Multiple-dose therapy with bovine colostrum confers significant protection against diarrhea in a mouse model of human rotavirus-induced gastrointestinal disease.

Rotavirus is the most important etiologic agent of severe gastroenteritis. Previously, we reported that skimmed and concentrated bovine late colostrum (SCBLC) obtained from normal unimmunized cows at 6 to 7d after parturition effectively prevented against human rotavirus (HRV)-induced severe gastroenteritis in vivo, when administered as a single dose 60 min before viral inoculation. In the present study, we examined the efficacy of multiple administrations of SCBLC at smaller dosages after viral inoculation in vivo. We demonstrate that multiple administrations within 24h after virus inoculation resulted in earlier recovery from diarrheal symptoms, in an administration frequency-dependent manner. Furthermore, we investigated whether isolated IgG anti-HRV activity in SCBLC was equivalent to that of IgG isolated from bovine mature milk as measured by in vitro activity assays. We found that IgG-containing fractions from SCBLC and mature milk exhibited approximately the same level of anti-HRV activity. We concluded that the SCBLC contains a high level of IgG against HRV-induced severe gastroenteritis, which will be possible to use in protective effects in immunocompromised hosts, such as children and the elderly. Multiple doses of SCBLC during the early stages of infection or lower dosage of SCBLC given as a single dose both resulted in relief of diarrheal symptoms.

Thymoquinone suppresses expression of inducible nitric oxide synthase in rat macrophages.

The objective of the present study was to determine the immunomodulatory role of thymoquinone (TQ) regarding its effect on the production of nitric oxide (NO) by rat peritoneal macrophages. Under certain conditions, macrophagesand certain other cells can produce high concentrations of NO from its precursor L-arginine via inducible nitricoxide synthase (iNOS)pathway. TQ has been established as the major component of the oil extracted from Nigella saliva plant seeds, which is being used frequently in herbal medicine. TQ (IC50 1.4-2.76 microM) dose- and time-dependently reduced nitrite production, a parameter for NO synthesis, in supematants of lipopolysaccharide (LPS)-stimulated (5 microg/ml) macrophages without affecting the cell viability. The protein level of iNOS in peritoneal macrophages was also decreased by TQ in a concentration-dependent manner. In addition, TQ inhibited the increase in iNOS mRNA expression induced by LPS indicated by reverse transcription-polymerase chain reaction (RT-PCR). These inhibitory effects of TQ were confirmed by immunofluorescence staining of iNOS in macrophages which showed decreased immunoreactivity for iNOS after treatment with TQ if compared with the control LPS-stimulated cells. These results suggest that TQ suppresses the production of NO by macrophages; an effect which may be useful in ameliorating the inflammatory and autoimmune conditions.

Avian-to-mammal transmission of an avian rotavirus: analysis of its pathogenicity in a heterologous mouse model.

It has been suggested that group A avian rotaviruses can be transmitted to mammals, but there is no direct evidence that such viruses induce disease in mammals. Suckling mice were orally inoculated with two avian rotaviruses. A pigeon rotavirus, PO-13, was found to induce diarrhea, but a turkey rotavirus, Ty-3, did not. The diarrhea induced by PO-13 was dependent on the age of the mouse. In histopathological examinations, antigens of PO-13 were sporadically detected in absorptive cells in the ileum, and lesions were observed as ballooning degenerations of absorptive cells in a region from the duodenum to the ileum. However, the rotavirus antigen was not detected in the majority of these degenerative cells. These results indicated that PO-13 could infect and induce diarrhea in suckling mice. This is the first evidence of an avian rotavirus being experimentally transmissible to a mammal.

Rescue of rabies virus from cloned cDNA and identification of the pathogenicity-related gene: glycoprotein gene is associated with virulence for adult mice.

In order to identify the viral gene related to the pathogenicity of rabies virus, we tried to establish a reverse genetics system of the attenuated RC-HL strain, which causes nonlethal infection in adult mice after intracerebral inoculation. A full-length genome plasmid encoding the complete antigenomic cDNA of the RC-HL strain and helper plasmids containing cDNAs of the complete open reading frame of the N, P, and L genes, respectively, were constructed. After transfection of these plasmids into BHK-21 cells infected with the T7 RNA polymerase-expressing vaccinia virus, infectious rabies virus with almost the same biological properties as those of the wild-type RC-HL strain was rescued. Using this reverse genetics system of the RC-HL strain, we generated a chimeric virus with the open reading frame of the glycoprotein gene from the parent Nishigahara strain, which kills adult mice after intracerebral inoculation, in the background of the RC-HL genome. Since the chimeric virus killed adult mice following intracerebral inoculation, it became evident that the open reading frame of the glycoprotein gene is related to the pathogenicity of the Nishigahara strain for adult mice.

Complete nucleotide sequence of a group A avian rotavirus genome and a comparison with its counterparts of mammalian rotaviruses.

The nucleotide sequences encoding four structural proteins (VP1-4) and six nonstructural proteins (NSP1-6) of avian rotavirus PO-13 were determined. Based on the results of earlier sequencing studies [Ito et al., 1995, Sequence analysis of cDNA for the VP6 protein of group A avian rota viruses. Arch. Vriol. 140, 605-612; Rohwedder et al., 1997, Chicken rotavirus Ch-1 shows a second type of avian VP6 gene, Virus Genes 15, 65-71; Rohwedder et al., 1997, Bovine rotavirus 993/83 shows a third subtype of avian VP7 protein, Virus Genes 14, 147-151], determination of PO-13 genome sequence has been completed. The PO-13 genome is 18845 nucleotides in length. It is 290 nucleotides longer than the genome of SA11. The amino acid sequence homology between PO-13 and mammalian rotaviruses ranged from 76-77% (VP1) to 16-18% (NSP1). The features of gene and amino acid sequence were compared with those of the corresponding protein of mammalian rotaviruses. Based on results of the phylogenetic analyses of NSP1, we speculate that an ancestral rotavirus could have separated into groups A, B and C rotaviruses at an early evolutionary stage and that group A rotavirus separated into mammalian and avian rotaviruses with host evolution.

A comparison of complete genome sequences of the attenuated RC-HL strain of rabies virus used for production of animal vaccine in Japan, and the parental Nishigahara strain.

In order to establish the molecular basis of the pathogenicity of the attenuated RC-HL strain of rabies virus used for the production of animal vaccine in Japan, the complete genome sequence of this strain was determined and compared with that of the parental Nishigahara strain which is virulent for adult mice. The viral genome of both strains was composed of 11,926 nucleotides. The nucleotide sequences of the two genomes showed a high homology of 98.9%. The homology of the G gene was lower than those of N, P, M and L genes at both nucleotide and deduced amino acid levels, and the percentage of radical amino acid substitutions on the G protein was the highest among the five proteins. These findings raise the possibility that the structure of the G protein is the most variable among the five proteins of the two strains. Furthermore, we found two clusters of amino acid substitutions on the G and L proteins. The relevance of these clusters to the difference in the pathogenicity between the two strains is discussed.

Rabies virus infection prevents the modulation by alpha(2)-adrenoceptors, but not muscarinic receptors, of Ca(2+) channels in NG108-15 cells.

In mouse neuroblastoma x rat glioma hybrid (NG108-15) cells, we examined whether rabies virus infection affects the voltage-dependent Ca(2+) current (I(Ca)) and agonist-induced I(Ca) inhibition. The viral infection had little effect on the current-voltage relationship for peak I(Ca) or on the late I(Ca) that remained at the end of a 200-ms step depolarization. Noradrenaline and carbachol, via alpha(2)-adrenoceptors and muscarinic receptors, respectively, reduced I(Ca) concentration dependently. The maximum effect of noradrenaline was attained at 10 microM with 19.4+/-1.8% inhibition of I(Ca), which was significantly decreased to 9.9+/-1.3% after viral infection. The decrease was not reversed with 100 microM noradrenaline, suggesting that it does not result from a decrease in agonist sensitivity of cells. The maximum effect of carbachol (300 microM; 27.7+/-2.9% inhibition) remained unchanged, despite carbachol sharing intracellular signaling pathways with noradrenaline. These results indicate that in NG108-15 cells, rabies virus infection does not alter the functional expression of voltage-dependent Ca(2+) channels, but it attenuates the alpha(2)-adrenoceptor-mediated I(Ca) inhibition, possibly through some change at the receptor level.

Mapping of epitopes and structural analysis of antigenic sites in the nucleoprotein of rabies virus.

Linear epitopes on the rabies virus nucleoprotein (N) recognized by six MAbs raised against antigenic sites I (MAbs 6-4, 12-2 and 13-27) and IV (MAbs 6-9, 7-12 and 8-1) were investigated. Based on our previous studies on sites I and IV, 24 consecutively overlapping octapeptides and N- and C-terminal-deleted mutant N proteins were prepared. Results showed that all three site I epitopes studied and two site IV epitopes (for MAbs 8-1 and 6-9) mapped to aa 358-367, and that the other site IV epitope of MAb 7-12 mapped to aa 375-383. Tests using chimeric and truncated proteins showed that MAb 8-1 also requires the N-terminal sequence of the N protein to recognize its binding region more efficiently. Immunofluorescence studies demonstrated that all three site I-specific MAbs and one site IV-specific MAb (7-12) stained the N antigen that was diffusely distributed in the whole cytoplasm; the other two site IV-specific MAbs (6-9 and 8-1) detected only the N antigen in the cytoplasmic inclusion bodies (CIB). An antigenic site II-specific MAb (6-17) also detected CIB-associated N antigen alone. Furthermore, the level of diffuse N antigens decreased after treatment of infected cells with cycloheximide. These results suggest that epitopes at site I are expressed on the immature form of the N protein, but epitope structures of site IV MAbs 6-9 and 8-1 are created and/or exposed only after maturation of the N protein.

Molecular epidemiology of rabies in Thailand.

For the purpose of making clear the dynamics of rabies viruses that are prevalent among dogs in Asia, especially Thailand, nucleoprotein (N) genes of isolates derived from Thailand were partially sequenced, and a phylogenetic analysis was performed on the basis of the sequencing data. Firstly, all 27 isolates from Thailand belonged to one group that was distantly related to an isolate from China and was separated into at least six lineages. On the other hand, the isolate from Japan was related to viruses from the Arctic. Secondly, in order to analyze the diversity of the N gene more conveniently, restriction fragment length polymorphism (RFLP) analysis was performed on the N gene of 27 isolates from Thailand. The RFLP analysis could distinguish the lineages of each isolate, and the lineages of additional 34 isolates were deduced by this method. On examination of the geographical distribution of the six lineages, based on the results of phylogenetic and RFLP analyses, it was clear that infection cycles of the rabies virus in Thailand have tended to be maintained endemically.

Modification of membrane currents in mouse neuroblastoma cells following infection with rabies virus.

1. The effect on membrane currents of infection of mouse neuroblastoma NA cells with rabies virus was studied by using the whole-cell patch clamp technique. 2. Three types of membrane currents, namely voltage-dependent Na+ current (I(Na)), delayed rectifier K+ current (I(K-DR)) and inward rectifier K+ current (I(K-IR)) were elicited in uninfected cells. 3. In cells 3 days after infection with the virus, no detectable change was observed in morphology and membrane capacitance, but I(Na) and I(K-IR) were significantly decreased in amplitude without any appreciable difference in the time course of current activation and inactivation. The voltage-dependence of I(Na) activation was significantly shifted in the positive direction along the voltage axis with a decreased slope. I(K-DR) remained almost unaltered after the viral infection. 4. The resting membrane potential, measured with a physiological K+ gradient across the cell membrane, was decreased (depolarized) after the viral infection. The depolarization was associated with the decreased amplitude of I(K-IR). 5. These results suggest that infection of mouse neuroblastoma NA cells with rabies virus causes reduction of functional expression of ion channels responsible for I(Na) and I(K-IR), and provide evidence for possible involvement of the change in membrane properties in the pathogenesis of rabies disease.

Use of protein AG in an enzyme-linked immunosorbent assay for screening for antibodies against parapoxvirus in wild animals in Japan.

Using protein AG in an enzyme-linked immunosorbent assay (ELISA), we tried to detect antibodies against parapoxvirus in 9 species of wild animals in Japan: the Japanese badger (Meles meles anakuma), Japanese black bear (Ursus thibetanus japonicus), Japanese deer (Cervus nippon centralis), Japanese monkey (Macaca fuscata), Japanese raccoon dog (Nyctereutes procyonoides viverrinus), Japanese serow (Capricornis crispus), Japanese wild boar (Sus scrofa leucomystax), masked palm civet (Paguma larvata), and nutria (Myocastor coypus). A total of 272 serum samples were collected over the period from 1984 to 1995 and were tested by the protein AG-ELISA, the agar gel immunodiffusion test, and an indirect immunofluorescence assay. The protein AG-ELISA was effective in a serological survey for parapoxvirus in wild animals, and antibodies were detected only in Japanese serows. A total of 24 of 66 (36.4%) Japanese serows reacted positively, and they were found in almost all prefectures in all years tested. These results suggest that epizootic cycles of parapoxvirus exist widely in Japanese serows and that they could be reservoirs for the virus in the field in Japan. Moreover, it is probable that they might carry the virus to domestic animals such as cattle, sheep, and goats.

A high-Mr glycoprotein fraction from cow's milk potent in inhibiting replication of human rotavirus in vitro.

Rotavirus is the major cause of infectious diarrhea in infants and young children all over the world. We have found that a high-M(r) glycoprotein fraction from cow's milk is potent in inhibiting replication of human rotaviruses in vitro. Since the activity seems to be unique and specific, this fraction may be useful as a novel agent for treatment or prevention of rotavirus diarrhea.

Organochlorine pesticides and polychlorinated biphenyl congeners in wild terrestrial mammals and birds from Chubu region, Japan: interspecies comparison of the residue levels and compositions.

In order to understand the residue levels of organochlorine compounds (OCs) and their accumulation patterns in wildlife inhabiting Chubu region, Japan, the concentrations of polychlorinated biphenyls (PCBs), hexachlorocyclohexane isomers (HCHs), DDT compounds (DDTs) and hexachlorobenzene (HCB) were measured in 8 species of terrestrial mammals and 10 species of birds. In view of feeding habits, the contamination levels of OCs were found to be higher in omnivorous mammals than in herbivorous ones, and in fish-eating ones and raptores than in omnivorous birds. In fox and dog, PCB-180 (2, 2', 3, 4, 4', 5, 5'-heptachlorobiphenyl) was the most dominant PCB congener, while in the other species PCB-153 (2, 2', 4, 4', 5, 5'-hexachlorobiphenyl) was the most persistent. The ratios of lower chlorinated PCB congeners (tri- to tetra-) to total PCBs were larger in fish-eating birds than in the other birds. The results indicate that the compositions of PCB congeners would reflect the differences of feeding habits and xenobiotic metabolizing systems among each species.

Serological surveillance of Lyme borreliosis in wild Japanese serows (Capricornis crispus).

Through serological surveillance of wild animals by enzyme linked immunosorbent assay with protein G (PG-ELISA), we obtained epidemiological data on Lyme borreliosis in Japanese wild animals. In this study, we carried out serological surveillance for Lyme borreliosis in wild Japanese serows (Capricornis crispus). Forty-six of 200 (23%) serum samples were positive, indicating that Lyme borreliosis is prevalent in wild Japanese serows. This positive rate was relatively higher than that of other animals and was similar/identical to that in other important hosts worldwide. Our results suggest that Japanese serows may be one of the important hosts of Lyme borreliosis in the central mountainous and forested areas of Japan.

Antigenic and functional analyses of glycoprotein of rabies virus using monoclonal antibodies.

Thirty-five monoclonal antibodies (MAbs) against glycoprotein (G protein) of the RC-HL strain of the rabies virus have been established. Using these MAbs, two antigenic sites (I and II) were delineated on the G protein of the RC-HL strain in a competitive binding assay. Of these, 34 MAbs recognized the epitopes on site II. Site II was further categorized into 10 subsites according to their patterns in a competitive binding assay. Each site II-specific MAb showed 5 to 23 nonreciprocal competitions. The reactivities of 35 MAbs to rabies and rabies-related viruses in an indirect immunofluorescent antibody test showed that six MAbs in group A binded to rabies and rabies-related viruses and eight MAbs in group E reacted only with rabies viruses, considering that the former represent the genus-specific of Lyssavirus and the latter are rabies virus-specific. From biological assays, 28 of the 35 MAbs showed neutralization activity, 31 showed hemagglutination inhibition (HI) activity, and 18 showed immunolysis (IL) activity. The MAbs recognizing neutralization epitopes fell into at least three groups: those exhibiting both HI and IL activity, those showing only HI activity, and those showing neither HI nor IL activity. All IL epitopes overlap with HA epitopes. Five of the nine MAbs which reacted with the antigen treated by sodium dodecyl sulfate in ELISA were not reduced, or reduced only slightly, in the titer. None of the MAbs reacted with 2-mercaptoethanol-treated antigen. Only one MAb that recognized site I reacted with the denatured G protein in a Western blotting assay, indicating that its epitope is linear. These results suggest that almost all of the epitopes on the G protein of the rabies virus are conformation-dependent and the G protein forms a complicated antigenic structure.

A virus-neutralizing epitope on the glycoprotein of rabies virus that contains Trp251 is a linear epitope.

We have established a hybridoma producing monoclonal antibody (MAb) against a linear epitope of glycoprotein (G protein) of the RC-HL strain of rabies virus. This MAb15-13 showed almost the same neutralizing activity to all of five rabies fixed strains, including RC-HL, and reacted to the denatured G protein in western blot analysis. To characterize and map this linear epitope, an antigenic variant NR15-13 was selected from RC-HL strain in the presence of neutralizing MAb15-13. The variant reacted with MAb15-13 in an immunofluorescent antibody test but was not neutralized by the antibody and the antibody did not bind to the variant G protein in a Western blot analysis. The variant NR15-13 had an amino acid substitution at position 251 of the G protein, where tryptophan of the parental RC-HL strain was replaced by arginine. Site-directed mutagenesis analysis using the expression system in simian COS7 cells revealed that a single amino acid substitution at 251-tryptophan by arginine on the G protein of the parental RC-HL strain abolished the antigenicity of the epitope for MAb15-13 in western blot analysis, and the replacement of 251-arginine by tryptophan recovered the activity. These results strongly suggest that tryptophan at position 251 on the G protein is essential for construction of the linear epitope against MAb15-13.

Sequence analysis of the VP6 gene in group A turkey and chicken rotaviruses.

cDNAs corresponding to the VP6 gene of the turkey rotavirus strains Ty-1 and Ty-3, and the chicken rotavirus strain Ch-1, were cloned and sequenced. The nucleotide and deduced amino acid sequence homology in the coding region of the VP6 gene in avian rotaviruses ranged from 78.1 to 93.9% and 86.1 to 98.7%, respectively. Both sequences of VP6 from avian rotaviruses exhibited a low degree of sequence homology (67.8-70.7% and 69.8-74.6%, respectively) compared with mammalian rotaviruses. Phylogenetic tree analysis showed that all avian rotaviruses were included in a single cluster and have separated early or from mammalian rotaviruses during evolution. The chicken rotavirus strain Ch-1 was a distant relative of other avian rotaviruses.

Identification of a phosphatase-sensitive epitope of rabies virus nucleoprotein which is recognized by a monoclonal antibody 5-2-26.

We have investigated a phosphatase-sensitive sequential epitope of the nucleoprotein (N), one of the phosphoproteins of rabies virus, which is recognized by the monoclonal antibody (MAb) #5-2-26. The epitope was shared in common by all of the rabies virus strains we tested, including the HEP, ERA, CVS and Japanese strains (Nishigahara and Komatsukawa). Thin layer chromatography of the acid hydrolyzates of 32P-labeled N protein showed that the protein contained phosphoserine and phosphothreonine at a molar ratio of about 4 to 1, while no phosphotyrosine was detected. Immunoprecipitation studies with several deletion mutants of the N protein showed that the epitope is located in a region spanning from amino acid 344 to 415. If the phosphatase-sensitive epitope is located at or near the phosphoamino acid, the location of the latter could be narrowed further to a region from amino acid 354 to 389 by comparing the amino-acid sequences among the viral strains. To examine this assumption, point mutation was introduced by amino-acid substitution with alanine at either of five potential phosphorylation sites (i.e., positions 354, 375, 377, 386 and 389) in the 354-389 region. Among those, only one substitution, at position 389, greatly affected the antigenicity. Substitution of serine-389 by threonine also reduced the antigenicity. These results strongly suggest that serine-389 is a phosphorylation site and essential for constructing or stabilizing the antigenic structure for MAb 5-2-26.

Monoclonal antibody #5-2-26 recognizes the phosphatase-sensitive epitope of rabies virus nucleoprotein.

We prepared monoclonal antibodies (MAbs) against the rabies virus N protein, among which one antibody (MAb 5-2-26) was shown to lack reactivity with the phosphatase-treated N protein. The MAb was able to recognize the sodium dodecyl sulfate (SDS)-denatured N protein. The MAb did not recognize the N-protein analogues produced in Escherichia coli (E. coli), indicating that the N-gene products were not normally processed in E. coli after translation. On the other hand, the MAb reacted normally with N-gene products produced in COS-7 cells, but not with those produced in the presence of K-252a (a protein kinase inhibitor of a broad spectrum). The MAb displayed weak cross-reactivity with the Triton-insoluble network structures composed of several components, while another phosphoprotein (M1) of the virus was not recognized at all. These results suggest that MAb 5-2-26 preferentially recognizes a phosphatase-sensitive linear epitope of N protein, which may enable further investigations to be conducted on the mechanism of N-protein phosphorylation and its role(s) in virus replication.

Bovine rotavirus 993/83 shows a third subtype of avian VP7 protein.

VP7 genes of rotavirus (RV) 993/83 isolated from a German calf with diarrhea and of RV PO-13 isolated from a Japanese pigeon were sequenced. Alignment of the deduced VP7 amino acid sequence showed 98.8% sequence identity, while only 70% and 84% identity was seen with VP7 from chicken RV Ch-2 and turkey RV Ty-1, respectively. Over the antigenic regions A, B, and C mammalian RV 993/83 showed more aa identity with mammalian G3 RVs than with chicken RV Ch-2, which could explain the strong one-way cross-neutralization observed between RV 993/83 and G3 RVs. Despite marked VP7 sequence diversity avian RVs could not be differentiated into distinct G types.