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4,600 | Antigenicity of mouse hepatitis virus strain 3 subcomponents in C57 strain mice | C57 strain mice were inoculated intraperitoneally with denatured mouse hepatitis virus strain 3 particles and virus surface projection, membrane and ribonucleoprotein subcomponents, obtained from detergent treated purified virus preparations. All immunised animals developed high levels of serum antibody directed against the respective antigens, detectable by enzyme-linked immunosorbent assay. Mice that had been immunised with denatured virus particles or surface projections were protected against infection with mouse hepatitis virus strain 3, whereas immunisation with virus membrane or ribonucleoprotein subcomponents failed to protect mice against virus challenge. |
4,601 | Typing of recent infectious bronchitis virus isolates causing nephritis in chicken | Four isolates of infectious bronchitis viruses (IBV) from chickens with nephritis, were characterized by polymerase chain reaction (PCR) and restriction enzyme fragment length polymorphism (RFLP), and were found to be genetically different from the other twelve strains which we previously studied. |
4,602 | Ultrastructure and immuno-cytochemistry of BHK-21 cells infected with a modified Bucyrus strain of equine arteritis virus | Morphogenesis of a modified Bucyrus strain of equine arteritis virus (EAV) in BHK-21 cells was studied. Bacillary tubules were first detected in the cytoplasm 8 h after infection, and mature virions 79 to 122 nm in diameter, 101 nm on average, were mostly observed in the cisternae of the rough endoplasmic reticulum (RER) at 12 h or later. They had isometrical cores and morphological subunits in the outer layer. Budding occurred from the RER and the outer nuclear membrane, but not from the cell surface. Structural linkage was detected between the tubule and the virus core. Aberrant strands were occasionally demonstrated within the nucleus 12 h after infection, and immunofluorescence and immunogold labeling revealed viral antigen also in the nucleus. |
4,603 | Mutations in Sendai virus variant F1-R that correlate with plaque formation in the absence of trypsin | With the emergence of new viruses, such as the SARS virus and the avian influenza virus, the importance of investigations on the genetic basis of viral infections becomes clear. Sendai virus causes a localized respiratory tract infection in rodents, while a mutant, F1-R, causes a systemic infection. It has been suggested that two determinants are responsible for the systemic infection caused by F1-R [Okada et al (1998) Arch Virol 143:2343–2352]. The primary determinant of the pantropism is the enhanced proteolytic cleavability of the fusion (F) protein of F1-R, which allows the virus to undergo multiple rounds of replication in many different organs, whereas wild-type virus can only undergo multiple rounds of replication in the lungs. The enhanced cleavability of F1-R F was previously attributed to an amino acid change at F115 that is adjacent to the cleavage site at amino acid 116. Secondly, wild-type virus buds only from the apical domain of bronchial epithelium, releasing virus into the lumen of the respiratory tract, whereas F1-R buds from both apical and basolateral domains. Thus, virus is released into the basement membrane where it can easily gain access to the bloodstream for dissemination. The microtubule disruption is attributed to two amino acid differences in M protein. To confirm that the F and M gene mutations described above are solely responsible for the phenotypic differences seen in wld-type versus F1-R infections, reverse genetics was used to construct recombinant Sendai viruses with various combinations of the mutations found in the M and F genes of F1-R. Plaque assays were performed with or without trypsin addition. A recombinant virus containing all F1-R M and F mutations formed plaques in LLC-MK2 cells and underwent multiple cycles of replication without trypsin addition. To clarify which mutation(s) are necessary for plaque formation, plaque assays were done using other recombinant viruses. A virus with only the F115 change, which was previously thought to be the only change important for plaque formation of F 1-R F, did not confer upon the virus the ability to form plaques without the addition of trypsin. Another virus with the F115 and both M changes gave the same result. Therefore, more than one mutation in the F gene contributes to the ability of F1-R to form plaques without trypsin addition. |
4,604 | Characterizing the Pregnancy Immune Phenotype: Results of the Viral Immunity and Pregnancy (VIP) Study | PURPOSE: The increased risk of morbidity and mortality from certain microbial infections and the demonstrated improvements in the clinical course of some autoimmune diseases support the existence of pregnancy-related alterations in immune status. Elucidating the changes in innate and adaptive immunity during gestation may improve pregnancy outcomes and facilitate the development of targeted therapies for autoimmune diseases. METHOD: The Viral Immunity and Pregnancy (VIP) study evaluated over 50 subjects longitudinally at three time points during pregnancy and at two time points post-delivery. Leukocyte enumeration was performed; functional responses of NK cells and CD4 T cells were analyzed, and soluble factors such as cytokines, defensins, and steroid hormones were measured in maternal blood. RESULTS: In comparison to the post-partum period, the latter part of pregnancy was characterized by significant increases in blood phagocytes and pDCs and decreases in the number and activity of NK and T cells. Alterations were found in antimicrobial proteins and serum cytokines. CONCLUSIONS: These data show that pregnancy is not a period of immunosuppression but an alteration in immune priorities characterized by a strengthening of innate immune barriers and a concomitant reduction in adaptive/inflammatory immunity in the later stages of pregnancy. |
4,605 | Attenuated Respiratory Syncytial Virus Vaccines in Asthmatic Children | Extract: Two live “attenuated” respiratory syncytial virus (RSV) vaccines were administered intranasally and by aerosol in placebo-controlled trials among asthmatic children hospitalized at National Jewish Hospital and Research Center (NJH). The first vaccine, a 26° -adapted RSV vaccine, was given to 28 children, and a placebo control was given to 25. Twenty-one of 28 vaccinees (75%) had “takes,” as judged by virus shedding and/or rises in serum antibody and/or rises in nasal secretion neutralizing activity. No excess in wheezing was attributable to the vaccine administration, although there was a high background level of acute respiratory symptoms in both vaccinees and control subjects. In an outbreak of natural RSV infection which occurred some months after the vaccine was given, there was some evidence that those who received the vaccine less than 4 months before exposure to wild virus were protected against reinfection. Protection was not evident when this interval was greater than 4 months. The second vaccine, a temperature-sensitive mutant strain (ts-1), was administered to 22 children, and placebo to 21. Thirteen children (59%) had “takes.” In vaccinees under 6 years of age, 7 of 7 had “takes.” Again, no excess wheezing was seen in vaccinees as compared with control subjects, although there was some evidence that upper respiratory symptoms were more frequent in younger vaccinees. Four of 10 vaccinees shed virus with temperature-sensitive characteristics somewhat different from those of the vaccine strain. Vaccine virus was demonstrated to spread to uninoculated or placebo control children. Natural RSV challenge did not occur during the period of study. Speculation: One of two live “attenuated” RSV vaccines may have produced a brief period of protection against natural infection. This finding offers hope that such live vaccines might prevent disease in selected children over a critical time period, such as infants for the 1st year of life, or allergic or asthmatic children during periods of epidemic prevalence. Asthmatic children did not appear to develop symptoms of wheezing after attenuated RSV infection. This finding suggests that the mechanism of wheezing in asthmatic children with RSV infection may be dependent on, among other factors, the virulence of the virus strain (perhaps its capacity to replicate in and, possibly, invade the lower respiratory tract), rather than on an allergic response to antigens introduced into, and limited to, the upper airway. In view of the spread of the ts-1 vaccine and its apparent loss of temperature sensitivity in some vaccinees, the vaccine may have had the potential for reversion to virulence and hence initiation of epidemic disease. These characteristics are undesirable in live respiratory virus vaccines and should, if possible, be avoided in the development of future such vaccines. |
4,606 | A genetic analysis of macrophage activation and specific antibodies in relation to the resistance of heterogeneous mouse populations to MHV3 infection | The genetically selected high antibody responder mice (H(III)) are susceptible and the low antibody responder mice (L(III)) are resistant to the experimental infection with Mouse Hepatitis Virus 3 (MHV3). The mortality rates of the F(1) hybrids and of the F(2) segregants showed the codominance of the susceptible and resistant characters. The direct individual intrapopulation correlation between the induction of antiviral state in macrophages activated by IFN gamma and the resistance to the virus infection, showed that an antiviral state could be induced in resistant mouse macrophages, whereas in susceptible mouse macrophages no restriction of virus replication could be observed. A direct inter- and intrapopulation correlation of pre-existing antibody titres against MHV3 with the mortality and a direct interpopulation correlation of those titres with the mean survival time of susceptible animals was shown. The data indicate, among the mechanisms of resistance against the virus infection, a role of IFN gamma macrophage-activation and of antibodies against MHV3 which may delay the mean survival time in susceptible animals. |
4,607 | Method for the preparation of a herpesvirus hominis fluorescent conjugate for direct immunofluorescence | A detailed method for the preparation, standardization and interpretation of a direct immunofluorescentherpesvirus hominis conjugate is presented. |
4,608 | Mouse hepatitis virus and host determinants of vertical transmission and maternally-derived passive immunity in mice | Transmission of mouse hepatitis virus (MHV) in utero following oronasal inoculation of pregnant mice was found to depend upon MHV strain and host genotype. Virulent, polytropic MHV-JHM was recovered from multiple maternal tissues, including liver and uterus, as well as placenta and fetus in susceptible BALB/cByJ mice. Fetuses were infected during all 3 trimesters of pregnancy. Low virulence, polytropic MHV-S infected fetuses in a low percentage of susceptible BALB/cByJ dams. Infection of resistant CD-1 mice with MHV-JHM was limited, with no fetal infection. Enterotropic MHV-Y was largely restricted to intestine of BALB/cByJ and CD-1 dams, with minimal dissemination and no fetal infection. Maternally-derived MHV IgG antibody was detectable in pup sera through 4 weeks of age. Antibody titers were generally lower in second litters of the same dam. Cross-fostering experiments showed that antibody was transferred via colostrum and not in utero, and that pups were capable of absorption through 2 weeks of age. Pups nursing immune dams were protected against MHV challenge at 1 and 2 weeks of age, compared to pups nursing naive dams. Immunity to MHV challenge was cross-protective against both antigenically homotypic and heterotypic strains of MHV. |
4,609 | Molecular pathogenesis of systemic lupus erythematosus | The study of patients with systemic lupus erythematosus revealed the close association of the disease with measles—or a related virus. High titres of antibodies to measles virus were found in patients that correlated with the course of the disease. Immunofluorescence tests revealed measles virus or a related antigen in lupus-affected tissues. Inclusion bodies consisting of paramyxovirus-like ribonucleoprotein structures were regularly detected in both affected tissues and leukocytes. Molecular hybridization of measles virus RNA with DNA from the affected tissues showed that DNA transcripts of measles or a closely related virus are integrated in the cellular nuclear DNA. Possible pathogenetic mechanisms of the disease are discussed. |
4,610 | Epitope mapping and the detection of transmissible gastroenteritis viral proteins in cell culture using biotinylated monoclonal antibodies in a fixed-cell ELISA | A fixed-cell ELISA was developed using swine testicle (ST) cells infected with the virulent Miller strain of transmissible gastroenteritis virus (TGEV) and purified biotinylated monoclonal antibodies (b-MAbs). Five of the b-MAbs were specific for the peplomer (E2), five reacted to the nucleocapsid (N), and one reacted to the E1 protein of the Miller strain of TGEV. Protein A-Sepharose purification of MAbs yielded protein concentrations ranging from 0.40 to 3 mg per ml of ascites. Separate pools of N-MAbs and E2-MAbs, and the E1-MAb were used to monitor synthesis of TGE viral antigen in ST cells from 0 to 16 h post-infection at various multiplicities of infection (MOI). Epitopes of N proteins appeared sooner and at a lower MOI than those for the E1 and E2 proteins. The fixed-cell ELISA was also used to examine relative binding affinities of TGEV MAbs. Concentrations of b-MAbs producing a half-maximal signal ranged from 0.11 to 3.8 µg/ml for E2-MAbs, from 0.05 to 0.82 µg/ml for N-MAbs, and 6 µg/ml for the E1-MAb. The assay was used to determine the 50% neutralization concentrations for four neutralizing E2-MAbs (0.1 µg/ml to 6.9 µg/ml) and one E1-MAb (1.2 µg/ml). Competition assays between b-MAbs and unlabeled competitors indicated that at least two major antigenic sites exist on the E2-protein and 2 to 3 antigenic sites are present on the N-protein of Miller TGEV. |
4,611 | Viremic dissemination of mouse hepatitis virus-JHM following intranasal inoculation of mice | Using a sensitive infant mouse bioassay to detect infectious virus, the pattern of mouse hepatitis virus (MHV) JHM dissemination in blood and other tissues was examined during the first 5 days following intranasal inoculation. MHV replicated in nasal turbinates of both susceptible BALB and resistant SJL mice from days 1 through 5, but BALB mice had higher titers on days 1 and 2. Viremia was detectable on days 1 through 5 in BALB mice, but only on days 3 and 5 in SJL mice. Transient virus replication occurred in the lungs of both mouse genotypes at 1 and 2 days, then ceased. This correlated with more consistently demonstrable virus in blood collected from the left atrium of the heart, compared to jugular vein, portal vein and right atrial blood. Virus was associated equally with the plasma and cellular fractions of blood on day 3, but was primarily in the buffy coat of the cellular fraction on day 5. Interferon-α/β was detected in serum and spleen, but not liver or brain of BALB mice or in any tissue of SJL mice. BALB serum and spleen interferon was first detected at 36h, peaked between 48 and 72h, and was undetectable by 108h. The distribution of virus in nose, cervical, axillary and mesenteric lymph nodes, spleen, Peyer's patch, thymus, bone marrow and liver was examined at 1, 2, and 3 days. The resulting pattern suggested lymphatic spread of virus to cervical lymph node and mesenteric lymph node as pathways of dissemination in addition to viremia. |
4,612 | Crossover regions in foot-and-mouth disease virus (FMDV) recombinants correspond to regions of high local secondary structure | The RNA genome of foot-and-mouth disease virus (FMDV) was analysed for the degree of inverted complementarity and thus potential secondary structure using the procedure of Pustell and Kafatos [Nucleic Acids Res (1982) 10: 4765–4782]. Regions of crossover in 42 FMDV recombinants [King et al. (1985) Virus Res 3: 373–384; Saunders et al. (1985) J Virol 56: 921–929] and regions lacking crossovers were assigned an average secondary structure score against which the number of observed recombinants was plotted. In general it was found that the mean value of potential secondary structure is significantly higher in crossover zones than in recombination-free zones. Recombination increased much more steeply with increasing secondary structure in the part of the genome coding for non-structural proteins than in the 5′ third of the genome coding for structural proteins. |
4,613 | Comparison of the structural protein coding sequences of the VR-2332 and Lelystad virus strains of the PRRS virus | The 3′-portion of the genome of a U.S. isolate of the porcine reproductive and respiratory syndrome (PRRS) virus, ATCC VR-2332, was cloned and sequenced. The resultant 3358 nucleotides contain 6 open reading frames (ORFs) with homologies to ORFs 2 through 7 of the European strain of the PRRS virus and other members of the free-standing genus of arteriviruses. Both VR-2332 and the European isolate (called the Lelystad virus) have been identified as infectious agents responsible for the swine disease called PRRS. Comparative sequence analysis indicates that there are degrees of amino acid identity to the Lelystad virus open reading frames ranging from 55% in ORF 5 to 79% in ORF 6. Hydropathy profiles indicate that the ORFs of VR-2332 and Lelystad virus correspond structurally despite significant sequence differences. These results are consistent with the biological similarities but distinct serological properties of North American and European isolates of the virus. |
4,614 | Morphogenesis of avian infectious bronchitis virus in primary chick kidney cells | Primary chick kidney cells were infected with avian infectious bronchitis virus (IBV) and examined by electron microscopy. Virus particles entered the cells by viropexis and distinction could be made between engulfment by cell processes (phagocytosis) and entry by micropinocytosis in coated transport vesicles. Virus maturation occurred by budding into either the cisternae of the endoplasmic reticulum or cytoplasmic vacuoles, and evidence was obtained to suggest that the viral surface projections could be attached during the budding process. Late in infection large numbers of virus particles were present, mainly in cytoplasmic vacuoles, and the majority were released by cell lysis. Release by fusion of vacuoles with the plasma membrane was also observed, and individual virions could be transported from the endoplasmic reticulum to the surface within coated vesicles. |
4,615 | Detection of human rhinovirus C in children with acute lower respiratory tract infections in South Korea | Recently, HRV-C was identified as a new species of HRV, but its spectrum of clinical disease is still not clear. The purpose of this study was to investigate the molecular epidemiology of HRVs in children with acute lower respiratory tract infections (LRTIs). A total of 54 HRV-positive samples that were negative for other respiratory viruses were sequenced. HRV-A was detected in 33, HRV-B in 4, and HRV-C in 17 of these samples. All HRV-C-positive patients showed favorable clinical outcomes. We confirmed the presence of HRV-C in children with LRTIs, but its association with clinical severity is not clear. |
4,616 | Oligodendroglial pathology in canine distemper | Canine distemper virus (CDV) causes a multifocal demyelinating disease in dogs. The mechanism of acute demyelination in distemper is still poorly understood. The initial demyelinating lesion in distemper is directly virus induced, since there is a clear correlation between the occurrence of demyelination and CDV replication in the cells of the white matter. Yet, there is little evidence for oligodendroglial infection. Changes of these cells have been reported in vitro and in vivo. The in vitro studies showed that – in contrast to other cells such as astrocytes and macrophages – oligodendrocytes hardly express CDV protein. However, we could show that these cells underwent a restricted infection with transcription of CDV RNA and that this phenomenon correlated with down-regulation of myelin gene transcription. The extension of these in vitro findings in vivo was obscured by the lack of reliable oligodendrocyte labelling techniques in canine brain tissue sections. In this study we combined immunohistochemistry with in situ hybridization to examine oligodendrocytes in demyelinating lesions and to investigate the question of oligodendrocyte infection in vivo. We could demonstrate that CDV infection leads to massive down-regulation of myelin gene expression in demyelinating lesions and that this effect correlates in part with a restricted infection of oligodendrocytes. |
4,617 | Pseudoviral hollow-cored vesicles in multiple sclerosis brain | Novel, superficially ‘virus-like’ hollow-cored particles 50–60 nm in diameter were found in the perivascular extracellular space of the brain from a patient who died with acute multiple sclerosis (MS). It is concluded that they are not virions but are derived from myelin undergoing vesicular demyelination. This case demonstrates the need for caution in the interpretation of unusual electron microscopic appearances. |
4,618 | Protection from mouse hepatitis virus type 3-induced acute disease by an anti-nucleoprotein monoclonal antibody | Fusion of MHV-3-immune splenocytes from MHV-3-resistant A/J murine strain, with NS myeloma cells produced several hybridomas. Among eight hybridoma clones, the 1E7A4H1 clone secreted kappa IgG2a apparently directed against the nucleoprotein of the MHV-3 virion. The monoclonal antibody was able to neutralize the in vitro cytopathic effect of MHV-3 on cultured L2 cells, and was detected by indirect immuno-fluorescence on MHV-3-infected cultured YAC cells. In addition, it conferred a significant protection against MHV-3-induced acute disease, if injected intraperitoneally to C57BL/6 mice before inoculation with MHV-3. |
4,619 | Bovine respiratory syncytial virus infection of bovine embryonic lung cultures: Enhancement of infectivity with diethylaminoethyl-dextran and virus-infected cells | The effects of incorporating diethylaminoethyl-dextran (DEAE-D) in the inoculum with bovine respiratory syncytial virus (BRSV) on the infectivity of BRSV was evaluated. A concentration of 40 µg DEAE-D/ml provided maximal enhancement of infection as determined by the time of onset of cytopathic effect (CPE), the percentage of cells infected by the inoculum, and the amount of virus produced. When DEAE-D was used in the inoculum, the CPE appeared a day earlier, the percentage of cells infected by the inoculum, as determined by the fluorescent antibody test, was increased 11 times, and the viral titer was increased 2 times as compared to results obtained without DEAE-D. Bovine respiratory syncytial virus-infected cultures contained much cell-associated virus which could be liberated by sonication to increase the titer of virus stocks. The use of BRSV-infected cells rather than supernates from BRSV-infected cells increased the rate at which a cytopathic effect developed, although it did not substantially increase the titer of virus which was harvested. The use of DEAE-D in the inoculum and the passage of BRSV-infected cells instead of viral suspensions was found to be the quickest and most effective method of consistently obtaining BRSV with a titer of about 10(5.5) TCID(50)/ml. |
4,620 | A novel, cell-specific attenuation of a herpes simplex virus type 1 infection in vivo | We have observed a cell-specific attenuation of herpes simplex virus type 1 strain 17syn+ in vivo that was dependent upon the cell type used to grow the virus. Direct corneal infection of rabbits with 17syn+ propagated in Vero cells caused 60% (6 of 10) to develop severe central nervous system (CNS) disease as evidenced by seizures and/or paralysis; all neurologically impaired rabbits died. In contrast, infection of rabbits with 17syn+ propagated in BHK-21 cells induced seizures and was fatal in 10% (1 of 10). The cell-specific attenuation of a 17syn+ occurred after one growth cycle in BHK-21 cells. To determine whether the decreased virulence of the BHK-21 cell-grown virus correlated with a less severe CNS inflammatory reaction, CNS tissues from rabbits infected with 17syn+ grown in Vero and BHK-21 cells were compared. Histopathological analyses revealed no differences in the location or severity of inflammatory lesions from rabbits infected with virus grown in either cell type. Virus-induced corneal disease was less dependent upon the cell type used to propagate the virus as there were no significant differences in the type or severity of observed corneal lesions. Possible explanations based on differences between Vero and BHK-21 cells are discussed. |
4,621 | Immunoreactivity of the central nervous system in cats with a Borna disease-like meningoencephalomyelitis (staggering disease) | The inflammatory cell composition and the expression of major histocompatibility complex (MHC) antigens in the central nervous system (CNS) of 13 cats with a spontaneous, Borna disease-like meningoencephalomyelitis (staggering disease) was investigated by immunohistochemistry with a panel of monoclonal and polyclonal antibodies. T lymphocytes were the predominating inflammatory cells within the adventitial space. CD4(+) T cells were more abundant than CD8(+) T cells. Scattered IgG-, IgA- and IgM-containing cells were found in the adventitial space and surrounding neuropil, often adjacent to neurons. There was a markedly increased MHC class II expression in cells morphologically resembling microglia. In several cats, Borna disease virus specific antigen was detected, but only in a few cells, mainly of macrophage character. Our findings indicate a long-standing inflammatory reaction in the CNS of cats with staggering disease, possibly triggered and sustained by a persistent viral infection. |
4,622 | Effects of insertion of multiple AP-1 binding sites into the U3 region of the long terminal repeat of feline immunodeficiency virus | An oligonucleotide containing multiple AP-1 binding sites was introduced into the regulatory sequence in the long terminal repeat (LTR) of feline immunodeficiency virus (FIV). Chloramphenicol acetyltransferase assay revealed that basal promoter activity of the mutated LTR was higher than that of the wild-type LTR in Crandell feline kidney (CRFK) cells. The mutated LTR was introduced into an infectious molecular clone of FIV and the clone was transfected into CRFK cells. The virus production of the mutant in the cells was as high as that of the wild-type when determined by the reverse transcriptase activity assay. The growth of the mutant virus obtained from the transfected CRFK cells was examined in feline T lymphoblastoid cell lines (MYA-1 and FeL-039 cells) and primary feline peripheral blood mononuclear cells (fPBMCs). The growth was delayed when compared with that of the wild-type virus in all the cells used. Upon examination by polymerase chain reaction, the length of the LTR of the mutant virus was shortened in both MYA-1 cells and fPBMCs. Sequence analysis revealed that the insertion was completely deleted 39 days after infection in the MYA-1 cells. |
4,623 | The polypeptide composition of avian infectious bronchitis virus | Avian infectious bronchitis virus grownin ovo was purified by differential centrifugation and isopycnic sedimentation in density gradients. The purified virus was analysed by SDS polyacrylamide gel electrophoresis and found to comprise up to sixteen polypeptides, four of which were glycopeptides. Bromelain treatment of the particles removed three polypeptides and two glycopeptides. |
4,624 | The influence of pH on the growth and stability of transmissible gastroenteritis virusin vitro | The influence of pH on the growth of transmissible gastroenteritis virus (TGEV) in adult pig thyroid cell culture, and on the stability of the virus was studied. At pH 6.5 the yield of virus was 10 fold higher than cultures held at pH 7.2 and 100 fold higher than those at pH 8.0. The adsorption, penetration and uncoating steps of the viral replicative cycle were shown to be unaffected by pH variation. Synthesis of TGEV RNA during the first 12 hours post infection was found to be unaffected by pH variation between the range 6.5–8.0. After 12 hours breakdown of this RNA appeared to occur in cultures held at pH 7.2 and 8.0 but not at pH 6.5. When incubated at 37° C for 24 hours the virus infectivity was found to be least affected by pH 6.5 but when kept at 4° C for the same length of time, the virus infectivity remained constant between pH 5.0 and pH 8.0. |
4,625 | Avian infectious bronchitis virus structural polypeptides: Effect of different conditions of disruption and comparison of different strains and isolates | Variations in the conditions used for disruption of purified virus involving differences in heat treatment and reducing agent concentration produced little affect on the polypeptide profiles of the Massachusetts 41 (M41) strain of avian infectious bronchitis virus obtained by polyacrylamide gel electrophoresis. Comparisons of the structural polypeptides of 12 IBV isolates, consisting of M41, six serologically related viruses and representatives of five other serotypes, showed that the viruses could be placed in three groups on the molecular weights of the major glycopolypeptides. These were 31,000 and 86,000 for M41, the six related viruses and the serologically distinct SE17; 27,000 and 89,000 for Iowa 97 and Holte and 27,000 and 91,000 for Connecticut and T strain. |
4,626 | Early infantile pertussis; increasingly prevalent and potentially fatal | We report nine cases of severe early pertussis in infants less than 7 weeks of age. Clinical features at this age are atypical and may be confused with more common illnesses such as bronchiolitis. All were very difficult to manage. Ventilation was required for apnoeas in five cases, seizures in two or respiratory failure in two. Complications included hypotension in seven cases, pulmonary hypertension in one, pneumothoraces in two, seizures in five and co-infection in five. Two cases were referred for extracorporeal membrane oxygenation and six died. Infection was confirmed either at post mortem or by culture from pernasal swabs. The mother or other close family members were symptomatic at the time and thought to be the source of infection. Conclusion The nine cases suggest a significant resurgence of the infection, which may be fatal in early life. If reporting continues to increase, the immunisation schedule will need to be reviewed and secondary transmission prevented where possible, to protect this vulnerable pre-immunisation group. |
4,627 | The possible value of certain cells for the propagation of respiratory viruses | A strain of HeLa cells, L 132 cells and roller tube cultures of human embryo respiratory epithelium were compared with standard laboratory systems for the propagation of certain rhinoviruses, parainfluenza viruses and some others. |
4,628 | Mouse hepatitis virus strain 3 infection of C57, A/Sn and A/J strain mice and their macrophages | Mouse hepatitis virus strain 3 replicated in C57, A/Sn and A/J strain mouse macrophages with the production of a clear cytopathic effect, although only C57 and A/Sn strains of mice were killed with similar MHV3 dilutions. We could not confirm a previous report showing thatin vitro cultured macrophages from A/J strain mice were resistant to MHV3 infection. |
4,629 | Antigenic variation in strains of avian infectious bronchitis virus | Fifteen british field strains of IBV were compared using cross serum neutralization tests in embryonated eggs with seven standard reference strains of IBV. While the British field strains were considered to form a relatively homogeneous group considerable antigenic variation did occur. It was considered that it was not feasible at this time to describe accurately a serotype classification for IBV, similar to that described for other virus groups. |
4,630 | Investigations on the RNA binding and phosphorylation of groundnut bud necrosis virus nucleocapsid protein | Groundnut bud necrosis virus belongs to the genus Tospovirus, infects a wide range of crop plants and causes severe losses. To understand the role of the nucleocapsid protein in the viral life cycle, the protein was overexpressed in E. coli and purified by Ni-NTA chromatography. The purified N protein was well folded and was predominantly alpha-helical. Deletion analysis revealed that the C-terminal unfolded region of the N protein was involved in RNA binding. Furthermore, the N protein could be phosphorylated in vitro by Nicotiana benthamiana plant sap and by purified recombinant kinases such as protein kinase CK2 and calcium-dependent protein kinase. This is the first report of phoshphorylation of a nucleocapsid protein in the family Bunyaviridae. The possible implications of the present findings for the viral life cycle are discussed. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00705-011-1110-0) contains supplementary material, which is available to authorized users. |
4,631 | Pathogenetic observations on pleural effusion disease in rabbits | A pathogenetic study of pleural effusion disease (PED) in rabbits was made, using the virulent PED agent or virus (PEDV) and an avirulent derivate of this isolate. Independent of infective dose within the range examined, the virulent isolate caused fatal clinical disease, whereas the avirulent isolate caused subclinical infection. The two isolates differed in rapidity of initial spread of infection and in the maximum virus titres in serum, but they both resulted in a similar low level persisting viraemia. Circulating virulent virus gradually became avirulent during the viraemia. Avirulent infection induced protective immunity to virulent challenge during the first week after primary infection, but full clinical protection was not established until after the fourth week. The findings, corrobated with other closely comparable observations, suggest that the emergence of PED as an intercurrent mortality problem during rabbit passage of pathogenicTreponema pallidum is the result of a specific selective pressure on a benign passenger virus. The expression of virulence of PEDV appears to be dependent on length of interval between passages. |
4,632 | Borna disease virus-infected astrocytes function in vitro as antigen-presenting and target cells for virus-specific CD 4-bearing lymphocytes | Astrocytes isolated from the brain of newborn Lewis rats and an astrocytic cell line were susceptible to infection with the neurotropic Borna disease virus in vitro. Since astrocytes also have been found to be infected in vivo it seemed appropriate to test this cell type for interaction with a Borna disease virus-specific CD4(+) T cell line. Borna disease virus-infected astrocytes were found to be capable of presenting virus-specific antigen to virus-specific T cells in vitro. However, the response was significantly enhanced if the purified 38/39 kDa Borna disease virus-specific protein was added exogenously to the cultures. Beside the function as antigen-presenting cells for various antigens including virus-specific protein and myelin basic protein, persistently infected astrocytes were also found to act as target cells for a CD4(+) T cell line as shown in conventional(51)Cr release assays after induction of MHC class II expression by gamma interferon. Infection of astrocytes alone did not cause expression of this self antigen. It could be shown that the ability of CD4(+) BDV-specific T cells to mediate lysis was in part dependent on the stage of activation. Lymphocytes “activated” before testing exerted high lysis after only 4h of coincubation with target cells, whereas “resting” T cells did not cause significant lysis until 12h of coincubation. The dependence of the interaction between effector and target cells on MHC class II antigen was demonstrated by the finding that antibodies to Ia antigens reduced lysis of target cells. |
4,633 | Applying the species concept to plant viruses | Plant virologists who maintain that the concept of species cannot be applied to viruses argue their case in terms of an obsolete concept of biological species defined by gene pools and reproductive isolation and applicable only to sexually reproducing organisms. In fact, various species concepts have been used by biologists and some of them are applicable to asexual organisms. The rationale for applying the species concept in virology is that viruses are biological entities and not chemicals: they possess genes, replicate, specialize, evolve and occupy specific ecological niches. The following definition is proposed: a virus species is a polythetic class of viruses constituting a replicating lineage and occupying a particular ecological niche. Such a definition of the species category does not and cannot provide a list of diagnostic properties for recognizing members of a particular virus species. It should also be stressed that a single property such as an arbitrary level of genome homology or the extent of serological relationship always fails to establish membership in a polythetic class. A binomial system of nomenclature is advocated in which the vernacular English name of the plant virus is adopted as the species name and the group name is assimilated to the level of genus. Adoption of this system would ensure that a universal classification system based on the classical categories of species, genus, and family becomes possible for all viruses. |
4,634 | Glial proteins in canine distemper virus-induced demyelination: A sequential immunocytochemical study | A temporal series of demyelinating lesions in experimental canine distemper virus (CDV) infection was examined with immunohistological techniques demonstrating myelin basic protein (MBP), myelin-associated glycoprotein (MAG), and glial fibrillary acidic protein (GFAP) on serial sections. The earliest lesions were characterized by decreased MBP and MAG and increased GFAP. During the further progression of the disease, MBP and MAG losses continued to match each other. There was no indication of MAG loss preceding the disappearance of MBP. In the more advanced lesions there was a marked decrease of GFAP positive cells. Since these findings differed considerably from similar immunohistochemical studies in progressive multifocal leukoencephalopathy (PML) where demyelination results from oligodendroglial infection, it was concluded that the oligodendroglial cell body is not the primary target of CDV. The marked astroglial changes were also considered to contribute to demyelination in CDV infection but the mechanism by which this happens remains unknown. |
4,635 | Detection and genetic characterization of a novel pig astrovirus: relationship to other astroviruses | Emerging viruses represent a continuous threat to human health and to farmed animals, as evidenced on multiple occasions by outbreaks of influenza, henipavirus and SARS. Knowledge about the diversity of viromes present in reservoir species can lead to a better understanding of the origin of emerging pathogens. In this study, we extend the knowledge of astrovirus diversity in pigs by reporting the genetic characterization of an unknown astrovirus lineage. Phylogenetic analyses provided evidence that this porcine astrovirus lineage is unique and does not appear to share a recent common ancestor with any known mamastrovirus. The data reported in this study extend the number of porcine astrovirus lineages to a total of five, all of which most likely represent distinct species of different origins. |
4,636 | The 5′-terminal 32 basepairs conserved between genome segments A and B contain a major promoter element of infectious bursal disease virus | The regions of the infectious bursal disease virus (IBDV) genome with regulatory function are not known. In the present study, progressively deleted lengths of the 5′ noncoding region of segment A were constructed in pGL3 vectors having SV40 enhancer or promoter, and a luciferase (LUC) reporter gene. Transient transfections of the constructs made in a promoter-less pGL3-Enhancer vector when transfected in Vero cells and the lysates assayed for LUC expression, allowed the localization of maximal activity to the 32-nucleotide stretch (precursor polyprotein ORF positions −131 to −100), which is highly conserved at the 5′ end of both genome segments. This fragment, when evaluated in parallel in an enhancer-less pGL3-Promoter vector demonstrated no activity. To determine if this region is recognized by IBDV replicative proteins, we engineered modifications in an enhancer-less pGL3-Promoter vector where the terminal 32-bp fragment, the full-length noncoding region, or the noncodig region with the 32-bp fragment deleted was positioned in either the plus-sense or the minus-sense orientation immediately downstream of the SV40 promoter and upstream of the LUC gene. Transfections of these constructs in IBDV-infected and uninfected Vero cells resulted in the endogenous generation of recombinant viral-LUC RNAs containing the 5′ terminal viral RNA sequences in either the plus-sense or the minus-sense orientation. LUC assays of the infected cell lysates showed up-regulated expression of LUC only with constructs containing the 32-bp fragment in the minus-sense orientation. Deletion of this 32-bp fragment abolished such LUC expression. We therefore conclude that the 5′-terminal 32 base pairs of genomic segment A contain a major promoter element in IBDV. In addition, our results show that IBDV replicative proteins recognize and transcribe single-stranded RNA in vivo. |
4,637 | Cell-to-cell movement of plant viruses: Insights from amino acid sequence comparisons of movement proteins and from analogies with cellular transport systems | Cell-to-cell movement is a crucial step in plant virus infection. In many viruses, the movement function is secured by specific virus-encoded proteins. Amino acid sequence comparisons of these proteins revealed a vast superfamily containing a conserved sequence motif that may comprise a hydrophobic interaction domain. This superfamily combines proteins of viruses belonging to all principal groups of positive-strand RNA viruses, as well as single-stranded DNA containing geminiviruses, double-stranded DNA-containing pararetroviruses (caulimoviruses and badnaviruses), and tospoviruses that have negative-strand RNA genomes with two ambisense segments. In several groups of positive-strand RNA viruses, the movement function is provided by the proteins encoded by the so-called triple gene block including two putative small membrane-associated proteins and a putative RNA helicase. A distinct type of movement proteins with very high content of proline is found in tymoviruses. It is concluded that classification of movement proteins based on comparison of their amino acid sequences does not correlate with the type of genome nucleic acid or with grouping of viruses based on phylogenetic analysis of replicative proteins or with the virus host range. Recombination between unrelated or distantly related viruses could have played a major role in the evolution of the movement function. Limited sequence similarities were observed between i) movement proteins of dianthoviruses and the MIP family of cellular integral membrane proteins, and ii) between movement proteins of bromoviruses and cucumoviruses and M1 protein of influenza viruses which is involved in nuclear export of viral ribonucleoproteins. It is hypothesized that all movement proteins of plant viruses may mediate hydrophobic interactions between viral and cellular macromolecules. |
4,638 | Inhibition by monensin of human cytomegalovirus DNA replication | Monensin, at concentrations which depended on the multiplicity of infection, was found to prevent DNA replication of human cytomegalovirus (HCMV) as well as production of viral progeny in human foreskin fibroblasts. The drug did not affect DNA replication of herpes simplex virus. Inhibition of consecutive HCMV DNA synthesis was also observed following delayed addition of the drug within 12–24 hours postinfection, but was fully reversible upon its removal. Viral replication proceeded, however, without impairment in cultures treated with monensin prior to infection. Induction of viral DNA polymerase activity was not impeded by the inhibitor. Analysis of protein- and glycoprotein synthesis revealed that monensin interfered with the production of a number of HCMV-specific polypeptides. Furthermore, evidence was obtained that the drug may hinder intracellular transport of a 135 kd glycopolypeptide. |
4,639 | Effect of pH on the growth and cytopathogenicity of avian infectious bronchitis virus in chick kidney cells | The growth of avian infectious bronchitis virus (IBV) in chick kidney cells at different pH values in the range 6.0–9.0 demonstrated that although the virus was released at a much faster rate at the higher pH values the titre tended to drop more quickly. At the acid pH values the virus was released more slowly but reached a maximum titre similar to that at the higher pH values and showed only minimum reduction in infectivity up to 49 hours post inoculation. The stability of virus in tissue culture medium was shown to be directly related to pH between pH 6.0–8.0, being more stable at the acid pH values. The degree of cytopathogenicity induced in chick kidney cells following infection with IBV was directly related to the pH at which the cells were incubated, occurring earlier and more extensively in cells at the higher pH values. Cell macromolecule synthesis in chick kidney cells was inhibited following infection with IBV and was apparently due to cell damage and death. |
4,640 | Identification of type-specific and cross-reactive neutralizing conformational epitopes on the major capsid protein of human papillomavirus type 31 | The majority of the neutralizing epitopes of papillomaviruses (PV) are conformation-specific and have not been fully characterised. Studies have, to date, been limited to a few HPV types only. We analysed the epitopes on the major capsid protein (L1) of Human papillomavirus (HPV) type 31 using monoclonal antibodies (MAbs) generated against HPV-31 virus-like particles (VLPs). The type-specific MAbs against HPV-31 were all found to be neutralizing and recognized conformation-dependent epitopes. Two other MAbs directed against a conformational epitope were found to be cross-reactive with other HPV types, and one of them was found to be cross-neutralizing. Cross-reactive antibodies were further investigated using wild-type HPV-16 L1 VLPs and two mutants. The results obtained suggested the existence of a cross-neutralizing conformational epitope at the N-terminal part of the FG loop of the major capsid protein, and the other four cross-reactive MAbs recognized epitopes also located at the N-terminal part of the FG loop. |
4,641 | Isolation of encephalomyocarditis virus among stillborn and post-weaning pigs in Quebec | Encephalomyocarditis (EMC) virus was isolated from aborted fetuses and lungs of suckling pigs from three Quebec pig farms that experienced outbreaks of reproductive failure in sows and respiratory problems in suckling and post-weaning piglets. Multifocal interstitial pneumonia and mild non-suppurative myocarditis and meningoencephalitis were the more significant histopathological lesions observed in piglets. Vero cells were found to be more sensitive than BHK-21 cells and pig cell lines for primary isolation of EMC virus. The Quebec EMC virus isolates were highly virulent for mice and were antigenically related to reference strain of EMC virus as demonstrated by indirect immunofluorescence, seroneutralization and Western immunoblotting. Specific virus neutralization antibody titers up to 1:12,800 were detected in samples of thoracic or abdominal fluids of the aborted fetuses. |
4,642 | Phylogenetic analyses of the putative M (ORF 6) and N (ORF 7) genes of porcine reproductive and respiratory syndrome virus (PRRSV): implication for the existence of two genotypes of PRRSV in the U.S.A. and Europe | The putative membrane (M) protein (ORF 6) and nucleocapsid (N) protein (ORF 7) genes of five U.S. isolates of porcine reproductive and respiratory syndrome virus (PRRSV) with differing virulence were cloned and sequenced. To determine the genetic variation and the phylogenetic relationship of PRRSV, the deduced amino acid sequences of the putative M and N proteins from these isolates were aligned, to the extent known, with other PRRSV isolates, and also other members of the proposed arterivirus group including lactate dehydrogenase-elevating virus (LDV) and equine arteritis virus (EAV). There was 96–100% amino acid sequence identity in the putative M and N genes among U.S. and Canadian PRRSV isolates with differing virulence. However, their amino acid sequences varied extensively from those of European PRRSV isolates, and displayed only 57–59% and 78–81% identity, respectively. The phylogenetic trees constructed on the basis of the putative M and N genes of the proposed arterivirus group were similar and indicated that both U.S. and European PRRSV isolates were related to LDV and were distantly related to EAV. The U.S. and European PRRSV isolates fell into two distinct groups, suggesting that U.S. and European PRRSV isolates represent two distinct genotypes. |
4,643 | Single radial haemolysis for the determination of antibody to reoviruses | A single radial haemolysis (SRH) for reovirus antibody determination was developed and compared to standard haemagglutination-inhibition (HI) and complement fixation (CF) techniques. SRH appeared more simple and sensitive than CF, but less sensitive and less specific than HI. |
4,644 | Pleural effusion disease in rabbits: Observations on viraemia, immunity and transmissibility | Baby rabbits surviving infection with pleural effusion disease virus (PEDV) developed viraemia persisting for at least six months. Only the infectious serum samples collected during the first 2 months of disease could transfer the typical PED. Six months after neonatal infection, virus concentration in serum was 10(2) to 10(4) rabbit-infectious doses per ml, the level of IgG appeared elevated, and serum rendered non-infectious by ether-treatment had a protective effect in passive immunisation experiments. No evidence of glomerulonephritis or deposits of immunoglobulins could be demonstrated in the kidneys. During the nursing period PEDV was transmitted from infected baby rabbits to two out of four dams, but not to control litter-mates. After the nursing period control rabbits, caged together with the viraemic rabbits for 60 to 150 days, remained free from PEDV infection. |
4,645 | Asymptomatic infection of mouse hepatitis virus in the rat | After intranasal inoculation of suckling rats mouse hepatitis virus multiplied mostly in the nasal epithelium; though there were no symptoms, antibodies were produced. Antibodies were also demonstrated in adult rats. These findings suggest that the rat may be a natural host for the virus. |
4,646 | Virus isolation and serum antibody responses after infection of cats with transmissible gastroenteritis virus | Transmissible gastroenteritis virus was administered orally to cats. No clinical disease resulted but infectious virus was isolated from faeces for up to 22 days after infection and serum antibody was detected by neutralisation and immunofluorescence tests. |
4,647 | Contamination of live attenuated vaccines with an infectious feline endogenous retrovirus (RD-114 virus) | Retroviruses are classified as exogenous and endogenous retroviruses according to the mode of transmission. Endogenous retroviruses (ERVs) are retroviruses which have been integrated into germ-line cells and inherited from parents to offspring. Most ERVs are inactivated by deletions and mutations; however, certain ERVs maintain their infectivity and infect the same host and new hosts as exogenous retroviruses. All domestic cats have infectious ERVs, termed RD-114 virus. Several canine and feline attenuated vaccines are manufactured using RD-114 virus-producing cell lines such as Crandell-Rees feline kidney cells; therefore, it is possible that infectious RD-114 virus contaminates live attenuated vaccines. Recently, Japanese and UK research groups found that several feline and canine vaccines were indeed contaminated with infectious RD-114 virus. This was the first incidence of contamination of ‘infectious’ ERVs in live attenuated vaccines. RD-114 virus replicates efficiently in canine cell lines and primary cells. Therefore, it is possible that RD-114 virus infects dogs following inoculation with contaminated vaccines and induces proliferative diseases and immune suppression, if it adapts to grow efficiently in dogs. In this review, we summarize the incidence of contamination of RD-114 virus in live attenuated vaccines and potential risks of infection with RD-114 virus in dogs. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00705-013-1809-1) contains supplementary material, which is available to authorized users. |
4,648 | Is serum procalcitonin a reliable diagnostic marker in children with acute respiratory tract infections? A retrospective analysis | INTRODUCTION: Acute respiratory tract infections (ARI) in children are often treated with antibiotics even without evidence of bacterial infection. Serum procalcitonin (PCT) is elevated in bacterial but not in viral infections. PATIENTS AND METHODS: We performed a retrospective analysis of children in the PID-ARI.net study on respiratory infections to address the question of whether plasma PCT could potentially distinguish between bacterial infections requiring antibiotic therapy and viral ARI. We analysed data on 327 children who had been included in the German PID-ARI.net study and in whom nasopharyngeal aspirates had been analysed with a 19-valent multiplex reverse transcription-polymerase chain reaction–enzyme-linked immunosorbent assay for viral and atypical bacterial pathogens. Serum PCT was determined using a quantitative immunoassay (BRAHMS Kryptor PCTsensitive, Henningsdorf, Germany). We then focussed specifically on those children who were treated with antibiotics and therefore had been suspected of having bacterial infection but who had a serum PCT level lower than 0.1 ng/ml. RESULTS: Out of 327 children, 132 had serum PCT levels below 0.1 ng/ml. Of these 132, 38 children had been treated with antibiotics. After exclusion of 26 patients (with critical illnesses, antibiotics on admission or for reasons other than ARI), 12 children remained for further evaluation. Of these 12 children, four had atypical pneumonia; four others had positive virus testing, and, in the last four, the aetiology of ARI remained unknown; evidence of bacterial infection could not be detected in any. CONCLUSIONS: Taken the results of this retrospective analysis, serum PCT values below 0.1 ng/ml might be a marker to identify children with acute respiratory tract infection in whom antibiotic treatment could be withheld. However, only a prospective intervention trial will prove the general safety of this limit. |
4,649 | Populations of herpes simplex virus glycoprotein gC with and without affinity for the N-acetyl-galactosamine specific lectin ofHelix pomatia | Two fractions of herpes simplex virus glycoprotein gC were isolated and characterized by means of immunosorbent-purification with monoclonal antibodies against gC and Helix pomatia lectin (HPA) affinity chromatography. About 25 per cent of the glycoprotein gC population demonstrated affinity for the lectin, compatible with presence of N-acetylgalactosamine as terminal sugar of the oligosaccharide. The HPA-binding populations of gC appeared as two electrophoretic bands with lower molecular weights than the non-binding gC. The gC subfraction without affinity for the HPA was subjected to treatments aiming to desialylize the carbohydrate moiety. Only 5 per cent of the initially non-reactive fraction of gC became reactive to HPA after the treatments, suggesting that masking of penultimate N-acetylgalactosamine by sialic acid was not a main reason for lack of HPA affinity. Results of treatment with alkaline Na BH(4) demonstrated presence of oligosaccharide-peptide linkages sensitive to β-elimination suggesting O-glycosidic type of linkage. The subfraction of gC demonstrating affinity for HPA as well as gC devoid of HPA binding capacity both revealed affinity for Con A. Therefore N-glycosidically as well as O-glycosidically linked oligosaccharides seemed to be present on the one and same glycoprotein. On the basis of the results presented we assume that the glycosylation of HSV glycoprotein gC may lead to, at least, two populations of the glycoprotein gC, one with terminal N-acetylgalactosamine residues of oligosaccharides 0-glycosidically linked to the polypeptide and the other without affinity for HPA. However, both populations of gC contain similar proportions of oligosaccharides of the high mannose or complex types with N-glycosidic carbohydrate-peptide linkages as indicated by their affinity for Con A. |
4,650 | Identification of epitopes associated with different biological activities on the glycoprotein of vesicular stomatitis virus by use of monoclonal antibodies | Thirteen monoclonal antibodies (MAbs) to the glycoprotein (G) of vesicular stomatitis virus (VSV) serotype Indiana were prepared and examined for their effects on various biological activities of VSV, including in vitro infection, hemagglutination, adsorption to cells, and mediation of cell fusion. Competitive binding assays with these MAbs revealed the presence of at least seven distinct antigenic determinants (epitopes) on the G protein. In some cases, overlappings among epitopes to various degrees were observed as partial inhibition or binding enhancement. The MAbs to all the epitopes but one (epitopes 1–6) reacted with the denatured G protein in a Western immunoblot analysis. Four of the epitopes (epitopes 2, 4, 5, and 7) were involved in neutralization and two (epitopes 1 and 2) in hemagglutination inhibition. None of the MAbs inhibited the adsorption of radiolabeled VSV to BHK-21 cells; the MAbs to epitope 2 slightly enhanced the virus adsorption. All neutralization epitopes except epitope 2 (epitopes 4, 5, and 7) were associated with inhibition of VSV-mediated cell fusion. These results show a direct spatial relationship between the epitopes recognized by the MAbs and functional sites on G protein and further insights into the structure and function of G protein. |
4,651 | Correlation of persistent mouse hepatitis virus (MHV-3) infection with its effect on mouse macrophage cultures | MHV3 has three distinct effects in different strains of mice: strain A mice are completely resistant, most strains (including C57BL, DBA/2, BALB/c and NZB strains) die of acute hepatitis whereas in certain strains (eg. C3H and A2G) the virus produces a persistent infection with neurological symptoms. In cultures of peritoneal macrophages from susceptible strains, MHV-3 replicated freely, with giant cell formation. No replication was observed in macrophages from strain A mice. In contrast to this full susceptibility or resistance, macrophage cultures from strains of mice in which persistent infections occur showed an intermediate susceptibility, as judged by the intensity of the cytopathic effect, the presence of viral antigens in the cytoplasm and levels of viral replication. Possible ways in which the intermediate susceptibility of macrophages and persistent infections might be related are discussed. |
4,652 | Targeted inhibition of immunodeficiency virus replication in lymphocytes through retroviral mediated gene transfer | To test the feasibility of gene therapy for AIDS patients, an animal model is needed to evaluate the efficacy and safety of this approach. Antiviral genes (encoding antisense RNA or viral protein) derived from Simian immunodeficiency virus (SIV) were efficiently targeted into CD4+ lymphocytes through retroviral-mediated gene transfer. After challenging with infectious viruses, the transduced lymphocytes that received antiviral genes were not only protected from SIV infection, but also from infection with HIV, for at least 25 days. Furthermore, little or no cytolytic effect (syncytium formation) was observed in the protected cells. These data demonstrated that SIV or HIV replication could be effectively blocked by antisense sequence(s) or negative dominant factors which were introduced into targeted cells through retroviral-mediated gene transfer. |
4,653 | Intestinal Absorption of Macromolecules during Viral Enteritis: An Experimental Study on Rotavirus-Infected Conventional and Germ-Free Mice | ABSTRACT: Epithelial transport and degradation of horseradish peroxidase (HRP), a macromolecular tracer, was studied in conventional and germ-free suckling mice following an experimental infection with rotavirus. Conventional and germ-free mice developed diarrhea from days 2 to 8 postinfection (pi), with growth failure. In mucosal homogenates, infectious virus detected by immunofluorescence on MA 104 cells was present from day 2 through day 8 pi in germ-free mice, but persisted longer (day 13 pi) in conventional mice. Only mild histological lesions were observed during diarrhea, but obvious macrovacuolation of epithelial cells and increased cellular density occurred during the convalescence period (days 9 to 13 pi). Intact and degraded HRP fluxes from mucosa to serosa were measured in vitro on segments of jejunum mounted in Ussing chambers. Both groups of mice developed increased HRP permeability during the experimental period, but at different times after inoculation: during the diarrheal period (days 2 and 3 pi) conventional mouse epithelium absorbed five times more HRP than noninfected controls and during the convalescence period (days 9 to 13 pi) HRP absorption in germ-free mice rose 10-fold as compared to its level before infection. In both cases, this increase in HRP permeability was entirely due to an increase in intact HRP absorption, probably via a transcellular route, and occurred without any alteration in degraded HRP transport. These results indicate that in mice, rotavirus infection causes a transient rise in gut permeability to undegraded proteins. The intestinal microflora seems to affect the timing, magnitude, and duration of this increased permeability. |
4,654 | The rubella virus nonstructural protease recognizes itself via an internal sequence present upstream of the cleavage site for trans-activity | The substrate requirement for rubella virus protease trans-activity is unknown. Here, we analyzed the cleavability of RV P200-derived substrates varying in their N-terminal lengths (72–475 amino acids) from the cleavage site by the RV protease trans-activity. Only substrates with at least 309 amino acid residues N-terminal to the cleavage site were able to undergo cleavage. Further, rubella sequence was found to be necessary in the N-terminal region of the substrate, whereas a heterologous sequence C-terminal to the cleavage site was tolerated. These results demonstrated a requirement for residues located between amino acids 994–1102 of the RV P200 polyprotein, besides its cleavage site for RV protease trans-activity. This region overlaps with the starting site of the essential cis-protease activity of RV P200 polyprotein. This is a novel observation for a viral protease of the family Togaviridae. |
4,655 | Papain-like proteinase of turnip yellow mosaic virus: a prototype of a new viral proteinase group | Sequence comparisons predicted a potential papain-like proteinase domain in the N-terminal cleavage product (NRP) of the large nonstructural replicase polyprotein (RP) of turnip yellow mosaic virus (TYMV). Replacement of the predicted catalytic amino acids, Cys-783 by Ser, or of His-869 by Glu, abolished cleavage of the 206K RP into a ∼150K NRP and a ∼78K C-terminal product in reticulocyte lysates, while other substitutions exerted no apparent influence on proteolysis. The proteinase-deficient mutant RPs could not be cleaved in trans by as much as an eight-fold molar excess of wild-type proteinase. Deletion experiments have excluded the possible influence on autoproteolysis of amino acid sequences 1–708 and 982–1204 flanking the proteinase domain. Thus, the proteinase of TYMV with a papain-like dyad of essential amino acids has been mapped just upstream from the putative NTPase domain. Statistically significant sequence similarities with the TYMV proteinase were found for the similarly located domains of the replicase polyproteins of carlaviruses, capilloviruses, apple stem pitting virus and apple chlorotic leaf spot virus as well as for those of other tymoviruses and for the domain located downstream from the putative NTPase domain of the large polyprotein of beet necrotic yellow vein furovirus. All these domains are not significantly similar to other known proteinases, although they conserve papain-like Cys- and His-containing motifs. Thus these domains constitute a compact group of related enzymes, the tymo-like proteinases, within the proposedpapainlike proteinase supergroup. The resulting alignment of 10 tymo-like proteinase sequences has revealed a third highly conserved residue — Gly (Gly821 in TYMV RP) followed by a hydrophobic residue. We speculate that all the tymo-like proteinase domains of the viral replicative proteins may share common biochemical and biological features. |
4,656 | Genetic characterization of feline bocavirus detected in cats in Japan | Feline bocavirus (FBoV) has been classified into three genotypes (FBoV1-FBoV3). FBoVs are mainly detected in feces. In the present study, we collected rectal swabs from cats in Japan and examined the samples for the presence of FBoV. The FBoV infection rate was 9.9 % in 101 cats. No significant association was observed between FBoV infection and clinical symptoms. Based on the full-length NS1 protein, the three strains of FBoVs detected in the present study shared high homologies with the genotype 2 FBoV POR1 strain. This is the first study to report FBoV in Japan. |
4,657 | The nucleotide sequence of the coat protein genes and 3′ non-coding regions of two resistance-breaking tobamoviruses in pepper shows that they are different viruses | The nucleotide sequence of the coat protein genes and 3′ non-coding regions of two different resistance-breaking tobamoviruses in pepper have been determined. The deduced coat protein of an Italian isolate of pepper mild mottle virus (PMMV-I) consists of 156 amino acids and its 3′ non-coding region is 198 nucleotides long. They have been found to be very similar in sequence and structure to those previously reported for a Spanish isolate (PMMV-S). In contrast, a Dutch isolate termed P 11 codes for a coat protein of 160 amino acids and its 3′ non-coding region is 291 nucleotides long, which may have arisen by duplication. The nucleotide and the predicted coat protein amino acid sequence analysis show that this isolate should be considered as a new virus within the tobamovirus group. The term paprika mild mottle virus (PaMMV) is proposed. |
4,658 | Polypeptide patterns of infectious bronchitis virus serotypes fall into two categories | Molecular weights of six major polypeptides of infectious bronchitis virus (IBV) are: 1. 75,000; 2. 50,000; 3. 45,000; 4. 35,000; 5. 28,000 or 24,000, and 6. 22,000 dalton. According to the mobility of protein 5 the polypeptide patterns of IBV serotypes fall into two main categories. |
4,659 | Temperature-sensitive mutants of mouse hepatitis virus type 3 (MHV-3): isolation, biochemical and genetic characterization | Mouse hepatitis virus 3 (MHV-3) is highly hepatotropic in sensitive mice. Temperature-sensitive mutants (ts mutants) induced by N-methyl-N′-nitrosoguanidine and 5-fluorouracil were isolated. Twelve mutants which were able to induce the formation of syncytia at 33°C but not at the restrictive temperature (39.5°C) were selected for detailed study. No viral RNA synthesis was detected after infection at the restrictive temperature with six of the mutants (RNA(−)) whereas six others were RNA(+), although they displayed RNA synthesis which was generally reduced. No differences have been detected in the size of the genome or the viral-intracellular RNA species found in wild type virus or ts mutant infected cells at permissive temperature. The pattern of virus-induced proteins analyzed after immunoprecipitation by SDS-PAGE was similar in wild type virus and RNA(+) mutant infected cells at 39.5°C. Complementation experiments between ts mutants enabled us to distinguish five groups. Three of the groups contained RNA(−) mutants and two of them RNA(+). Plaques made by mutants in one group displayed characteristic features that distinguished them from the wild type. |
4,660 | Specific tropism of Japanese encephalitis virus for developing neurons in primary rat brain culture | Among all the neural cells in fetal rat brain culture developing neurons showed the highest rate of infection by Japanese encephalitis virus (JEV). JEV specifically bound to these cells as measured by immuno-staining. These results indicate that developing neurons are the major target of JEV, and that the initial specific binding of virus to these cells may be one of the reasons for the neurotropism of JEV. |
4,661 | Rhinovirus detection using probes from the 5′ and 3′ end of the genome | This study investigated the abilities of cDNA probes from the 5′ and 3′ ends of the genome of human rhinoviruses (HRV-) 14, 9, and 1B to detect RNA from 59 rhinovirus serotypes. The results show that probes from the 5′ end of the genomes of HRV-14, 9, and 1B detected a large number of serotypes but the detection rate was variable and depended on the degree of homology with the particular probe. In contrast, all the 3′ end probes were specific for the homologous virus. However, along HRV-9 probe detected a large number of serotypes. It was concluded that such cDNA probes would not detect all serotypes with equal efficiency. Synthetic oligonucleotides corresponding to short but highly conserved regions in the 5′ non coding region may overcome this problem. |
4,662 | The behaviour in vitro of attenuated recombinant influenza viruses | Influenza strains produced by recombination and tested as possible live vaccine candidates were studied in organ cultures of trachea. Two strains which proved to be too virulent in human volunteers regularly caused damage to the ciliated epithelium and viruses grew to high titre. Two strains which proved to be attenuated for volunteers did not cause appreciable damage, although they replicated to low titre in the epithelium. Similar results were obtained with influenza A virus attenuated by passage in the presence of horse sera. The method may be of value for detecting virulent live influenza vaccine candidates without risking severe illness in volunteers. |
4,663 | Low-pH-induced fusion of Vero cells infected with Junin virus | Junin virus (JV) infected Vero cells were used to investigate virus capacity to induce cell-cell fusion. Polykaryocyte formation due to JV was found to be pH and temperature-dependent. A reduced fusion activity was detected on BHK-21 cells. Different JV-strains exhibited a similar extent and pH dependence of their fusion activity. Neutralizing antibodies against the main viral glycoprotein (GP38) inhibited syncytium production and GP38 conformational changes in response to acid treatment were detected by an immunoprecipitation assay. |
4,664 | Analysis of synonymous codon usage patterns in torque teno sus virus 1 (TTSuV1) | Torque teno sus virus 1 (TTSuV1) is a novel virus that has been found widely distributed in the swine population in recent years. Analysis of codon usage can reveal much about the molecular evolution of TTSuV1. In this study, synonymous codon usage patterns and the key determinants in the coding region of 29 available complete TTSuV1 genome sequences were examined. By calculating the nucleotide content and relative synonymous codon usage (RSCU) of TTSuV1 coding sequences, we found that the preferentially used codons were mostly those ending with A or C nucleotides; less-used codons were mostly codons ending with U or G nucleotides, and these were mainly affected by composition constraints. Although there was a variation in codon usage bias among different TTSuV1 genomes, the codon usage bias and GC content in the TTSuV1 coding region was lower, which was mainly determined by the base composition in the third codon position and the effective number of codons (ENC) value. Moreover, the results of correspondence analysis (COA) indicated that the codon usage patterns of TTSuV1 isolated from different countries varied greatly and had significant differences. In addition, Spearman’s rank correlation analysis and an ENC plot revealed that apart from mutation pressure, which was critical in determining the codon usage pattern, other factors were involved in shaping the evolution of codon usage bias in TTSuV1, such as natural selection. Those results suggested that synonymous codon usage patterns of TTSuV1 genomes were the result of interaction between mutation pressure and natural selection. The information from this study not only provides important insights into the synonymous codon usage pattern of TTSuV1, but also helps to identify the main factors affecting codon usage by this virus. |
4,665 | Effects of origin and state of differentiation and activation of monocytes/macrophages on their susceptibility to porcine reproductive and respiratory syndrome virus (PRRSV) | In this study, the susceptibility of porcine peripheral blood monocytes (BMo), peritoneal macrophages (PMφ) and alveolar macrophages (AMφ) to PRRSV was examined. To test the effect of differentiation and activation on their susceptibility, AMφ and BMo were aged, cultivated in either adhesion or suspension and treated with bacterial lipopolysaccharide (LPS) and phorbol myristate acetate (PMA). It was found that freshly isolated PMφ and BMo were non-permissive to PRRSV. PMφ remained refractory but a few BMo became susceptible after 1 day cultivation. AMφ were permissive with a significant increase of their susceptibility after one day cultivation. In a binding assay, it was demonstrated that the attachment of biotinylated PRRSV to AMf is much more efficient than to PMφ and BMo. Two monoclonal antibodies (Mabs) 41D3 and 41D5 which block PRRSV infection of AMφ and are directed against a candidate receptor for PRRSV only reacted with the cell membrane of AMφ. PMA treatment of AMφ blocked PRRSV replication in the cells in a dose-dependent manner. The blocking effect of PMA decreased after 9 h continuous pre-treatment and diminished after 24 h continuous pre-treatment. PMA treatment did not affect the binding of PRRSV and MAb 41D3 and 41D5 to AMφ. Direct or indirect treatment of AMφ and BMo with LPS or cultivation in suspension did not significantly affect their susceptibility. These results provide clear evidence that PRRSV has a strongly restricted tropism for only some sub-populations of porcine monocytes/macrophages and that some specific states of differentiation and activation of monocytes/macrophages considerably affect their susceptibility. |
4,666 | Monoclonal antibodies to a virulent strain of transmissible gastroenteritis virus: comparison of reactivity with virulent and attenuated virus | Twelve hybridomas secreting monoclonal antibodies (MAbs) against Miller virulent strain of transmissible gastroenteritis virus (TGEV) were generated and characterized. In a cell culture immunofluorescence (CCIF) assay, three MAbs directed against peplomer protein (E 2) had perinuclear fluorescence and four unclassified MAbs showed cell membrane fluorescence. Six of these seven MAbs neutralized both attenuated and virulent TGEV, and the seventh (an unclassified MAb) neutralized only the latter virus. Two MAbs able to bind the cell membrane of infected cells had low neutralizing antibody titers (8 to 72) but were able to distinguish between virulent and attenuated TGEV (9- to 72-fold differences in neutralizing titers). Two E 2-specific MAbs had higher neutralizing antibody titers (782 to 34,117) and showed 4- to 13-fold differences in titers against the attenuated and virulent TGEV strains. Five MAbs which were specific for nucleocapsid (N) protein had cytoplasmic, particulate fluorescence in CCIF, and did not neutralize TGEV. Comparison of CCIF antibody titers of MAbs to the virulent and attenuated strains of TGEV indicated that differences existed in titers of most E 2 and all N-specific MAbs, with titers consistently higher against virulent TGEV (homologous strain). Hyperimmune antisera prepared in gnotobiotic pigs against the attenuated, virulent and a recent isolate of TGEV immunoprecipitated the 3 major structural proteins of both the attenuated and virulent TGEV strains. Relative mol. wt. differences in the E 1 and E 2 proteins between the two virus strains were revealed using either the hyperimmune pig sera or MAbs. In addition to the 48 K N protein, a 44 K protein was coimmunoprecipitated by the hyperimmune sera and MAbs, but mainly from lysates of attenuated TGEV. |
4,667 | Identification and characterization of RNA-dependent RNA polymerase activity in recombinant Japanese encephalitis virus NS5 protein | The complete nonstructural NS5 gene of Japanese encephalitis virus (JEV) was amplified and cloned into an expression vector. The NS5 protein was expressed in Escherichia coli and purified by His-tag based affinity chromatography. This recombinant NS5 protein exhibited RNA-dependent RNA polymerase (RdRp) activity in vitro in the absence of other viral or cellular factors. The RNA polymerase activity was dependent on divalent cations, and Mn(2+) was found to be 20 times more effective than Mg(2+) in coordinating the catalytic reaction of RdRp, while Ca(2+) inhibited enzyme activity. The optimal reaction conditions for the in vitro RdRp reaction were established. Characterization of the RdRp reaction products demonstrated that the JEV NS5 protein can initiate RNA synthesis through a de novo initiation mechanism in our in vitro reaction system. Comparing the efficiency of different RNA templates, we found that JEV NS5 protein was more efficient in using negative-strand RNA templates, indicating that the JEV NS5 protein is involved in regulating the ratio of positive- to negative-strand RNA. Four amino acid sequence motifs crucial for RdRp activity were also identified using site-directed mutagenesis analysis. All substitutions of the conserved residues within these motifs led to a complete inactivation or severe loss of enzyme activity. |
4,668 | Biological and macromolecular properties of murine cells persistently infected with MHV-JHM | A persistently-infected neuroblastoma culture [Neuro-2A (JHMV)] was established with the murine hepatitis virus JHM [MHV-JHM]. After 100 days of passage, the endogenous virus [Neuro-2A (JHMV) end] released by this culture was unable to induce the syncytia typical of MHV-JHM and the endogenous virus was not temperature-sensitive. The Neuro-2A (JHMV) culture was cured of virus production by passage under neutralizing antibody [Neuro-2A (JHMV) Ab]. The Neuro-2A (JHMV) and the Neuro-2A (JHMV) Ab cultures were as susceptible to heterologous infection with mengovirus and vesicular stomatitis virus as the uninfected Neuro-2A culture. However, the Neuro-2A (JHMV) and Neuro-2A (JHMV) Ab cultures were partially resistant to homologous superinfection by MHV-JHM and the closely related MHV-A59. Virus related to MHV-JHM was rescued from the antibody-cured cells by cell fusion. The synthesis of MHV-JHM specific antigens by Neuro-2A (JHMV) cells, Neuro-2A (JHMV) Ab cells and 17 Cl-1 cells infected by Neuro-2A (JHMV) end was studied by SDS-PAGE. The genomic RNAs of MHV-JHM and Neuro-2A (JHMV) end were compared by oligonucleotide mapping. The results of the protein and RNA studies indicated that the genome of Neuro-2A (JHMV) end was substantially modified from the genome of MHV-JHM, but the modifications did not significantly alter the molecular size of the viral-specific proteins. |
4,669 | Analysis of virus-specific RNA species and proteins in Freon-113 preparations of the Borna disease virus | Treatment of homogenates from Borna disease virus (BDV)-infected brain tissue or cell cultures with Freon-113 yielded infectious particles with a buoyant density of 1.16–1.22 g/ml. Positive- and negative-stranded BDV-specific RNA species as well as three virus-specific proteins, known to be present in BDV-infected cell extracts, were demonstrated in these Freon-treated fractions. When the Freon-purified virus preparations were treated with RNase A prior to RNA extraction, only negative-stranded, genomic RNA was detected in Northern blot hybridizations using sense and antisense RNA probes. These data substantiate that BDV is a negative-stranded RNA virus. |
4,670 | Pathogenesis of diarrhoea caused by astrovirus infections in lambs | Experimental infection of 2-day-old gnotobiotic lambs with lamb astrovirus produced mild diarrhoea after an incubation period of about 48 hours. No other clinical symptoms developed. Infection was studied by immunofluorescent and histological examination of tissues from the lambs. Astroviruses infected only mature villus epithelial cells and subepithelial macrophages in the small intestine, where they produced partial villus atrophy. Infected enterocytes were replaced with cuboidal cells from the crypts, and the lesion gradually healed by 5 days after infection. No serological relationship was detected by immunofluorescence between lamb astrovirus antigen in gut sections and antisera to either calf or human astrovirus. |
4,671 | Comparison of mouse hepatitis virus strains for pathogenicity in weanling mice infected by various routes | The pathogenicity for mice of nine strains of mouse hepatitis virus was studied in mice free from the virus by the intracerebral, intraperitoneal, intravenous and intranasal routes of inoculation. |
4,672 | Virus isolation and titration at 33‡ and 37‡ C | Various prototype viruses and original specimens were comparatively titrated in cell cultures at 33‡ and 37‡ C. Higher titers at 37‡ were consistently obtained with adenoviruses; for other viruses (enteroviruses, herpesvirus hominis, vaccinia virus, parainfluenza viruses) the titers were mostly identical at either temperature. Original specimens and prototype strains showed the same behavior. The habit to cultivate viruses from throat swabs at 33‡ C is unsatisfactory for adenoviruses. |
4,673 | NCI-H292 as an alternative cell line for the isolation and propagation of the human paramyxoviruses | Primary rhesus monkey kidney (MK) cells have long been the cells of choice for isolation and propagation of the human paramyxoviruses (parainfluenza 1, 2, 3, 4A, 4B, and mumps). However, problems with the supply and cost of MK cells and the presence of endogenous viruses, including herpes B virus and SV-5, necessitated a search for an alternative cell line. Continuous cell cultures of human origin (L132, A-549, HuT-292, HEK, G-293, G-401, A-498, A-704, CAKI-1, RD) and simian origin (LLC-MK2, BSC-1, MA-104, Vero) were evaluated for their capacity to support the growth of the human paramyxoviruses, as followed by cytopathic effect, hemadsorption, hemagglutination, and EIA. NCI-H292 (HuT-292) human lung mucoepidermoid carcinoma cells (ATCC # CRL-1848) proved to be the most sensitive line for cultivating all serotypes and strains of the paramyxoviruses. These cells were also shown to be a suitable substitute for MK in primary isolation of paramyxoviruses from clinical specimens. RPMI-1640 with 1.5µg/ml trypsin was the preferred maintenance medium; alternatively, Eagle's MEM supplemented with 1.5µg/ml trypsin and 0.1% ITS was satisfactory. NCI-H292 cells are a continuous line with excellent growth characteristics, although the genetic polyploidy of the cells may limit the number of passages of usable cells. |
4,674 | Heterogeneity of infectious bronchitis virus grown in eggs | Egg-grown infectious bronchitis virus, strain Beaudette, was concentrated and centrifuged on sucrose density gradients to separate the virus into five peaks with densities of 1.144, 1.160, 1.172, 1.191 and 1.218 g/cm(3). All peaks retained infectivity, complement fixation activity and were labelled with(3)H-uridine. Morphologically the densest peak consisted of very large virus particles and amorphous material, the other peaks consisted of mainly intact particles although small differences in size and pleomorphism were seen. Polyacrylamide gel electrophoresis of material from the density gradient peaks revealed four major polypeptides and at least 10 minor polypeptides. The proportions of the polypeptides were approximately similar for all peaks with the exception of the densest peak in which the major polypeptides were greatly reduced. The four major polypeptides had approximate molecular weights of 1. 52,000, 2. 45,000, 3. 34,000, 4. 32,000. The major polypeptides 1 and 4 were shown to be glycosylated as were two of the minor polypeptides. |
4,675 | A comparative analysis of measles virus RNA by oligonucleotide fingerprinting | Isolates from two cases of acute measles, one case of acute measles encephalitis and three patients with subacute sclerosing panencephalitis were compared. This comparison was based upon the electrophoretic analysis of T(1) oligonucleotides from single-stranded, full-length RNA isolated from cytoplasmic nucleocapsids. Although all viruses have oligonucleotides in common, each isolate generated a unique pattern of oligonucleotides. However, no group of oligonucleotides was observed which would allow a differentiation between viruses isolated from acute infections and those isolated from CNS diseases; indicating that probably all measles viruses differ in their nucleotide sequence, regardless of origin. |
4,676 | Maternally-derived passive immunity to enterotropic mouse hepatitis virus | Maternally-derived antibody to enterotropic mouse hepatitis virus (MHV) strain Y was transferred to pups by both intrauterine (IgG) and lactogenic (IgA and IgG) routes. Antibody present in the gastric whey of pups suckling immune dams dropped to undetectable levels by weaning age (21 days post partum). MHV-specific IgG was found in the serum of passively immune pups up to 10 weeks of age. Immune dams transferred equal levels of antibody to 3 consecutive litters of pups, without evidence of decline. Immunoblots showed that IgA and IgG in whey and serum were directed against nucleoprotein N and glycoprotein S. MHV-specific IgM was not detected in any sample. |
4,677 | Improvement of arbovirus HA antigens by treatment with a colloidal silica gel and sonication | A remarkable increase in HA titers for weakly haemagglutinating Norwegian arbovirus strains, Uukuniemi and Runde viruses, was achieved by including treatment with the colloidal silica gel Aerosil in the antigen preparation scheme. By combining this procedure with sonication, the titers of sucrose-aceton extracted, infected suckling mouse brains could be increased several hundred times. Good antigens also were obtained from virus grown in BHK 21/c 13 cell cultures and concentrated by polyethylene glycol 6000/NaCl. Rubella virus HA antigen and HB(s)Ag were adsorbed to the gel, and excluded from a preparation by treatment with Aerosil. This indicates a limitation to the universal use of the method, presumably related to the particle size. |
4,678 | Differences in antigenicity of E2 in Semliki Forest virus particles and in infected cells | Using six monoclonal antibodies to epitopes a-f on the glycoprotein E2 of Semliki Forest virus (SFV) we found antigenic differences between E2 in infected cells and in virus particles, respectively, if glycosylation was impaired by 2-deoxy-D-glucose or inhibited by N-methyl-1-deoxynojirimycin. Furthermore we concluded that a conformational change of E2 takes place on virus budding. |
4,679 | Genetic grouping for the isolates of avian infectious bronchitis virus in Taiwan | In order to differentiate recent isolates of avian infectious bronchitis virus (IBV) in Taiwan, polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and direct sequencing methods were used to type 25 IBV Taiwan isolates. Two conserved sequences that flank the hypervariable region I (HVR I) in the N-terminus of S1 protein gene were chosen as primers. Sequences of 228–231 base pairs (bp) were amplified by PCR from 25 Taiwan isolates and 4 reference strains (H120, Conn, JMK, Holte). PCR products were digested with 5 restriction endonucleases,BsoFI,DdeI,MboII,AluI,RsaI, and different IBV isolates were grouped according to their RFLP patterns. The RFLP patterns of the 4 reference strains in this study matched the published sequences in GenBank. Except 1 vaccine strain, the other 24 Taiwan isolates were different from these 4 and 18 other IBV strains whose sequences were published. The data from PCR-RFLP and sequencing of IBV genomes showed that the 24 Taiwan isolates can be divided into 2 distinct groups, I and II. Seven RFLP patterns are identified in group I and only 1 in group II. |
4,680 | Partial DNA cloning and sequencing of a canine parvovirus vaccine strain: application of nucleic acid hybridization to the diagnosis of canine parvovirus disease | The cloning and sequencing of anEco RI-Pst I fragment derived from the replicative form of a canine parvovirus (CPV) vaccine strain are reported. The variability of the 5′ end of NS 1 protein gene in the genome is confirmed by comparison with previously determined DNA sequences. A 15 nucleotide deletion was also observed in this vaccine strain. In order to improve CPV diagnosis, radioactively labelled RNA or DNA and biotin labelled DNA obtained by random priming of the recombinant plasmid were used as probes mainly on gut or stool samples from naturally infected dogs. Results of filter hybridization correlated well with histopathological diagnosis of parvovirus infection and with hemagglutination tests performed on dog faeces. We propose that nucleic acid hybridization may be an alternative diagnostic method to ascertain the presence of CPV, especially in frozen samples. |
4,681 | Prevalence of neutralising antibodies to Berne virus in animals and humans in Vellore, South India | In Southern India the prevalence of neutralising antibody to Berne virus was high in sera obtained from cattle (49%), horses (38%), and sheep (36%). Neutralising antibody was not detected in sera from humans and monkeys. |
4,682 | PA-X: a key regulator of influenza A virus pathogenicity and host immune responses | PA-X, a fusion protein belonging to influenza A viruses (IAVs), integrating the N-terminal 191 amino acids of PA gene and the ribosomal frame-shifting product that lengthens out to 41 or 61 amino acids. Since its discovery in 2012, multiple functions have been attributed to this small protein, including a process, where wide-spread protein synthesis in infected host cells is shut down (called host shutoff), and viral replication, polymerase activity, viral-induced cell apoptosis, PA nuclear localization, and virulence are modulated. However, many of its proposed functions may be specific to strain, subtype, host, or cell line. In this review, we start by describing the well-defined global host-shutoff ability of PA-X and the potential mechanisms underlying it. We move on to the role played by PA-X in modulating innate and acquired immune responses in the host. We then systematically discuss the role played by PA-X in modulating the virulence of influenza viruses of different subtypes and host origins, and finish with a general overview of the research advances made in identifying the host cell partners that interact with PA-X. To uncover possible clues about the differential effects of PA-X in modulating viral virulence, we focus on systemically evaluating polymorphisms in PA-X from various viral subtypes and hosts, including avian and human H5N1, H5N6, H9N2, and H7N9 viruses. Finally, we conclude with a proposition regarding the possible future research directions for this important protein. |
4,683 | Detection of astroviruses in gut contents of nude and normal mice | Gut contents of nude and normal mice were examined by electron microscopy in association with an outbreak of diarrhea in a colony of nude mice. Virus-like particles with a morphology consistent with previous descriptions of astroviruses of other species were demonstrated in a high percentage of the animals. |
4,684 | Physicochemical properties of transmissible gastroenteritis virus hemagglutinin | Transmissible gastroenteritis virus was readily adsorbed onto chicken erythrocytes at 4°C. The hemagglutinin thus adsorbed could be eluted from the erythrocytes by incubating in phosphate buffered saline at 37°C. The on chicken erythrocytes for the hemagglutinin was inactivated by neuraminidase and potassium periodate, but not by trypsin, 2-mercaptoethanol and formalin. The hemagglutinin was inactivated by trypsin, papain, pepsin, α-amylase, phospholipase C, neuraminidase, formalin, 2-mercaptoethanol, potassium periodate, ethylether, chloroform, Tween-80 and β-propiolactone, but not by sodium deoxycholate and trichlorotrifluoroethane, suggesting that the active component of the hemagglutinin involved glycoproteins. The hemagglutinin was stable at 37°C or lower temperatures but not at 60°C or higher temperatures. The hemagglutinin activity was resistant to ultraviolet irradiation, while the infectivity was very susceptible. The hemagglutinin and the infectivity were readily sedimented by ultracentrifugation at 45,000 × g for 60 minutes. In rate zonal centrifugation of the hemagglutinin preparation on a sucrose density gradient, the hemagglutinin activity showed a sharp peak at 1.19 g/ml coinciding with the peak of infectivity. The activity in the peak fraction seemed to be structually associated with virus particles. |
4,685 | Identification and sequence determination of the capsid protein gene of feline calicivirus | We have determined 4380 bases of the sequence from a cDNA clone containing the 3′ end of feline calicivirus strain F9. We find four candidate open reading frames of which three are complete and comprise 245, 317 and 2012 nucleotides. The fourth continues toward the 5′ end. We have expressed the largest complete open reading frame inE. coli. Sera raised to this antigen react specifically with the capsid protein and its intracellular precursor molecule. N-terminal sequence analysis of purified, mature capsid protein confirms this assignment and has identified the position at which precursor is cleaved. |
4,686 | Mouse cytomegalovirus is infectious for rats and alters lymphocyte subsets and spleen cell proliferation | The Smith strain of mouse cytomegalovirus (MCMV) was infectious for infant and mature DA strain laboratory rats as judged by development of neutralizing antibodies and specific spleen cell proliferation on stimulation with MCMV antigen. An i. p. inoculum of 10(6) PFU of MCMV was fatal for more than two-thirds of infant mice (1–7 days of age), and disseminated viral infection was documented by isolation of virus from body organs. In contrast, weanling and adult rats did not become ill as a result of infection with a larger inoculum of 10(7) PFU. However, these older MCMV infected rats did show transient reversals of T helper/suppressor cell ratios and alterations of immune cell function as detected byin vitro spleen cell proliferation assays. Seven days after MCMV infection, there was a generalized increase in(3)H-thymidine incorporation by spleen cells in both resting (unstimulated) cultures and cultures exposed to mitogens (Con A, PHA, LPS) and to MCMV antigen. At 14 days, the spleen cell proliferation in the unstimulated cultures returned to normal but was depressed compared to controls in response to Con A. These observations show that laboratory rats are susceptible to MCMV infection and that assymptomatic infection may occur and cause transient alterations in lymphocyte subsets and in their reactivity to mitogens. |
4,687 | Semliki Forest virus-induced polykaryocyte formation is an ATP-dependent event | Infection of Aedes albopictus cells with Semliki Forest virus (SFV) leads to polykaryocyte formation below pH 6.2. This syncytium formation is accompanied by a decrease of the cellular ATP level. Addition of inhibitors of oxidative phosphorylation leads to a rapid, total depletion of ATP in infected cells at pH 6 and results in an inhibition of polykaryocyte formation. However, when cells were exposed for only a few minutes to pH 6 in the presence of the inhibitors and then kept at pH 7.2, the ATP level partially recovered to values sufficient for syncytium formation. Similar results were obtained after ATP depletion induced by 2-deoxyglucose. Thus, it can be concluded that SFV-induced syncytium formation is an ATP-dependent event. |
4,688 | Autoimmunity caused by host cell protein-containing viruses | Autoreactive T cells specific for myelin basic protein (MBP), a major component of central nervous system (CNS) protein, are frequently found in blood and cerebrospinal fluid of patients with postinfectious encephalomyelitis. This autoimmune syndrome is a CNS complication after infections with a number of different enveloped viruses, e.g. mumps, measles, rubella, influenza and varicella. However, the pathophysiological mechanism leading to this breaking of natural self tolerance in the course of viral infection remains an enigma. A long-lasting hypothesis has suggested that incorporation of cellular (self) proteins into the envelope of budding viruses might be a possible mechanism leading to autosensitization. In a model study we demonstrate here that vesicular stomatitis virus (VS V), grown in myelin protein-expressing cell cultures, is highly efficient in triggering T cell responses to MBP in vitro and can prime autoreactive T cell immune responses in vivo. On the basis of these findings, we suggest that incorporation of CNS membrane components into the viral envelope and subsequent priming of self-reactive immune responses might be the common pathogenic mechanism underlying the postinfectious encephalomyelitis syndrome. |
4,689 | Rabies viruses infect primary cultures of murine, feline, and human microglia and astrocytes | Recent studies have reported the detection of rabies viral antigens and virions in astrocytes and microglia of rabies-infected animals. As a first step toward understanding whether these glial cells may be involved in rabies virus replication, persistence, and/or pathogenesis, we explored their potential to be infected in vitro. Primary cultures of murine, feline, and human microglia and astrocytes were infected with several different rabies viruses: two unpassaged street virus isolates, a cell culture-adapted strain, and a mouse brain-passaged strain. Infection, as determined by immunofluorescence, was detected in 15 of the 16 (94%) virus-glial cell combinations. Replication of infectious virus, determined by infectivity assay, was detected in 7 of the 8 (88%) virus-cell combinations. These results show that astrocytes and microglia can be infected by rabies viruses, suggesting that they may have a potential role in disease, perhaps contributing to viral spread, persistence and/or neuronal dysfunction. |
4,690 | Characterization of avian paramyxovirus type 6 isolated from a Eurasian teal in the intersection of migratory flyways in Russia | The complete genome sequence was determined for avian paramyxovirus (APMV-6) serotype 6 strain teal/Chany/455/2009, isolated from a teal (Anas crecca) in Siberia. Siberia is crossed by four major migration flyways and represents the major breeding area for many wild bird species in the Palearctic. Strain teal/Chany/455/2009 is genetically closely related to Kazakh and Chinese strains and belongs to the genetic group of duck/Hong Kong/18/199/77-like APMV-6 viruses. We show that the virus has low pathogenic potential according to genetic markers and animal model experiments. |
4,691 | The nucleic acid of infectious bronchitis virus | The nucleic acid of infectious bronchitis virus (IBV), like that of other enveloped viruses, consists of discontinuous single stranded RNA. However, unlike many other viruses, there is extreme heterogeneity in the sizes of the RNA fragments, as revealed by centrifugation in sucrose gradients or electrophoresis in polyacrylamide gels. Two principal classes of RNA fragments are present: a. A larger class comprising 74.9–85.4 per cent of total RNA and consisting of fragments having molecular weights ranging from 0.5×10(6) to considerably greater than 3.0×10(6) daltons and, b. A smaller class comprising 9.1–19.7 per cent of total RNA with the size approximately that of ribosomal 4S RNA. All IBV RNA's were fully susceptible to ribonuclease and had a buoyant density in caesium sulphate identical to that of tobacco mosaic virus RNA. No difference in the RNA profile for IBV was observed from the use of different methods of virus purification. The single-stranded RNA's of poliovirus and tobacco mosaic virus remained undegraded after preparation in the presence of IBV. |
4,692 | Determination of different antigenic sites on the adenovirus hexon using monoclonal antibodies | Eighteen mouse ascitic fluids containing monoclonal antibodies (MAbs) directed against crystallized hexon of adenovirus (AV) type I were used to map the antigenic structure of the capsomer in reciprocal competitive binding ELISA. With the help of peroxidase-labelled MAbs at least nine epitopes (epitope clusters) located on three distinct antigenic sites were identified on the hexon. Epitope on antigenic site I recognized by two MAbs could be the genus specific antigenic determinant based on the broad reactivity patterns of the MAbs. Epitopes on the antigenic site II recognized by fifteen MAbs could be divided into seven epitope clusters according to the competition patterns. Antigenic site III recognized by one MAb completely differs from the antigenic site I and on the basis of one-way blocking with all the MAbs specific for antigenic site II, should be also different from the latter one. The data suggest that the seven epitope clusters of antigenic site II contain partially overlapping epitopes and may be a part of a large single immunodominant antigenic region on AV1 hexon as well as on hexons of heterologous types. |
4,693 | Replication of sialodacryoadenitis virus of rat in LBC cell culture | Sialodacryoadenitis virus of rat readily propagated and induced marked cytopathic effect in a rat cell line, LBC cell culture, which provides a sensitive, practical assay system for viral infectivity and neutralizing antibody, and a satisfactory source of the virus. |
4,694 | A new typing method for the avian infectious bronchitis virus using polymerase chain reaction and restriction enzyme fragment length polymorphism | Two primers with the length of 22 bases each and 400 bases apart on the spike protein gene of avian infectious bronchitis virus (IBV) were prepared. Using these primers, the genome RNA from twelve strains of the various serotypes were reverse-transcribed to cDNA and amplified by polymerase chain reaction (PCR). With all strains, 400 base DNA was amplified, indicating that there were no apparent insertions or deletions in this region. However, the amplified DNA showed different cleavage patterns by the restriction enzymes. These 12 strains were classified into 5 groups. The strain typing based on a comparison of the cleavage patterns was consistent with the previous serological typing. This study thus provides a simple and rapid method for typing of IBV. |
4,695 | Sequence of the coding regions from the 3.0 kb and 3.9 kb mRNA: Subgenomic species from a virulent isolate of transmissible gastroenteritis virus | Subgenomic mRNA from a virulent isolate of porcine transmissible gastroenteritis virus (TGEV) was used to produce cDNA clones covering the genome region from the 3′ end of the pelomer gene to the start of the integral membrane protein gene. The nucleotide sequence of this area was determined using clone pTG11 and a previously reported cDNA clone pTG22. Three open reading frames (ORFs) were identified encoding putative polypeptides of relative molecular masses (M(r)) 6,600, 27,600, and 9,200. The sequence encoding the M(r) 9,200 polypeptide was found to be present on the “unique” 5′ region of the 3.0 kb mRNA species whereas the other two ORFs mapped on the 3.9 kb mRNA species. Differences between the ORFs from this strain of TGEV and those from a previously reported avirulent strain of TGEV were compared. |
4,696 | Comparative biological characterization of mouse adenovirus strains FL and K 87 and seroprevalence in laboratory rodents | The growth, stability and seroprevalence in laboratory rodents of the two known strains of mouse adenovirus were compared. The FL strain of mouse adenovirus grew in both L 929 murine fibroblasts and in CMT-93 murine rectal carcinoma cells, whereas the K 87 strain grew only in CMT-93 cells. The bulk of the FL progeny virus was released from the host cells. K 87 virus was largely cell-associated. Both virus strains were stable at 37° C in liquid medium. The K 87 strain was completely inactivated after 5–15 minutes at 56° C, whereas FL infectivity was still detected after two hours at this temperature. Both virus strains were stable in the dessicated state for 14 days, although FL viability was more dependent on the presence of protein in the virus diluent. Seroepidemiologic data suggest that viruses antigenically related to mouse adenovirus are more prevalent among laboratory rats than among laboratory mice and that the virus(es) infecting rats differ from those infecting mice. Results of retrospective serologic testing suggest an association between mouse adenovirus and an outbreak of disease in a mouse breeding colony. |
4,697 | Modulation of lethal and persistent rat parvovirus infection by antibody | Two day-old athymic (rnu/rnu) and euthymic (rnu/+) rat pups nursing immune or non-immune dams were inoculated oronasally with the Yale strain of rat virus (RV-Y). All athymic and euthymic pups (57/57) from immune dams remained clinically normal, whereas 51 of 66 athymic and euthymic pups from non-immune dams died within 30 days. Infectious RV was detected by explant culture in 12 of 15 surviving pups of both genotypes from non-immune dams 30 days after inoculation, but in none of the 57 surviving pups from immune dams. RV-Y DNA was detected by Southern blotting in kidneys of surviving athymic pups from non-immune dams but was not detected in pups from immune dams. Euthymic pups from immune dams appeared not to produce endogenous antibody to RV after virus challenge, whereas euthymic pups from non-immune dams produced high-titered RV immune serum. Pups of both genotypes given immune serum prior to or with RV were fully protected from disease and persistent infection, whereas pups given immune serum 24 hours after RV were partially protected. These studies show that RV antibody offers significant protection against lethal and persistent RV infection. |
4,698 | Growth of fastidious adenovirus serotype 40 in HRT 18 cells: Interactions with E 1 A and E 1 B deletion mutants of subgenus C adenoviruses | Growth of fastidious adenovirus serotype 40 (Ad 40) in several cell lines was investigated. Ad 40 was able to readily propagate in human intestinal cell line, HRT 18. Coinfection assays were made in non-permissive and permissive cells between Ad 40 and Ad 5dl 312 or dl 1520, mutants deleted in E 1 A and E 1 B regions, respectively, to test the ability of Ad 40 to complement these mutants and vice versa. Ad 40 could enhance Ad 5dl 312 DNA synthesis in HRT 18 and HeLa cells, although its own DNA disappeared in the presence of this mutant in HRT 18 cells. In coinfection with dl 1520, Ad 40 DNA synthesis was inhibitied by dl 1520 in HRT 18 cells and dl 1520 DNA synthesis was inhibited by Ad 40 in 293 cells. This might reflect the presence of unusual products encoded by Ad 40 E 1 B region. |
4,699 | Replication of two porcine parvovirus isolates at non-permissive temperatures | Previous studies have shown that replication in vitro of the porcine parvovirus (PPV) isolate, KBSH, was restricted at 39°C but not at 37°C. In contrast, replication of the Kresse isolate was restricted at 37°C but not at 39°C. In this study, Kresse and KBSH isolates were passaged up to ten times in swine testicle (ST) cells at non-permissive temperatures, and at subsequent passage viral protein synthesis, viral DNA synthesis, and progeny virus were evaluated. KBSH became adapted for replication at 39°C upon serial passages, displaying an appreciable increase in viral progeny, viral polypeptides, and viral DNA concentration. This finding was also observed with Kresse virus isolate continuously passaged at 37°C. Neither isolate became adapted for replication at 32°C. In an attempt to examine the effect of in vitro passage at non-permissive temperatures on pathogenicity in swine, KBSH passaged 10 times either at 37°C or 39°C was inoculated into swine fetuses. Two of four fetuses inoculated with 39°C-passaged KBSH were dead and hemorrhagic or mummified. All four fetuses inoculated with 39°C-KBSH contained viral antigen and viral DNA. In contrast, fetuses inoculated with 37°C-passaged KBSH, or with cell culture fluid were normal in appearance. Viral antigen and viral DNA were not demonstrated in fetuses inoculated with 37°C-KBSH or cell culture fluids. These findings suggest the possibility that the ability to replicate at 39°C is associated with virulence in swine fetuses. |
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