Rescue of a foreign gene by Sendai virus.

A simple protocol for the rescue of a synthetic genome into a paramyxovirus has been developed. First, a synthetic Sendai virus-like RNA, containing the antisense coding region of the chloramphenicol acetyltransferase gene replacing the coding region of the Sendai virus genome, was transcribed from a cDNA. When introduced into cells that are infected with Sendai virus, this RNA construct was transcribed, replicated, and packaged into infectious virions. The addition of infected cell extract to the RNA prior to transfection markedly enhanced levels of chloramphenicol acetyltransferase expression and rescue. However, this enhancement is not due to encapsidation of the RNA into nucleocapsids as the RNA remains nuclease-sensitive. Uninfected cell extract also enhances expression and rescue efficiency, implying involvement of a cellular factor(s) with the synthetic viral-like RNA construct that allows for enhanced polymerase recognition. This system should allow for the dissection of the various cis-acting RNA signals within the paramyxovirus genome.     

Prevention of human rotavirus infection with chicken egg yolk immunoglobulins containing rotavirus antibody in cat

A study was made on the passive protection against rotavirus-induced diarrhea. Chickens were immunized with bovine rotavirus (serotype 1) and the egg yolk immunoglobulins containing a high titer anti-rotavirus neutralizing antibody (CEYI) was obtained. The CEYI was then orally administered to specific-pathogen-free cats, and the cats were infected with human rotavirus. The cats treated with the CEYI remained clinically healthy after challenge, whereas diarrhea occurred in the placebo-fed cats as control. Virus antigens were detected in feces in all the diarrheal cases in the placebo-fed cats but were only sporadically detected in the CEYI-fed cats. However, the cats were only protected against rotavirus infection by the presence in the gut at the time of infection of the antibody. These results suggested that continuous administration of the CEYI is capable of preventing children from diarrhea induced by human rotavirus infection and viral shedding.     

Protection studies in colostrum-deprived piglets of a bovine rotavirus vaccine candidate using human rotavirus strains for challenge

Studies were performed to evaluate the potential use of the bovine RIT 4237 rotavirus strain as a vaccine candidate against infection with human rotaviruses. Initial experiments revealed that colostrum-deprived piglets were susceptible to infection with several human strains, except for those belonging to subgroup 1. Subsequently, different immunization procedures with RIT 4237 were studied in this animal model. It was found that a two-dose administration, either given intramuscularly (twice) or once intramuscularly and once intragastrically, was necessary to induce a significant serum antibody response. Finally, the protective effect of the latter vaccination schedules against subgroup 2 and 3 rotavirus strains of human origin was evaluated by artificial challenge. In both cases, prior administration of live RIT 4237 significantly decreased fecal shedding of the challenge virus when compared with control animals.     

Isolation of a gene encoding a chaperonin-like protein by complementation of yeast amino acid transport mutants with human cDNA.

A human cDNA library in lambda-yes plasmid was used to transform a strain of Saccharomyces cerevisiae with defects in histidine biosynthesis (his4-401) and histidine permease (hip1-614) and with the general amino acid permease (GAP) repressed by excess ammonium. We investigated three plasmids complementing the transport defect on a medium with a low concentration of histidine. Inserts in these plasmids hybridized with human genomic but not yeast genomic DNA, indicating their human origin. mRNA corresponding to the human DNA insert was produced by each yeast transformant. Complementation of the histidine transport defect was confirmed by direct measurement of histidine uptake, which was increased 15- to 65-fold in the transformants as compared with the parental strain. Competitive inhibition studies, measurement of citrulline uptake, and lack of complementation in gap1- strains indicated that the human cDNA genes code for proteins that prevent GAP repression by ammonium. The amino acid sequence encoded by one of the cDNA clones is related to T-complex proteins, which suggests a "chaperonin"-like function. We suggest that the human chaperonin-like protein stabilizes the NPR1 gene product and prevents inactivation of GAP.     

Immune protection of infants against rotavirus gastroenteritis by a serotype 1 reassortant of bovine rotavirus WC3

The safety and protective efficacy of a serotype 1 reassortant of bovine rotavirus WC3, disignated strain WI79-9, was evaluated in a double-blind placebo-controlled trial. Rotavirus reassortant WI79-9 contains a gene segment 9 coding for the surface structural protein vp7 of a human serotype 1 rotavirus, with all other gene segments derived from WC3 rotavirus, which had previously been shown to be safe and immunogenic in infants. Infants 2-11 months of age were given two doses of vaccine (10(7.3) plaque-forming units/dose) or of placebo 28 days apart. Adverse reactions to the vaccine were not detected. The incidence of serum plaque reduction neutralization antibody responses to two doses of vaccine was serotype 6, 97%; serotype 3, 68%; and serotype 1, 22%. Active surveillance during the subsequent rotavirus season revealed 8 cases of rotavirus gastroenteritis in 39 placebo control infants and no cases in 38 WI79-9 vaccine recipients (protection = 100%, P = .003). Six cases of rotavirus gastroenteritis were caused by type 1 and two by type 3 virus. Although vaccination with WI79-9 affected only the incidence of rotavirus gastroenteritis, the vaccinated infants exhibited a significantly reduced incidence of total days of diarrhea, fever, and illness associated with gastroenteritis in general.     

Rotavirus immunoglobulin a responses stimulated by each of 3 doses of a quadrivalent human/bovine reassortant rotavirus vaccine

A quadrivalent precursor to the pentavalent rotavirus vaccine candidate RotaTeq was evaluated in a 3-dose, 439-subject study. To determine immunogenicity, the quantity of rotavirus immunoglobulin A (IgA) in stool specimens obtained, at 1 of 10 study sites, from 37 placebo and 37 vaccine recipients was measured. None of the placebo recipients showed a clinically important (>/=3-fold) increase in stool rotavirus IgA, whereas 31 vaccine recipients showed an increase after at least 1 dose of vaccine. In total, 16, 19, and 15 vaccine recipients had increases after 1, 2, and 3 doses, respectively, indicating that a 3-dose regimen increased the immune response elicited by this vaccine.     

Effect of vaccination on serotype-specific antibody responses in infants administered WC3 bovine rotavirus before or after a natural rotavirus infection

In an evaluation of WC3 bovine rotavirus (serotype 6) vaccine in infants, some subjects experienced a natural serotype 1 rotavirus infection before vaccination and others after. Therefore, the effects of both WC3 and natural rotavirus strains as either primary or boosting immunogens on serotype-specific neutralizing antibody responses could be determined. After primary natural infection (symptomatic or asymptomatic), neutralizing antibody titers were highest to serotype 1 but were consistently high to serotype 3, and low titers (greater than or equal to 20) to serotypes 2 and 4 were often detected. Previous vaccination with WC3 had little effect on the magnitude of these responses. In contrast, subjects infected with serotype 1 strains before vaccination experienced large (average, 12-fold) rises in neutralizing antibody to human serotypes 1-4 when vaccinated with WC3. Thus, although WC3 and the natural strains are distinct serotypes, their epitopes were sufficiently similar that reinfection with WC3 could boost neutralizing antibody titers to human serotypes in subjects primed by a previous natural infection.     

人类轮状病毒感染新生小鼠肠道外组织的病理变化

目的了解人类轮状病毒(RV)全身扩散后对机体的影响,为临床的防治提供有价值参考。方法选用对人类RV敏感的健康新生昆明小鼠,经灌胃与腹腔注射两种途径接种RV,3d后处死小鼠,光镜下观察各脏器病理变化;电镜下观察肝脏的病理变化;各脏器原位杂交,原位PCR检测RV,并与健康小鼠比较。结果光镜下:RV口服组小肠绒毛、胃固有层、心肌细胞有改变;RV腹腔注射组除上述改变外,肝,肾也有改变;电镜下:肝细胞线粒体肿胀、凝集病变尤为明显,细胞核固缩-崩解,粗面内质网扩张,肝细胞中存在大量脂滴和空泡;毛细胆管明显扩张,微绒毛脱落。原位杂交:RV口服组小肠上皮细胞,RV腹腔注射组肠、肾近曲小管上皮细胞呈阳性;原位间接PCR:RV口服组小肠绒毛、肠腺细胞、肾近曲小管和集合管上皮细胞呈阳性,RV腹腔注射组肠、肾、肝、心脏、胰呈阳性。结论RV一旦向全身扩散,可侵犯除肠之外的多个器官组织,提示人类RV也可能具有一定的泛嗜性。     

Comparison of clinical features of rotavirus infection with rotavirus antigen titers in feces obtained during the acute phase

When fecal specimens obtained from infants with acute gastroenteritis during the acute phase between January 1986 and March 1987 were examined by enzyme immunoassay (EIA), 72 cases were found to have the rotavirus antigen. Such 3 clinical signs as diarrhea (D), fever (F) and vomiting (V) were all observed in 38 cases. These cases showing DFV syndrome demonstrated the highest geometric mean titer of rotavirus antigen among the rotavirus antigen-positive cases. Geometric mean titers (GMTs) were calculated to be as high as more than 10(3.1) in 26 of the 38 DFV cases (68%) and in 11 of 34 cases with DV, DF, FV or D syndromes (32%), showing a significant difference between the 2 groups (p less than 0.005). GTMs were slightly higher in cases having fever of 38 degrees C or more, than in those having fever of less than 38 degrees C.     

Protection of adults rechallenged with a human rotavirus

Previous studies of adults challenged with human rotavirus (CJN strain) showed that 74% became infected and 55% of those infected experienced illness. Protection against infection correlated with rotavirus antibody, most significantly (P = .005) serum rotavirus IgG. In this study, 20 previously challenged subjects were reinoculated with the same virus 9-12 months after their initial challenge. Only 1 of 8 subjects not infected after the initial challenge and 2 of 12 infected after the first inoculation became infected after reinoculation; none became ill. Titers of rotavirus antibodies (serum, jejunal, and stool) at the time of reinoculation were about as high as or higher than they were before the initial inoculation. This correlated with greater protection, but the extent of protection was significantly greater (P less than .0001) than predicted based on a previous model relating protection and preinoculation titers of serum rotavirus IgG. Thus, inoculation with human rotavirus provided homotypic protection for at least 9-12 months, and protection remained correlated with higher concentrations of rotavirus antibody. However, the specific relationship between protection and rotavirus antibody was altered after the initial inoculation.     

Enzootic bovine rotavirus is not a source of infection in Panamanian cattle ranchers and their families

Vaccination of humans against rotavirus (RV) diarrhea may be accomplished by oral immunization with attenuated animal strains known to be antigenically very similar to human strains. To define better the degree of infectivity in nature of these animal strains for humans, we conducted surveillance for RV infection/diarrhea in 180 farm workers, their 161 family contacts, and the 566 animals (512 cattle, 35 pigs, and 19 sheep) on 14 farms in rural Panama. No correlation between the high infection rates in farm workers (72%) and their family contacts (78%) and in cattle (56%) could be demonstrated. Heads of families with four or more children with RV infection experienced a twofold greater rate of RV infection compared with heads of families of similar size without RV infection. Despite the close similarity between human and bovine RV, in Panama intrafamilial (particularly child-to-child or child-to-parent) rather than interspecies transmission appeared to be the most important route for the spread of this highly infectious virus.     

Effects of antibody to rotavirus on protection of adults challenged with a human rotavirus

Effects of preinoculation rotavirus antibody titers on the probability of infection and illness were evaluated in adults challenged orally with different doses of a virulent human rotavirus (CJN strain). Preinoculation titers considered were serum neutralizing antibody, serum rotavirus IgA, serum rotavirus IgG, jejunal neutralizing antibody, jejunal rotavirus IgA, and stool rotavirus IgA. Doses of virus of either 9 x 10(1) or 9 x 10(3) focus-forming units were administered to 19 subjects each. Twenty-six were infected; 15 experienced illness. The probability of either outcome was unrelated to dose. Stool rotavirus IgA titers could not be correlated to either infection or illness, but the mean titers of the other five antibodies were significantly or nearly significantly lower in subjects infected or ill, when compared with those negative for either outcome. When analyzed by stepwise logistic regression, only serum rotavirus IgG remained significantly (P = .005) related to the probability of infection, and only jejunal neutralizing antibody remained significantly (P = .01) related to the probability of illness.     

Splicing-defective mutants of the yeast mitochondrial COXI gene can be corrected by transformation with a hybrid maturase gene.

We have developed a recombinant vector, termed pMIT, for transient expression of genes delivered to yeast mitochondria by biolistic transformation. Using that vector, we introduced a hybrid RNA maturase (splicing) gene into mitochondria of rho 0 petite cells and showed the gene to be functional in crosses. The hybrid maturase is an in-frame fusion between the N-terminal half of the maturase encoded by intron 1 of the COXI (cytochrome oxidase) gene and the C-terminal half of a similar maturase encoded by COXI intron 2. pMIT transformants can provide a functional maturase in crosses to restore respiration and COXI polypeptide synthesis to a respiratory-deficient strain defective in the synthesis of a maturase encoded by COXI intron 1; the transformant will also restore respiration to two splicing-defective cis mutants of COXI introns 1 and 3. We detect a 68-kDa polypeptide comparable in abundance to other major mitochondrial translation products as a likely product of the hybrid maturase gene. Transformants containing an internal 218-amino acid deletion mutation of the hybrid maturase gene no longer express a functional maturase in crosses and produce the expected shortened polypeptide of approximately 40 kDa; however, those transformants still restore respiration to the COXI cis mutants. These studies show the utility of the pMIT transformation system for the expression and reverse genetic analysis of yeast mitochondrial genes.     

Adenine nucleotide metabolism during the aggregation of human platelets by bovine factor VIII.

Human platelets, prelabelled with [3H]-adenine in vitro, were aggregated biphasically by bovine fibrinogen/factor VIII, and the amounts and specific radioactivity of ADP and ATP as well as the radioactivity of ATP, ADP, AMP, IMP and hypoxanthine + inosine were determined intracellularly and extracellularly. During the second phase of aggregation, ATP and ADP were released (50% of total) with a specific radioactivity 15-20 times lower than that of the ATP and ADP retained in the cells. The specific radioactivity of the ATP and ADP in the cells increased during release. Radioactive ATP was converted to radioactive hypoxanthine during release. These findings correlated well with those obtained with collagen, and indicate that bovine factor VIII induces the platelet release reaction whereby the storage (non-metabolic) pool of ATP and ADP is released while the metabolic pool is retained.     

Epidemiological aspects of rotavirus infection in hospitalized Venezuelan children with gastroenteritis.

The prevalence of rotavirus infection in hospitalized Venezuelan children with gastroenteritis was studied during the period November 1975 to December 1976. Rotaviruses were the pathogens most frequently associated with gastroenteritis, being found in 121 of 293 (41.3%) patients and in only 3 of 66 (4.5%) controls. Other viruses (adenoviruses, enteroviruses, and small icosahedrical viruses) were detected at a lower frequency both in cases and controls. Rotaviruses were detected at a lower frequency both in cases and controls. Rotaviruses were readily detected throughout the year, which may correspond to the absence of seasonal temperature variation in a tropical country such as Venezuela. Children of all age groups examined (0-5 yr) were susceptible to rotavirus infection. The frequency of infection was slightly higher in the age group 13-24 mo, and significantly lower in children younger than 6 mo old. Rotaviruses were readily detected even after 12 days from the onset of illness. These results indicate that rotaviruses may be a major cause of infantile acute gastroenteritis in Venezuela.     

Rescue of mutants of the tumor suppressor p53 in cancer cells by a designed peptide

We designed a series of nine-residue peptides that bound to a defined site on the tumor suppressor p53 and stabilized it against denaturation. To test whether the peptides could act as chaperones and rescue the tumor-suppressing function of oncogenic mutants of p53 in living cells, we treated human tumor cells with the fluorescein-labeled peptide Fl-CDB3 (fluorescent derivative of CDB3). Before treatment, the mutant p53 in the cell was predominantly denatured. Fl-CDB3 was taken up into the cytoplasm and nucleus and induced a substantial up-regulation of wild-type p53 protein and representative mutants. The mutants, His-273 and His-175 p53, adopted the active conformation, with a dramatic decrease in the fraction of denatured protein. In all cases, there was p53-dependent induction of expression of the p53 target genes mdm2, gadd45, and p21, accompanied by p53-dependent partial restoration of apoptosis. Fl-CDB3 sensitized cancer cells that carried wild-type p53 to p53-dependent gamma-radiation-induced apoptosis. Although Fl-CDB3 did not elicit a full biological response, it did bind to and rescue p53 in cells and so can serve as a lead for the development of novel drugs for anticancer therapy.     

Constitutive production of human interferons by mouse cells with bovine papillomavirus as a vector.

Mouse C127I cells were transformed with a chimeric plasmid consisting of bovine papillomavirus and human interferon (HuIFN) gene (either IFN-gamma or IFN-alpha 5) placed under the control of simian virus 40 early promoter. The transformed cells retained 30-50 copies each of the hybrid plasmid extrachromosomally and secreted a high level of HuIFNs constitutively up to 3-4 X 10(5) international units/ml. The secreted HuIFN-gamma had no detectable activity on mouse cells, whereas HuIFN-alpha 5 was slightly active on mouse cells. Each IFN was neutralized either by anti-HuIFN-gamma or anti-HuIFN-alpha antiserum. The partially purified 35S-labeled HuIFN-gamma produced by the transformed cells showed heterogeneous bands with apparent Mrs of 22,000-25,000 on NaDodSO4/polyacrylamide gel electrophoresis. This value is similar to that of natural IFN-gamma and larger than the molecular weight calculated from the amino acid sequence, which suggests that HuIFN-gamma secreted by transformed mouse cells was glycosylated. The 35S-labeled IFN-alpha 5, immunoprecipitated with anti-HuIFN-alpha antiserum from the supernatant of the transformed cells, had a molecular weight of mature protein. These results suggest that the bovine papillomavirus vector can be used to produce a large quantity of foreign proteins in mammalian cells.