Expression of bovine rotavirus VP8 and preparation of IgY antibodies against recombinant VP8.

Group A rotavirus is the leading cause of acute gastroenteritis in cattle and swine. Although, vaccination against this virus is an effective strategy for prevention, additional strategy to control disease is necessary. Egg yolk immunoglobulin (IgY)-based passive immunization could be a better option in preventing this disease. Bovine rotavirus (BRV) is group A rotavirus and possesses a genome of 11 segments of double-stranded RNA. The outer layer of capsid is composed of two proteins (VP7 and VP4), which induce virus neutralizing antibodies. Trypsin cleavage of VP4 produces VP8 (28 kDa) and VP5 (60 kDa) fragments. Since a number of studies have demonstrated the induction of neutralizing antibodies using VP8 subunit vaccines, we have produced IgY against the recombinant VP8. The cDNA spanning the VP8 subunit was amplified from bovine rotavirus-infected cells and cloned into pET21d(+) expression vector to generate recombinant VP8. The resulting carboxy-terminal His-tagged VP8 proteins were expressed in Escherichia coli strain BL21(DE3) by isopropyl ß-D-1-thiogalactopyranoside (IPTG) induction. The recombinant proteins were purified using Ni-NTA agarose beads, and the purified protein was used as the immunizing agent to produce polyclonal antibodies in chicken. The resulting polyclonal antisera specifically recognized VP8 in Western blot assay and were able to neutralize BRV replication in cell cultures. These results demonstrate that IgY can be used in immunological assays and, in addition, in passive immunization of newborn calves against BRV.

Mouse tetherin enhances moloney murine leukemia virus-induced syncytium formation.

Tetherin (also referred to as BST-2 or CD317) is an antiviral cellular restriction factor that inhibits the release of many enveloped viruses. It is a 30-36 kDa type II transmembrane protein, expression of which is induced by type I interferon. Mouse tetherin inhibits nascent cell-free particle release. However, it is unclear whether mouse tetherin restricts cell-to-cell spread of moloney murine leukemia virus (Mo-MLV) or whether is the mouse tetherin involved in syncytium formation. To examine cell-to-cell spread and syncytium formation of Mo-MLV in the presence or absence of mouse tetherin, R peptide (the cytoplasmic tail of the transmembrane protein (TM); 16 amino acids) truncated Env expressing vector was constructed. It contained enhanced green fluorescent protein (EGFP) in the proline rich region (PRR) of Env. This R(-)Env full-length molecular clone could rule out virus-cell transmission due to the slightly reduced R(-)Env protein incorporation into the viral particles. When NIH3T3 cells stably expressing mouse tetherin were transfected with R(-)Env full-length molecular clone, syncytium formation was significantly enhanced in the tetherin-expressing cells. These data suggest that tetherin-mediated retention of R-defective virions on the cell surface could enhance syncytium formation. In addition, we found that the R(-)Env full-length molecular clone containing EGFP in the PRR of Env to be a useful tool allowing fast and convenient detection of syncytia by fluorescence microscopy.

Use of IgY antibody to recombinant avian reovirus σC protein in the virus diagnostics.

Avian reovirus (ARV) is an important agent of several diseases causing considerable losses in poultry farming. An outer capsid protein (σC) of ARV, is known as a virus-cell attachment protein essential for virus infectivity. In this study, the σC gene of ARV was cloned and expressed in Escherichia coli. The expressed recombinant protein was used as immunogen for raising a specific IgY antibody in laying hens. At 14 weeks post immunization, the antibody titers in serum and egg yolk reached 302,000 and 355,000, respectively. The IgY antibody was capable to neutralize ARV in BHK-21 cells and it strongly reacted in ELISA with ARV but not with heterologous viruses. The IgY antibody detected ARV in field samples of infected animal tissues in dot blot assay. These results suggest that an efficient, economic and rapid diagnostics of ARV can be performed routinely using the IgY antibody against a recombinant ARV σC protein.

Tetherins of various species inhibit the release of porcine endogenous retrovirus from human cells.

Pigs are considered as suitable xenotransplantation organ donors. However, the risk of pathogen transmission from pigs to human is a major concern in the transplantation of porcine tissues since it had been shown that porcine endogenous retroviruses (PERVs) can infect human cells. Tetherin has recently been described as a host restriction factor that blocks the release of virus particles from cells infected with some enveloped viruses. We compared tetherins derived from various species in their activity against PERVs by using a pseudotype assay. The results showed that (i) mammalian tetherins inhibit spread of PERVs, (ii) murine and rhesus tetherins are weaker inhibitors than canine and feline ones, (iii) human tetherin is induced by interferon alpha (IFN-α) and (iv) IFN-α treatment of 293T-PERV-PK-CIRCE cells reduced PERV release. We conclude that transgenic overexpression of tetherin combined with its induction by IFN-α may reduce the risk of PERV dissemination in xenotransplantation.

Microelectrode recording: lead point in STN-DBS surgery.

BACKGROUND: Microelectrode recording is an integral part of many surgical procedures for movement disorders. We evaluate the Lead point compared to the NeuroTrek system. We used NeuroTrek in 18 Parkinsonian patients, Lead point-4 in 12 patients, during STN-DBS surgery. We compared MR-Stir image with Microelectrode recording. METHOD: The MicroGuide system with its integrated screen display provides the user with all the information needed during the surgery on its screen. Microelectrode recordings showed characteristic neuronal discharges on a long trajectory (5-6 mm), intraoperative stimulation induces dramatic improvement of Parkinsonian motor symptoms. FINDINGS: Microrecording data of the Leadpoint showed high background activity, and firing rate of 14-50 Hz. The discharge pattern is typically chaotic, with frequent irregular bursts and pauses. DISCUSSION: The microelectrode recording of the neuroTrek and Lead point-4 showed unique results of the typical STN spike. The DBS effect is maximized associated by MER mapping.

The mouse gap junction gene connexin29 is highly expressed in sciatic nerve and regulated during brain development.

A novel mouse gap junction gene, coding for a presumptive protein of 258 amino acids (molecular mass: 28 981 Da), has been designated connexin29. This single copy gene was mapped to distal mouse chromosome 5 and shows 75% sequence identity to a human connexin30.2 sequence in the database. Connexin29 mRNA (4.4 kb) is highly expressed in mouse sciatic nerve and less abundant in spinal cord as well as in adult brain, where it increased 12-fold between day 7 and 14 post partum. Our expression data suggest that the new connexin gene is active in myelin-forming glial cells.

Functional expression of the new gap junction gene connexin47 transcribed in mouse brain and spinal cord neurons.

A new mouse gap junction gene that codes for a protein of 46,551 Da has been identified and designated connexin47 (Cx47). It mapped as a single-copy gene to mouse chromosome 11. In human HeLa cells and Xenopus oocytes, expression of mouse Cx47 or a fusion protein of Cx47 and enhanced green fluorescent protein induced intercellular channels that displayed strong sensitivity to transjunctional voltage. Tracer injections in Cx47-transfected HeLa cells revealed intercellular diffusion of neurobiotin, Lucifer yellow, and 4',6-diamidino-2-phenylindole. Recordings of single channels yielded a unitary conductance of 55 pS main state and 8 pS substate. Cx47 mRNA expression was high in spinal cord and brain but was not found in retina, liver, heart, and lung. A low level of Cx47 expression was detected in ovaries. In situ hybridizations demonstrated high expression in alpha motor neurons of the spinal cord, pyramidal cells of the cortex and hippocampus, granular and molecular layers of the dentate gyrus, and Purkinje cells of the cerebellum as well as several nuclei of the brainstem. This expression pattern is distinct from, although partially overlapping with, that of the neuronally expressed connexin36 gene. Thus, electrical synapses in adult mammalian brain are likely to consist of different connexin proteins depending on the neuronal subtype.

A single amino acid change in the murine leukemia virus capsid gene responsible for the Fv1(nr) phenotype.

The nr allele at the mouse Fv1 restriction locus governs resistance to B-tropic and some N-tropic murine leukemia viruses (MLVs). Sequence analysis and site-specific mutagenesis of N-tropic MLVs identified a single amino acid difference responsible for this restriction that is distinct from the site that governs N or B tropism. Viruses with other substitutions at this site were evaluated for altered replication patterns.

Rapid subgrouping of nonpolio enterovirus associated with Aseptic Meningitis by RFLP (Restriction Fragment Length Polymorphism) assay.

In Korea, there was a big outbreak of Aseptic Meningitis due to enterovirus infection in 1993. Since virus isolation and neutralizing tests are too laborious and time-consuming for the detection of enterovirus from clinical specimen, we have developed a new molecular identification method for rapid subgrouping of isolates from patients with aseptic meningitis. For the rapid subgrouping of isolates, RT-PCR (Reverse Transcription-Polymerase Chain Reaction) and RFLP (Restriction Fragment Length Polymorphism) assays were used. We have selected two oligonucleotide primers from the conserved 5'-UTR/VP2 and VP1 regions. A 652 bp (base pair) product was amplified from the 5'-UTR/VP2 region of reference viruses and the isolates. For the subgrouping of the isolates by RFLP assay, we have used 12 reference viruses (Echovirus, E6, E9, E11, E12, Coxsackievirus, CB1, CB3, CB4, CB5, Coxsackievirus, CA9, CA16, CA21, CA24), which are the common viral agents associated with aseptic meningitis. By using subgroup-specific restriction enzymes BsmAI, , HinP1I, and PleI, the isolates were classified into Echovirus subgroups. We have also shown that subgrouping of the isolates by RFLP assay based on the VP1 region is possible.

Molecular biological characterization of enterovirus variant isolated from patients with aseptic meningitis.

In Korea, there was a big outbreak of aseptic meningitis in 1993. Six clinical isolates of enterovirus were obtained from patients with aseptic meningitis and were identified as echovirus type 9 by serotyping with a pool of neutralizing antisera. For molecular characterization of the isolates, the nucleotide sequences of 5'-noncoding region (NCR), VP4, VP2, VP1, 2A and 2C regions of the isolates were compared with the corresponding regions of echovirus type 9 Hill and Barty strains. Unlike Hill strain, Barty strain contained a C-terminal extension to the capsid protein VP1 with an RGD (argnine-glycine-aspartic acid) motif. To determine whether similar structural features were present in our isolates, their nucleotide sequences including the VP1 region were analyzed. All isolates exhibited the VP1 extension with the RGD motif. We concluded the Korean isolates in the year of 1993 as the echovirus type 9 Barty strain although the isolates showed 15-20% nucleotide sequence differences in the several genomic regions.

GB virus C/hepatitis G virus infection among Korean patients with liver diseases and general population.

GB virus C and hepatitis G virus (GBV-C/HGV) have been identified from the patients with acute or chronic liver diseases as possible agents of non-B, non-C hepatitis by two different groups, independently. To investigate whether GBV-C/HGV plays a role among Korean patients with liver diseases, GBV-C/HGV RNA were evaluated in 337 sera by the reverse transcription polymerase chain reaction (RT-PCR) using specific primers derived from 5'-noncoding region of GBV-C/HGV genome. GBV-C/HGV RNA was identified in 11/337 (3.3%). They consisted of 1/160 (0.6%) and 10/177 (3.3%) among the general population and patients with liver diseases, respectively (P < 0.01). Nucleotide sequences of all PCR amplicons were determined by the dideoxy chain termination method and analyzed by molecular evolutionary methods. The phylogenetic tree showed all sequences could be divided into three genotypes. These results indicate that: (1) GBV-C/HGV already exist in Korea; (2) GBV-C/HGV may play some role as an etiologic factor among the Korean patients with liver diseases; (3) GBV-C/HGV infection is rare among Korean general population; and (4) there are at least three different types of GBV-C/HGV in Korea.

Nucleotide sequencing of a part of the 5'-noncoding region of echovirus type 9 and rapid virus detection during the acute phase of aseptic meningitis.

A part of the 5'-noncoding region of echovirus type 9 isolates was sequenced, and an attempt was made for rapid virus detection in clinical samples obtained from 22 subjects hospitalized with aseptic meningitis. The sequence identity of 440-bp products amplified from the region by RT-PCR was 87.7% between the standard echovirus type 9(Hill strain) and the isolates. Specific IgM antibodies to Hill strain were positive in 45.5% by immunofluorescent antibody staining of virus-infected cells. A high detection rate of PCR products was observed in cerebrospinal fluids (CSFs; 54.5%) at admission, and in peripheral mononuclear cells (PMCs; 72.7%) at the end of hospitalization. Viral genomes were detectable for 2 days in serum samples, and for 6 days in PMC samples after onset of disease. When specific IgM antibody titers were less than 1:40, the amplification rate of viral genome from serum samples was 50.0%. These results indicate that the combination of specific IgM determination and viral genome amplification from CSFs will be a rapid and reliable method for early diagnosis.

Microsatellite instability-associated mutations associate preferentially with the intestinal type of primary gastric carcinomas in a high-risk population.

Most colon cancers exhibiting microsatellite instability (MI), a mutator phenotype of mismatch repair failure, are associated with mutations of the transforming growth factor-beta receptor type II genes (TGF-beta RII). Of intestinal- and diffuse-type gastric carcinomas, the former have been thought to arise from intestinal metaplasia in which gastric mucosa resembles intestinal mucosa. To evaluate the preferential histological type of MI-associated mutations in the development of gastric carcinoma, mutations of TGF-beta RII, p53, and p16 were analyzed for the two types of primary gastric carcinomas showing MI. Of 50 primary gastric carcinomas, including 33 intestinal types and 17 diffuse types, 15 cases (30%) demonstrated MI at 1 or more of the 11 microsatellite markers tested. The 15 MI cases were classified into two groups, widespread MI and low-level MI, based on the number of markers exhibiting the instability. Eleven were widespread MIs, and the remaining four cases were low-level MIs. Ten of the 11 (91%) widespread MIs were of the intestinal type, and 1 case (9%) was of the diffuse type. Of the 11 widespread MIs, 10 cases (91%) demonstrated frameshift mutations within the polyadenylate tract of the TGF-beta RII. The frameshift mutation was rarely detected at p53 and p16 (1 of 11, 9%). In contrast, the four low-level MI cases had no frameshift mutations within the repeat sequences of TGF-beta RII, p53, and p16, but two of the four cases demonstrated base substitution mutations within p53. Our results suggest that mismatch repair failure can mutate the TGF-beta RII and may provide one of the pathways for the development of the intestinal-type gastric carcinoma in high-risk populations.

Genomic characterization of M and S RNA segments of hantaviruses isolated from bats.

Genomic analysis of three Hantaan-like virus isolates from bats was performed. Cleavage patterns of reverse transcription (RT)-polymerase chain reaction (PCR) products and nucleotide sequences of G2 region of M RNA segment and N protein region of S RNA segment of the isolates were compared to that of Hantaan 76-118 strain. Genomic characteristics of the bat isolates were identical to that of Hantaan virus.