammation of coronary arteries leading to coronary aneurysms and thromboses. Infiltration of immune cells into the intima and adventitia are observed in autopsy tissues of patients with KD. Using semi-quantitative RT-PCR and cell-based ELISA, we demonstrated that tumor necrosis factor-a induced the expression of intercellular adhesion molecules-1 and E-selectin, as well as monocyte chemoattractant protein-1, in a time- and dose-dependent manner in primary human coronary artery endothelial cell cultures. This increase was inhibited by salicylic acid (NaSal), and involved the transcription factor NF-kappaB. Based on these data, we suggest a pathogenetic mechanism for KD, whereby immune cells are attracted to sites of inflammation, undergo extravasation, release enzymes that assist in vascular remodeling, thereby weakening the endothelium and hastening the process of aneurysm formation. NaSal, in addition to preventing thrombosis and lowering fever in KD, may also function in down-regulating adhesion molecules during the inflammatory stage of KD.

RT-PCR detection of the expression of the polymerase gene of a novel reptilian herpesvirus in tumor tissues of green turtles with fibropapilloma.

An alpha-herpesvirus has recently been associated with green turtle fibropapilloma (FP). To further understand the etiological role of this newfound green turtle herpesvirus (GTHV) in the pathogenesis of FP, expression of GTHV polymerase ( pol) gene was determined in tumors and normal-appearing nontumor tissues and organs from five green turtles suffering multiple fibropapillomas, using reverse transcription-polymerase chain reaction (RT-PCR). Amplification of RNA prepared from tumor tissues evidenced the substantial expression of GTHV DNA pol gene in all specimens tested (15/15). However, GTHV pol gene expression in normal-appearing tissues and organs of affected animals was limited (4/45), and GTHV mRNA was detected only in periorbital tissue (1/2), gall bladder (2/5) and lung (1/5) by nested RT-PCR. By contrast, RT-PCR evaluation of RNA isolated from non-tumored turtles revealed undetectable expression of this herpesvirus gene. cDNA sequence analysis revealed that GTHV gene sequences were identical in different tumors. Our data represent the first evidence of the replication of this putative turtle herpesvirus in affected green turtles and fibropapilloma tissues are always active sites of GTHV mRNA synthesis. These findings extend and substantiate the pathogenic association of GTHV with FP.

Highly active antiretroviral therapy (HAART)-associated lactic acidosis: in vitro effects of combination of nucleoside analogues and protease inhibitors on mitochondrial function and lactic acid production.

Lactic acidosis is a rare but potentially life-threatening and poorly understood sequelae among HIV-infected patients on highly active antiretroviral therapy (HAART). Mitochondrial DNA depletion and inhibition of respiratory complexes have been hypothesized to be involved in HAART-associated lactic acidosis. Although mitochondrial toxicity and increased plasma lactates are associated with long-term exposure to nucleoside analogue reverse transcriptase inhibitors (NRTI), reports of lactic acidosis are now emerging among HIV-infected patients exposed to combination therapy that includes not only NRTI but also protease inhibitors (PI). We therefore investigated the effects of clinically relevant NRTI and PI combinations on mitochondrial membrane potential, uncoupling of mitochondrial respiration from oxidative phosphorylation and lactic acid production. Our study demonstrated that treatment of HepG2 cells with a combination of nucleoside analogues and PI, decreased mitochondrial membrane potential (delta psi m) within 24 hr, followed by increased lactic acid production after 9 days of treatment. However, loss of delta psi m and increased lactates were not associated with mitochondrial uncoupling or ATP production. Our findings suggested that not only NRTI but also PI are capable of increasing lactic acid production in vitro, and probably involve early biochemical changes in mitochondrial function such as loss of mitochondrial membrane potential.

Clinical isolates of Streptococcus pneumoniae resistant to levofloxacin contain mutations in both gyrA and parC genes.

Thirty Streptococcus pneumoniae clinical isolates resistant to levofloxacin were analyzed for the quinolone resistance-determining DNA sequences to identify point mutations and were tested for in vitro susceptibility to multiple drug classes. Of these isolates, 29 had mutations in both gyrA and parC genes of DNA gyrase and topoisomerase IV, respectively. In GyrA, an amino acid change from Ser-81-->Phe was detected in 27 isolates and a Glu-85-->Lys change was found in the remaining three. Of the 29 isolates for which ParC data were available, Ser-79-->Tyr or Phe were the predominant mutations observed. MICs for levofloxacin were 4-16 mg/l, whereas those for moxifloxacin were 1-2 mg/l. Twenty-four (80%) isolates were susceptible to erythromycin, 25 (83%) to azithromycin, 26 (87%) to clarithromycin, 27 (90%) to clindamycin, 20 (67%) to penicillin, 21 (70%) to ceftriaxone and 30 (100%) to amoxycillin/clavulanate. These results confirm the presence of double mutations among clinical isolates of S. pneumoniae from diverse geographical regions of North America and also suggest that quinolone resistance may develop independently of resistance to other drug classes.

Predicted amino acid sequences for 100 JCV strains.

DNA sequence variation between JCV genotypes is confined largely to noncoding intergenic regions and introns. Nevertheless, evidence suggests that the amino acid sequence variations among the 8 genotypes of JCV can influence the potential for neurovirulence of the virus. In the current study, the amino acid sequences for 100 JCV genomes were translated and grouped into genotype families. Subtype consensus sequences were determined and the type-specific amino acid sequence variants were identified.

Reconstructing population history using JC virus: Amerinds, Spanish, and Africans in the ancestry of modern Puerto Ricans.

The roots of the Hispanic populations of the Caribbean Islands and Central and South America go back to three continents of the Old World. In Puerto Rico major genetic contributions have come from (1) Asians in the form of the aboriginal Taino population, an Arawak tribe, present when Columbus arrived on the Island, (2) Europeans, largely Spanish explorers, settlers, government administrators, and soldiers, and (3) Africans who came as part of the slave trade. Since JC virus (JCV) genotypes characteristic of Asia, Europe, and Africa have been identified, and excretion of JCV in urine has been proposed as a marker for human migrations, we sought to characterize the JCV strains present in a Caribbean Hispanic population. We found that the strains of JCV present today in Puerto Rico are those derived from the Old World populations represented there: Types 1B and 4 from Spain, Types 3A, 3B, and 6 from Africa, and Type 2A from Asia. The Type 2A genotype represents the indigenous Taino people. This JCV genotype was represented much more frequently (61%) than would be predicted by the trihybrid model of genetic admixture. This might be attributable to characteristics of JCV Type 2A itself, as well as to the nature of the early relationships between Spanish men and native women. These findings indicate that the JCV strains carried by the Taino Indians can be found in today's Puerto Rican population despite the apparent demise of these people more than two centuries ago. Therefore, molecular characterization of JCV provides a tool to supplement genetic techniques for reconstructing population histories including admixed populations.

The molt-inhibiting hormone in the American crayfish Procambarus clarkii: its chemical synthesis and biological activity.

The molt-inhibiting hormone of the American crayfish Procambarus clarkii (Prc-MIH), a 75-residue polypeptide containing three disulfide bridges, was synthesized by chemical ligation of two peptides, i.e., synthetic Prc-MIH(1-39) and Prc-MIH(40-75)-NH(2), and by subsequent folding to form the native disulfide-containing peptide molecule. The synthetic peptide was comparable to the natural Prc-MIH in inhibiting ecdysteroid secretion by in vitro bioassay and shared features with the natural Prc-MIH in some biochemical analyses. These results indicate that the chemical ligation method can be used for the synthesis of Prc-MIH. Furthermore, it was demonstrated that synthetic Prc-MIH has hyperglycemic activity, although the activity was weaker than that of the authentic crustacean hyperglycemic hormone in the American crayfish. To examine the structural requirement of the Prc-MIH for eliciting biological activity, an antibody raised against the C-terminal region (residues 55-75) and two synthetic peptides, i.e., a core region (residues 1-54) containing three disulfide bridges and the C-terminal region, were utilized. It is suggested that Prc-MIH exerts its activities through coordination between the core region and the C-terminal region.

Rapid acquisition of entire DNA polymerase gene of a novel herpesvirus from green turtle fibropapilloma by a genomic walking technique.

A 4837-bp sequence of a newfound green turtle herpesvirus (GTHV), implicated in the etiology of green turtle fibropapilloma, was obtained from tumor tissues of a green turtle with fibropapilloma using a genomic walking method based on restriction enzyme digestion, self-ligation and inverse polymerase chain reaction (IPCR). The 4837-bp sequence was 56.23% G/C rich and contained three nonoverlapping open reading frames (ORF). The largest ORF (3507-bp) encoded the DNA polymerase gene (pol gene), which exhibited a high degree of homology at both amino acid and nucleotide levels with the DNA pol genes of human and animal herpesviruses, with a predicted protein of 1169 amino acids and molecular weight of 132.6 kilodaltons. The ATG at 518 to 520 was the first initiation codon in the ORF and was presumed to be the first methionine codon of the pol gene. Phylogenetic analysis, based on the amino acid sequence of the GTHV DNA pol gene and the corresponding regions of other known human and animal herpesviruses, indicated that GTHV belonged to the Alphaherpesvirinae subfamily. The upstream ORF of the pol gene encoded the N-terminal region of the GTHV homologue of the DNA-binding protein gene, whereas the downstream ORF was the C-terminal region of a gene which was homologous to ORFs conserved in human and animal herpesviruses, i.e., herpes simplex virus 1 (HSV1) gene UL31, Epstein-Barr virus (EBV) gene BFLF2, equine herpesvirus 1 (EHV1) gene 29, and alcelaphine herpesvirus 1 (AHV1) hypothetical protein 69 gene. The arrangement of these three genes in GTHV genome was identical to that seen in other alphaherpesviruses. The sequence and location of the DNA pol gene in the GTHV genome should greatly facilitate future studies of the viral life cycle.

Tropical spastic paraparesis.

Human T-cell lymphotropic virus type I (HTLV-I) is the cause of endemic tropical spastic paraparesis (TSP) or HTLV-I-associated myelopathy (HAM). Because TSP/HAM is not a fatal disease, the neuropathology of this disease, albeit relatively well understood, is based on the examination of just a few incidental cases. We summarise our experience with the neuropathology of tropical spastic paraparesis/HTLV-I associated myelopathy (TSP/HAM). We studied three cases of TSP/HAM from different parts of the world. We demonstrated peculiar lamellated structures, called "multilamellar bodies" (MLB). It is tempting to suggest that MLB may represent specific ultrastructural markers of TSP/HAM. The pathology of the anteriorand posterior horns was similar and comprised axonal degeneration, accompanied by extensive astrocytic gliosis. Lymphocytic infiltration, particularly observed as "cuffs" around blood vessels, was scattered among other cellular elements. Ultrastructurally, myelin sheaths were relatively well preserved, and some demyelinated but not remyelinated fibres were observed. Moreover, axons with abnormal accumulations of neurofilaments, suggestive of axonal degeneration, were detected. Several axons contained Hirano bodies. In many samples glial processes replaced most of the remaining neuropil.

Conversion of pyrrole to pyrrole-2-carboxylate by cells of Bacillus megaterium in supercritical CO2.

Pyrrole was converted to pyrrole-2-carboxylate in supercritical CO2 using cells of Bacillus megaterium PYR 2910, and the yield of the carboxylation reaction in supercritical CO2 was 12 times higher than that under atmospheric pressure.

New JC virus (JCV) genotypes from papua new guinea and micronesia (type 8 and type 2E) and evolutionary analysis of 32 complete JCV genomes.

The JC virus (JCV) is a ubiquitous human polyomavirus that frequently resides in the kidneys of healthy individuals and is excreted in the urine of a large percentage of the population. Geographic-specific JCV variants, isolated from urine and from brain of progressive multifocal leukoencephalopathy (PML) patients, have been grouped into seven distinct genotypes based on whole genome analysis and by individual polymorphic nucleotides (typing sites) in the VP1 coding region. Mutations in the archetypal regulatory region, sometimes consisting of deletions and/or duplications, are also useful taxonomic characters for further characterizing and subdividing genotypes. Investigation of JCV variation in Papua New Guinea (PNG) revealed three distinct variants called PNG- 1, PNG-2, and PNG-3. These variants exhibited consistent coding region and regulatory region mutations. Evolutionary analysis of 32 complete JCV genomes including six new viral genomes from the western Pacific suggests that the new PNG JCV variants are closely associated with the broad group of Type 2 strains of JCV found throughout Asia, forming a monophyletic group with the Northeast Asian strains (Type 2A). Within the Type 2 clade, however, the PNG JCV variants cluster as two distinct groups and are therefore described here as new JCV genotypes designated Type 2E and Type 8.

Genetic diversity of Apodemus agrarius-borne hantaan virus in Korea.

Hantaan (HTN) virus, the etiologic agent of clinically severe hemorrhagic fever with renal syndrome (HFRS), was first isolated in 1976 from lung tissues of striped field mice (Apodemus agrarius) captured in Songnae-ri, Kyungki-do, Korea. To clarify the genetic diversity and phylogenetic relationship among Korean strains of HTN virus, viral sequences of the partial S and M segments were amplified from lung tissues of 24 seropositive striped field mice captured between 1989 and 1998 at 11 sites in South Korea. The 771-nucleotide (nt) S segment sequences (coordinates 432 to 1202) of HTN virus strains from Yangju-kun differed by 10 to 40 nt (1.3 to 5.2%) from virus strains from Pocheon-kun, Songnae-ri and Nonsan-kun. Similar degrees of genetic variation were found in the G1 and G2 glycoprotein-encoding M segment. Phylogenetic trees, based on the partial S and M segments and generated by the maximum parsimony and neighbor-joining methods, demonstrated that virus strains from various geographic regions in South Korea showed a tendency to form two phylogenetic subgroups and were evolutionarily distinct from HTN virus strains from the People's Republic of China.

Fecal excretion of a novel human circovirus, TT virus, in healthy children.

The role of TT virus (TTV) as a human pathogen is unclear, as is the mode of TTV transmission. To determine the prevalence of TTV infection and the possible fecal-oral route of transmission, we analyzed fecal specimens from 67 healthy, nontransfused children for TTV DNA sequences by heminested PCR, using the NG and T primer sets. The overall prevalence of TTV fecal excretion was 22.4% (15 of 67), with the T primer set (19.4%) being more sensitive than the NG primer set (10.4%). TTV prevalence based on gender or ethnicity showed no significant differences. None of seven children in the 0- to 6-month age group had detectable TTV in feces. Of three sets of siblings, two unrelated sets of twins, ages 33 and 37 months, were negative for fecal TTV DNA, while the third set of siblings, ages 99 and 35 months, was positive. The absence of TTV in the feces of children younger than 6 months and the high prevalence (40%) in children 7 to 12 months of age is consistent with age-specific acquisition of TTV infection by the nonparenteral route. TTV genotypes 1, 3, 4, and 5 were represented in our study population. TTV-positive siblings had TTV genotypes 1 and 4, suggesting unrelated environmental sources of TTV infection. This observation suggests a possible time frame for TTV acquisition in children which coincides with increased interaction with their environment and increased susceptibility to infectious agents.

Detection of herpesviral sequences in tissues of green turtles with fibropapilloma by polymerase chain reaction.

An alpha-herpesvirus has been associated recently with green turtle fibropapilloma (FP). To further clarify the role of this newfound green turtle herpesvirus (GTHV) in the pathogenesis of FP, various normal-appearing tissues and organs (including skin, eye, brain, heart, liver, spleen, intestine, lung, kidney, nerve, gonad, tongue, gall bladder, urinary bladder, thyroid and peripheral blood mononuclear cells (PBMC) from blood) and tumor tissues from 19 green turtles (Chelonia mydas) with FP, and tissues from three green turtles without FP, collected during 1997 to 1999 in the Hawaiian Islands, were tested for GTHV sequences by nested polymerase chain reaction (PCR), using GTHV-specific oligonuclotide primers. GTHV sequences were detected in all tumors (51/51) and most tissues (133/167) of tumored turtles. By contrast, such sequences were undetectable in tissues (0/28) of three non-tumored turtles. Analysis of GTHV sequences detected in different tissues and tumors revealed a low degree of genetic diversity (<1%). The wide distribution of this newfound herpesvirus in tumors and tissues of tumored green turtles and its absence in tissues of non-tumored turtles, argues for an etiologic role in FP.

Human polyomavirus JC variants in Papua New Guinea and Guam reflect ancient population settlement and viral evolution.

The peopling of the Pacific was a complex sequence of events that is best reconstructed by reconciling insights from various disciplines. Here we analyze the human polyomavirus JC (JCV) in Highlanders of Papua New Guinea (PNG), in Austronesian-speaking Tolai people on the island of New Britain, and in nearby non-Austronesian-speaking Baining people. We also characterize JCV from the Chamorro of Guam, a Micronesian population. All JCV strains from PNG and Guam fall within the broad Asian group previously defined in the VP1 gene as Type 2 or Type 7, but the PNG strains were distinct from both genotypes. Among the Chamorro JCV samples, 8 strains (Guam-1) were like the Type 7 strains found in Southeast Asia, while nine strains (Guam-2) were distinct from both the mainland strains and most PNG strains. We identified three JCV variants within Papua New Guinea (PNG-1, PNG-2 and PNG-3), but none of the Southeast Asian (Type 7) strains. PNG-1 strains were present in all three populations (Highlanders and the Baining and Tolai of New Britain), but PNG-2 strains were restricted to the Highlanders. Their relative lack of DNA sequence variation suggests that they arose comparatively recently. The single PNG-3 strain, identified in an Austronesian-speaking Tolai individual, was closely related to the Chamorro variants (Guam-2), consistent with a common Austronesian ancestor. In PNG-2 variants a complex regulatory region mutation inserts a duplication into a nearby deletion, a change reminiscent of those seen in the brains of progressive multifocal leukoencephalopathy patients. This is the first instance of a complex JCV rearrangement circulating in a human population.

Identification of a small, naked virus in tumor-like aggregates in cell lines derived from a green turtle, Chelonia mydas, with fibropapillomas.

Serial cultivation of cell lines derived from lung, testis, periorbital and tumor tissues of a green turtle (Chelonia mydas) with fibropapillomas resulted in the in vitro formation of tumor-like cell aggregates, ranging in size from 0.5 to 2.0 mm in diameter. Successful induction of tumor-like aggregates was achieved in a cell line derived from lung tissue of healthy green turtles, following inoculation with cell-free media from these tumor-bearing cell lines, suggesting the presence of a transmissible agent. Thin-section electron microscopy of the cell aggregates revealed massive collagen deposits and intranuclear naked viral particles, measuring 50+/-5 nm in diameter. These findings, together with the morphological similarity between these tumor-like cell aggregates and the naturally occurring tumor, suggest a possible association between this novel virus and the disease. Further characterization of this small naked virus will clarify its role in etiology of green turtle fibropapilloma, a life-threatening disease of this endangered marine species.

Amplification and analysis of DNA flanking known sequences of a novel herpesvirus from green turtles with fibropapilloma Brief report.

A 1,632-bp fragment, flanking the original 483-bp region of the DNA polymerase gene of a novel herpesvirus found in tissues of green turtles (Chelonia mydas) with fibropapilloma, was amplified from the circularized EcoRI-cut DNA extracted from tumor tissues by inverse PCR. The resultant 2,019-bp partial sequence of the DNA polymerase gene of the newfound herpesvirus, including the original 483-bp region, showed a high degree of homology at both the nucleotide and amino acid levels with that of other human and animal herpesviruses. Phylogenetic analysis confirmed that this novel herpesvirus belonged to the Alphaherpesvirinae subfamily.

Establishment, cryopreservation, and growth of 11 cell lines prepared from a juvenile Hawaiian monk seal, Monachus schauinslandi.

Eleven cell lines were prepared from skin, snout, liver, kidney, lung, heart, brain, spleen, thyroid, urinary bladder, and periorbital soft tissue of a juvenile Hawaiian monk seal (Monachus schauinslandi). The cell grew at 37 degrees C in RPMI 1640 medium supplemented with 20% fetal bovine serum. These cell lines have been subcultured 11-27 times since their initiation in May 1997. Growth of the monk seal cells was serum-dependent and plating efficiencies ranged from 4-24%. These monk seal cells grew well in M199, L-15 and MEM commonly used for cultivation of animal and mammalian cells and retained 87% cell viability following storage for 2.5 years in liquid nitrogen. Karyotyping indicated that these monk seal-derived cell lines remained diploid with a chromosome count of 34 at their early passage (passage 9-13). These cell lines were tested for herpesvirus by polymerase chain reaction using degenerate oligonucleotide primers designed from the highly conserved region of herpesviral DNA polymerase gene and no specific detection occurred. These newly established cell lines are currently being used for the investigation of an eye disease occurring in captive monk seal pups in Oahu and will be available for future isolation and study of monk seal viruses.