Histopathological Studies on the Neuropathogenicity of the Iriki and OBE-1 Strains of Akabane Virus in BALB/cAJcl Mice.

The OBE-1 strain of Akabane virus infects the fetus via the dam, resulting in abortion or congenital abnormalities in ruminants. In contrast, the Iriki strain of Akabane virus is highly virulent and causes encephalomyelitis by post-natal infection. To clarify the difference in pathogenicity between the two strains, BALB/cAJcl mice were inoculated either intraperitoneally or intracerebrally (IC) with either strain from 3 days to 8 weeks of age. Pathological examination revealed non-suppurative encephalitis in mice inoculated by either route with the Iriki strain. Virus antigens were distributed widely throughout the brain when the virus was inoculated into newborn mice, but distribution was limited to the brainstem in mice inoculated when 8 weeks old. However, brain lesions were observed only in mice inoculated with OBE-1 by the IC route when the mice were 3 days old, but these lesions were mild. To examine the manner of viral spreading, the Iriki strain was inoculated IC or intrastriatally into 8-week-old mice. Viral antigens were distributed prominently throughout the spinal cord as well as the brainstem and various cerebral nuclei, and were present with less prominence in the connective fibres. Virus antigens were also distributed in the subventricular zone, where neuronal stem cells exist. These results show that the neuroinvasiveness of the Iriki strain diminishes with age, while neurovirulence is maintained; however, for the OBE-1 strain both neuroinvasiveness and neurovirulence diminish with age. Furthermore, Akabane virus infects neuronal cells in the brainstem and spreads to the spinal cord via an unidentified transneuronal pathway.

Can health systems be enhanced for optimal health services through disease-specific programs? Results of field studies in Viet Nam and Cambodia.

Developing better health systems is the key to delivering optimal health services, although more evidence of effective strategies to do so is needed. Field surveys were conducted in Viet Nam and Cambodia to identify best practices in addressing health system bottlenecks to scale up disease control programs. The two countries were compared over time using a framework for analysis developed by the authors. In Viet Nam, a health system was in place for decades at the central to municipal levels, although it was fragile until the 1990s, when the government started taking measures. In Cambodia, the previous health system had been destroyed during previous internal conflict. In the post-conflict period, the health system was rebuilt with support for programs followed by centralization of health services. In different settings, different measures were taken to deal with similar bottlenecks. In Cambodia, vertical programs were dominant, so the government sought to centralize drug management to deal with shortages of essential drugs, while Viet Nam sought to mobilize resources to ensure drug distribution at all levels. This study shows there is no single successful approach to health systems, and a systemic approach needs to be taken because elimination of one bottleneck may reveal another. Efforts to enhance disease-specific programs may not always contribute to overall enhancement of the health system, and the best possible approach may not be the same in different countries. Further study is needed to explore common issues and principles for effective strategies to enhance health systems in different contexts.

Thoracic endovascular aortic repair for aortic complications after esophagectomy for cancer: report of three cases.

Aortic complications after esophageal cancer surgery are rare and usually fatal. Here, we report three patients who underwent thoracic endovascular aortic repair (TEVAR) for aortic complications after esophagectomy for cancer. In the first case, aortic rupture was caused by pyothorax due to residual tumor after esophagectomy. In the second case, aortic rupture was caused by pyothorax due to anastomotic leakage. In the third case, a pseudoaneurysm was caused by surgical injury during esophagectomy. TEVAR was safe and effective for severe aortic complications when graft infection was avoided. The first case died of sepsis on the 84th postoperative day, and the other two cases have survived 4 years and 2 years to date.

Interaction of Rhei Rhizoma extract with cytochrome P450 3A and efflux transporters in rats.

Traditional Chinese herbal medicines are frequently prescribed in pharmacotherapy in Japan. In the present study, we evaluated the possible interaction of several herbal extracts including Rhei Rhizoma extract with cytochrome P450 (CYP) 3A and efflux transporters such as P-glycoprotein and multidrug resistance-associated protein (MRP) 2. Rhei Rhizoma extract (100 microg/ml) significantly suppressed the CYP3A-mediated 6beta-hydroxylation of testosterone in hepatic microsomes, and increased the extent of bioavailability of midazolam, a typical CYP3A substrate, in rats. Also, Rhei Rhizoma extract (300 microg/ml) significantly suppressed P-glycoprotein-mediated efflux transport of rhodamine 123 (Rho123) in rat everted intestine. In an in-vivo study, Rhei Rhizoma extract added to intestinal perfusate at a concentration of 300 microg/ml significantly suppressed the intestinal exsorption of Rho123, though it exerted no effect on the biliary excretion of Rho123. Furthermore, the in-vitro and in-vivo MRP2-mediated intestinal efflux of 2,4-dinitrophenyl-S-glutathione was significantly suppressed by Rhei Rhizoma extract (1000 microg/ml). In conclusion, Rhei Rhizoma extract, which is taken orally at doses of 0.5-1 g each or 1-3 g daily in clinical practice, may cause pharmacokinetic herb-drug interactions in the process of the intestinal and/or hepatic CYP3A-mediated drug metabolism and P-glycoprotein- and/or MRP2-mediated efflux transport in the intestine.

[Long-term results of aortic root replacement with a composite graft].

We analyzed the long-term results of aortic root replacement with a composite graft. Since 1992, 127 patients had undergone aortic root replacement with a composite graft. There were 92 men and 35 women with a mean age of 56.5 years. There were 69 patients with annuloaortic ectasia, 17 aortic dissections, and 41 ascending aortic dilatation due to aortic valve disease. Marfan syndrome was diagnosed in 19 patients. As surgical procedure, button technique was used in 90 patients, Cabrol technique in 11, and Piehler technique in 26. Open distal anastomosis was performed in 82 patients to avoid clamp injury of rest aorta. Early mortality was 3.1% and no major morbid events had occurred. Follow-up was completed in 95.9% of the patients and the mean follow-up period was 6.1 years. Actuarial survival at 5, 10, and 15 years was 86.2%, 83.4%, and 83.4%, respectively. Actuarial freedom from aortic valve reoperation at 10 and 15 years was 99.2% and 95.7%, respectively. The results of aortic root replacement with a composite graft were excellent. This procedure should be the 1st choice for surgical treatment of the aortic root disease.

Characterization of temperature-sensitive Akabane virus mutants and their roles in attenuation.

Akabane virus (AKAV) of the genus Orthobunyavirus in the family Bunyaviridae is an important animal pathogen; however, studies on AKAV biology are scarce. Therefore, we generated temperature-sensitive (ts) mutants of AKAV in order to study its pathogenesis. The ts AKAV mutants were generated by incubating the virulent OBE-1 strain with the chemical mutagen 5-fluorouracil. Each ts mutant was inoculated intracerebrally into mice to assess its virulence, and the genomic sequences of the attenuated mutants were also determined. Three of the twelve ts mutants studied showed a mortality rate of less than 10%. Although no mutation was detected in the S RNA segment of these three mutants, amino acid substitutions were observed in both the M and L RNA segments. Three of the mutants and the wild-type virus demonstrated a similar pattern of immunoreactivity in an ELISA with anti-Gc monoclonal antibodies. On the other hand, using a minireplicon system, the level of L protein activity of each ts mutant decreased as the temperature increased. These results suggest that the L RNA segment could be involved in the virulence of AKAV, which increases our understanding of how the viral gene products contribute to pathogenesis.

Sequence determination and functional analysis of the Akabane virus (family Bunyaviridae) L RNA segment.

Akabane virus (AKAV) causes epizootic congenital deformities in cattle, sheep, and goats. Due to the lack of a complete genome sequence, the molecular biological properties of this virus are not known. We have cloned and sequenced the functional large (L) RNA segment of AKAV, and shown that it has polymerase activity using a minireplicon system with RNA polymerase I. The complete L RNA segment is 6868 nucleotides long and encodes an L protein of 2251 amino acids, which functions as an RNA-dependent RNA polymerase. A minireplicon reporter plasmid was constructed by flanking either the firefly luciferase or the green fluorescent protein gene in the antisense orientation with the 5'- and 3'-terminal noncoding regions of the small RNA segment. HmLu-1 cells were transfected with the reporter plasmid, and the L protein and nucleoprotein (N protein) expression plasmids. The reporter activity was upregulated in a dose-dependent manner with increasing concentration of either the L or N protein expression plasmid. Furthermore, the reporter activity could be downregulated by the AKAV NSs protein as well as by other orthobunyaviruses. These results show that the AKAV minireplicon system is a powerful tool for studying transcription and for rescuing infectious viruses from cloned cDNAs.

Screening and determination of gene function using randomized ribozyme and siRNA libraries.

Rapid progress in the sequencing of the genomes of model organisms, such as the mouse, rat, nematode, fly, and Arabidopsis, as well as the human genome, has provided abundant sequence information, but functions of long stretches of these genomes remain to be determined. RNA-based technologies hold promise as tools that allow us to identify the specific functions of portions of these genomes. In particular, catalytic RNAs, known also as ribozymes, can be engineered for optimization of their activities in the intracellular environment. The introduction of a library of active ribozymes into cells, with subsequent screening for phenotypic changes, can be used for the rapid identification ofa gene function. Ribozyme technology complements another RNA-based tool for the determination of gene function, which is based on libraries of small interfering RNAs (siRNAs).

Genetic analysis of calicivirus genomes detected in intestinal contents of piglets in Japan.

Enteric caliciviruses, noroviruses, and sapoviruses are emerging pathogens responsible for diarrhea or gastroenteritis in their respective hosts. In this study, swine enteric caliciviruses were detected in ten samples of intestinal contents from 24 piglets in Japan by reverse transcription-polymerase chain reaction using a broadly reactive primer pair (P290/289) that targeted the highly conserved RNA polymerase regions of the enteric caliciviruses. From the positive samples, the entire viral genome of strain K7/JP and 3'-end parts of the genomes of strains K5/JP and K10/JP were cloned and sequenced. K7/JP had an RNA genome of 7144 bases, excluding its 3' poly (A) tail. The K7/JP genome possessed two open reading frames and characteristics common to sapoviruses. In phylogenetic analysis using amino acid sequences of VP1, K5/JP was demonstrated to be close to the noroviruses previously detected in pigs, and K7/JP and K10/JP were considered to be classified as a new genogroup of sapoviruses.

Intracellular-diced dsRNA has enhanced efficacy for silencing HCV RNA and overcomes variation in the viral genotype.

RNA interference (RNAi) can be used to inhibit viral replication in mammalian cells and therefore could be a powerful new antiviral therapy. Small interfering RNA (siRNA) may be effective for RNAi, but there are some technical problems that must be solved in each case, for example, predicting the effective siRNA target site and targeting heterogeneous sequences in a virus population. We show here that diced siRNA generated from long double-stranded RNA (dsRNA) is highly effective for inducing RNAi in HuH-7 cells harboring hepatitis C virus (HCV) replicons and can overcome variations in the HCV genotype. However, in mammalian cells, long dsRNA induced an interferon response and caused cell death. Here we describe an improvement of this method, U6 promoter-driven expression of long hairpin-RNA with multiple point mutations in the sense strand. This can efficiently silence HCV RNA replication and HCV protein expression without triggering the interferon response or cell death normally caused by dsRNA. In conclusion, intracellular-diced dsRNA efficiently induces RNAi, and, despite the high rate of mutation in HCV, it should be a feasible therapeutic strategy for silencing HCV RNA.

Genetic recombination at different points in the Npro-coding region of bovine viral diarrhea viruses and the potentials to change their antigenicities and pathogenicities.

Cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strain KS86-1 cp was isolated from a cow persistently infected with non-cytopathogenic (ncp) BVDV strain KS86-ncp after development of mucosal disease by superinfection with cp BVDV strain Nose. cp BVDV strains 799cp and 839cp were also isolated from independent cattle that developed mucosal disease by superinfection with cp BVDV KS86-1cp. In the present study, genetic analysis revealed that the genes of cp BVDV strains 799cp and 839cp were chimeras between the genes of the persisting ncp BVDVs and that of superinfecting KS86-1cp. The genetic recombination that generates 799cp occurred between the identical points in the N(pro) gene region, whereas genetic recombination that generates 839cp occurred between different points in the N(pro) gene region. Both 799cp and 839cp were inherited Jiv gene of KS86-1cp strain and envelope protein genes of the persisting viruses. In addition, neutralization test disclosed that antigenicities of 799cp, 839cp, and KS86-1cp were also similar to each persisting virus. These findings indicate that exogenous cp BVDV containing insertion of Jiv gene in the 5 terminal region can induce genetic recombination with the original ncp BVDV at different points in the N(pro) gene region, and those viruses have high potential to change those antigenicities and pathogenicities by RNA recombination.

Enhancement of apoptosis via an extrinsic factor, TNF-alpha, in cells infected with cytopathic bovine viral diarrhea virus.

Isolates of bovine viral diarrhea virus (BVDV) are divided into cytopathic (cp) and noncytopathic (ncp) biotypes according to their effect on cultured cells. Calves persistently infected with ncp BVDV are known to develop lethal mucosal disease (MD) after superinfection by cp BVDV. Although the UV-irradiated supernatant of cp BVDV-infected cells has been reported to have no capacity to induce cell death, we found that it could enhance cell death through apoptosis. Up-regulation of tumor necrosis factor alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS) mRNAs was detected specifically in cp BVDV-infected primary cell cultures. Suppression of TNF-alpha via antisense oligonucleotide transfection or incubation with a polyclonal antibody against TNF-alpha resulted in attenuation of apoptosis induced by cp BVDV, suggesting that TNF-alpha participates in apoptosis execution. Although TNF-alpha is one of the iNOS-inducible factors, the iNOS up-regulation was not regulated by TNF-alpha. And iNOS was revealed to serve as anti-apoptotic factor, contrary to our expectation. In addition, the expression level of both TNF-alpha and iNOS mRNAs in the ncp BVDV-infected cells was kept lower than that in the mock-infected cells, suggesting that ncp BVDV reduced or interfered with the factor triggering the expression of both mRNAs. These characteristic mRNA transcriptions would help to explain why BVDV acts differently in cells as well as in vivo, depending on its biotype. To elucidate viral factors inducing TNF-alpha and iNOS may be critical to understand the mechanism of MD development, which closely correlates with cp BVDV-induced apoptosis.

A novel antigenic variant of Canine parvovirus from a Vietnamese dog.

Nine isolates of Canine parvovirus (CPV) were obtained from Vietnamese dogs and cats. One canine isolate showed a unique antigenic property which indicates a novel antigenic variant of CPV-2b when examined with hemagglutination inhibition tests using our monoclonal antibodies, 21C3 and 19D7, which were recently developed. This isolate had an amino acid substitution of residue 426, Asp to Glu, and the same substitution has recently been found in CPV from Italian dogs. This study first showed that such substitution caused an antigenic difference demonstrable by monoclonal antibodies and that a similar evolution may have occurred in CPV in Vietnam.

Obstruction of St Jude medical valves in the aortic position: a consideration for pathogenic mechanism of prosthetic valve obstruction.

Between 1995 and 2000, 8 patients with St. Jude Medical (SJM) valves in the aortic position required 9 redo valve replacement for prosthetic valve obstruction. Obstruction of the prosthetic valve was diagnosed by simultaneous echocardiography and cineradiography, and process of restricted leaflet movement that progressed to hemodynamic impairment was observed by serial studies in three recent patients. An oral anticoagulation was considered to be adequate in all patients except one patient who had withdrawal of warfrain. Pannus was the sole cause of valve obstruction in seven events in 6 patients, and both thrombus and pannus in 2 patients. Pannus overgrowth was found on the inflow aspect of the SJM valve, and involved the ends of the straight edge of the leaflets over pivot guards. These results suggest that pannus might play the primary role in development of obstruction of aortic SJM valves in patients on adequate oral anticoagulation.

[Long-term results of aortic root replacement for Marfan syndrome].

Between January 1974 and December 2000, aortic root replacement was performed for 46 patients with Marfan syndrome. The hospital mortality rate amounts to 8.6% (4 patients out of 46). Fifteen (35.7% of survivor) of 42 survivor died during the period of follow-up. Eighteen of 42 survivor were developed cardiovascular events of 21 times and 11 patients had to be reoperated on at least once for a total of 12 reoperations. Nine of 15 late death patient died in relation to aortic dissection and pseudoaneurysm of distal anastomosis and coronary anastomosis. Actuarial survival rate was satisfactory for 10 years after aortic root replacement, but sharply decreased after postoperative 10 years. The use of non-sealed graft, classical Bentall method, reoperation and inclusion technique were risk factors for the late death.

Gene expression and molecular evolution.

The combination of complete genome sequence information and estimates of mRNA abundances have begun to reveal causes of both silent and protein sequence evolution. Translational selection appears to explain patterns of synonymous codon usage in many prokaryotes as well as a number of eukaryotic model organisms (with the notable exception of vertebrates). Relationships between gene length and codon usage bias, however, remain unexplained. Intriguing correlations between expression patterns and protein divergence suggest some general mechanisms underlying protein evolution.

Unruptured aneurysm of the right coronary sinus of Valsalva with type B aortic dissection.

A seventy-two year-old man, who complained of severe back pain, was referred to our hospital. Digital subtraction angiogram (DSA) delineated an extracardiac unruptured aneurysm of the right coronary sinus of Valsalva and acute type B aortic dissection. Patch plasty of the right coronary sinus with reimplantation of the right coronary artery using a Dacron graft was performed. Postoperative DSA confirmed successful reconstruction of the aortic root and the patent right coronary artery.