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Biophysical studies of vesicular stomatitis virus.

McCombs, Robert M. (Baylor University College of Medicine, Houston, Tex.), Matilda Benyesh-Melnick, and Jean P. Brunschwig. Biophysical studies of vesicular stomatitis virus: J. Bacteriol. 91:803-812. 1966.-The infectivity and morphology of vesicular stomatitis virus (VSV) were studied after density gradient centrifugation in cesium chloride (CsCI), potassium tartrate (KT), and sucrose. Centrifugation in CsCl revealed two equally infectious bands corresponding to densities of 1.19 and 1.22 g/ml, and a third (density, 1.26 g/ml) band of low infectivity. Two bands (densities of 1.16 and 1.18 g/ml) were observed in the KT gradient, in which the lighter band contained most of the infectivity. Centrifugation in sucrose resulted in a single broad infectious band (density, 1.16 g/ml). The typical rod-shaped VSV particles were found mainly in the lighter bands obtained in CsCl (1.19 g/ml) and KT (1.16 g/ml) and in the single sucrose gradient band (1.16 g/ml). Bent particles equally as infectious as the rod-shaped particles were a constant finding in the CsCl preparations, and were observed mainly in the second band (density, 1.19 g). Numerous strands 15mmu wide were found in the third CsCl (density, 1.26 g/ml) and the second KT (1.18 g/ml) bands. Similar strands could be liberated from VSV particles after treatment with deoxycholate. Internal transverse striations were found to be a regular feature of VSV particles examined with the pseudoreplication negative-staining technique. For crude virus stocks, the physical particle-to-infectivity ratio ranged from 73 to 194. Several morphological similarities between VSV and myxoviruses were observed, including 10 mmu surface projections, pleomorphic morphological forms, and 15 mmu seemingly nucleoprotein strands.

Biophysical Phenomena↗

Expression of MxA protein in blood lymphocytes discriminates between viral and bacterial infections in febrile children.

Interferons (IFNs), which are induced by viruses, form an essential part of host's defense systems against viral infections. The antiviral actions of IFNs are mediated by several IFN-inducible gene products, one of which is Mx protein. To evaluate whether MxA protein expression in lymphocytes could function as an indicator of endogenous IFN production in children with acute febrile illness, we analyzed MxA protein levels in peripheral blood lymphocytes by flow cytometry in the acute phase of the disease. Children with a laboratory-confirmed viral infection [respiratory syncytial virus (RSV) in 21, adenovirus in 10, rotavirus in 5, and influenza, herpes simplex, or EBV in 7 other cases] had significantly higher (p < 0.002) MxA protein levels (median fluorescences in different virus groups ranged from 707 to 765) compared with children with a bacterial infection (n = 12, median fluorescence 548). To characterize further MxA protein expression during infections, cells from 41 patients were stimulated in vitro with exogenous IFN-alpha, and the level of MxA protein expression was determined. The rise in MxA staining levels was significantly higher in the group with bacterial infections compared with those with viral infection (p < 0.005), further indicating that the MxA protein levels were already elevated in vivo in patients with viral infections. This study suggests that elevated MxA protein expression levels can be used in the differential diagnosis of bacterial versus viral disease in febrile children.

Adenoviridae Infections↗

Antiviral effects of geranylgeranylacetone: enhancement of MxA expression and phosphorylation of PKR during influenza virus infection.

A cyclic polyisoprenoid compound, geranylgeranylacetone (GGA), has been used as antiulcer drug. GGA is also a potent inducer of heat shock proteins (HSPs). HSPs are considered to induce an antiviral effect; however, the detailed mechanism is unknown. To determine whether GGA might show antiviral activity and what the mechanism is, the effect of GGA against influenza virus (strain PR8) infection in vivo and in vitro was investigated. The results demonstrated that GGA treatment strongly suppressed the deleterious consequences of PR8 replication and was accompanied by an increase in HSP70 gene expression in mice. Results from in vitro analyses demonstrated that GGA significantly inhibited the synthesis of PR8-associated proteins and prominently enhanced expression of human myxovirus resistance 1 (MxA) followed by increased HSP70 transcription. Moreover, GGA augmented the expression of an interferon-inducible double-strand RNA-activated protein kinase (PKR) gene and promoted PKR autophosphorylation and concomitantly alpha subunit of eukaryotic initiation factor 2 phosphorylation during PR8 infection. It is proposed that GGA-induced HSP70 has potent antiviral activity by enhancement of antiviral factors and can clinically achieve protection from influenza virus infection.

Animals↗

Effect of poly I:C on the expression of Mx proteins and resistance against infection by infectious salmon anaemia virus in Atlantic salmon.

Mx proteins are induced by type I interferons (IFN alpha and beta) in mice and humans and inhibit the replication of orthomyxoviruses and some other single-stranded RNA viruses. Recently, Mx genes have been cloned from Atlantic salmon. Mx transcripts were shown to be induced in head-kidney, liver and gills of the fish by the synthetic double-stranded RNA polyinosinic polycytidylic acid (poly I:C). In the present work we have studied expression of Mx protein in organs of Atlantic salmon treated with poly I:C. A quantitative immunoblot method was established to monitor expression of Mx protein and to compare relative amounts of Mx protein in different organs. Treatment of Atlantic salmon with poly I:C increased the relative amount of Mx protein in liver, stomach, hindgut, head-kidney and spleen. In gills the levels of Mx protein were similar in control fish and poly I:C treated fish. Immunohistochemistry of tissue sections from liver, head-kidney and gills from poly I:C treated fish was in accordance with the immunoblotting data and showed staining for Mx protein in several different cell types. Classification of infectious salmon anaemia virus as an orthomyxovirus makes it a putative target for Atlantic salmon Mx protein. Atlantic salmon treated with poly I:C showed reduced cumulative mortality compared to the control fish when challenged with infectious salmon anaemia virus (ISAV) by intraperitoneal injection. This demonstrates that poly I:C has some protective effect against ISAV in vivo.

Anemia↗

Role and fate of PML nuclear bodies in response to interferon and viral infections.

Interferons (IFNs) are a family of secreted proteins with antiviral, antiproliferative and immunomodulatory activities. The different biological actions of IFN are believed to be mediated by the products of specifically induced cellular genes in the target cells. The promyelocytic leukaemia (PML) protein localizes both in the nucleoplasm and in matrix-associated multi-protein complexes known as nuclear bodies (NBs). PML is essential for the proper formation and the integrity of the NBs. Modification of PML by the Small Ubiquitin MOdifier (SUMO) was shown to be required for its localization in NBs. The number and the intensity of PML NBs increase in response to interferon (IFN). Inactivation of the IFN-induced PML gene by its fusion to retinoic acid receptor alpha alters the normal localization of PML from the punctuate nuclear patterns of NBs to micro-dispersed tiny dots and results in uncontrolled growth in Acute Promyelocytic Leukaemia. The NBs-associated proteins, PML, Sp100, Sp140, Sp110, ISG20 and PA28 are induced by IFN suggesting that nuclear bodies could play a role in IFN response. Although the function of PML NBs is still unclear, some results indicate that they may represent preferential targets for viral infections and that PML could play a role in the mechanism of the antiviral action of IFNs. Viruses, which require the cellular machinery for their replication, have evolved different ways to counteract the action of IFN by inhibiting IFN signalling, by blocking the activities of specific antiviral mediators or by altering PML expression and/or localization on nuclear bodies.

Animals↗

Lyssavirus infection activates interferon gene expression in the brain.

To investigate the innate immune response within the brain to lyssavirus infection, key transcripts indicative of innate defences were measured in a mouse model system. Following infection with Rabies virus, transcript levels for type 1 interferons (IFN-alpha and -beta), the inflammatory mediator interleukin 6 (IL-6) and the antiviral protein Mx1 increased in the brains of mice. Intracranial inoculation resulted in the early detection of virus replication and rapid expression within the brain of the innate immune response genes. Transcripts for type 1 IFNs declined as the disease progressed. Peripheral, extraneural inoculation delayed the host response until virus entered the brain, but then resulted in a large increase in the level of IFN-beta, IL-6 and Mx1 transcripts. Induction of this response was also observed following infection with the related European bat lyssaviruses, a group of zoonotic viruses capable of causing fatal, rabies-like disease in mammalian species.

Animals↗

A naturally occurring variant of porcine Mx1 associated with increased susceptibility to influenza virus in vitro.

Mx1 has been implicated in resistance to the influenza virus. We have now identified four alleles of the Mxl gene in domesticated breeds of pigs. Two of the alleles encode deletion variants (a 3-bp deletion in exon 13 and an 11-bp deletion in exon 14), which might be expected to interfere with Mx activity. The porcine Mxl genes corresponding to wild type, the 3-bp deletion mutant, and the 11-bp deletion mutant were cloned and expressed in NIH3T3 cells, and the antiviral activity for influenza virus was assayed. Virus yield was observed to be 10-100-fold greater with the 11-bp deletion allele than that for wild type and the 3-bp deletion alleles. The results suggest that the 11-bp deletion type is lacking antiviral activity able to contribute to the interference of influenza virus replication.

Amino Acid Sequence↗

Antiviral activity in sera of patients with influenza infection.

An interferon like activity was investigated in the sera of patients, very early during an Influenza A or B infection. A such substance was significantly more frequent among the patients who shed a Myxovirus Influenza than among the patients without virus isolation in the nasopharynx. The following relations are discussed : either is the interferon production merely associated without effect on the virus level? Or, on the other hand, in some cases is the rate or virus multiplication strictly directed by this interferon production? This study don't allowed to choose one of these hypothesis.

Animals↗

Interferon mediated antiviral activity against salmonid fish viruses in BF-2 and other cell lines.

Double-stranded RNA and type I interferon-like activity induce an antiviral state in vertebrate cells and in several fish cell lines by increasing the expression of proteins that inhibit virus replication. We compared the protection induced by the polyinosinic:polycytidylic acid (poly I:C) or poly I:C plus transfection agents against the infectious pancreatic necrosis virus (IPNV) and the infectious hematopoietic necrosis virus (IHNV) in BF-2 cells, with that induced in RTG-2, CHSE-214, or SAF cells. In addition, we examined the reduction in the infective titers of these viruses and the correlation with Mx protein expression as IFN marker. Furthermore, the suitability of BF-2 cells for the evaluation and optimization of immune responses in an IPNV-IHNV co-infection was assessed. The results demonstrated strong anti-IPNV and anti-IHNV activity (around 90% of infected cells surviving) in BF-2 cells transfected with poly I:C, in which a loss of 1log(10) or 3log(10) of the IPNV or IHNV infective titers, respectively, was observed. No antiviral activity was evident in the cells incubated with poly I:C alone. The protection recorded in the co-infection experiments was comparable with those of the single infections. The SAF cell line exhibited the lowest antiviral capacity (45%), which was also increased after transfection with poly I:C. In addition, medium from transfected BF-2 provided protection against IPNV (1log(10) loss of infective titer) and IHNV (2log(10) loss of infective titer) in new monolayers, indicating that these cells secreted the factors that induce antiviral activity. A correlation between antiviral activity and Mx protein expression was observed in all the cells. These results indicate that poly I:C transfection could improve IFN-like production in these cell lines. However, the antiviral effectiveness of poly I:C differed between cell lines. On the basis of our findings, we conclude that the BF-2 cell line is a useful model in which to study the role of IFN-induced cytokines in resistance against single or double infections with salmonid fish viruses.

Animals↗

Negative contrast electron microscopic techniques for diagnosis of viruses of veterinary importance.

Negative contrast electron microscopy (NCEM) was utilized as a routine tool in the diagnosis of viral infections of domestic and wild animals. Viruses identified by this technique were observed in infected culture systems or clinical specimens from several species including horses, cattle, sheep, dogs, cats, pigs, deer, Rocky Mountain bighorn sheep, antelope, and several avian species. Viruses were identified by NCEM based on their size, morphology, and symmetry and consisted of adenoviruses, herpesviruses, paramyxoviruses, myxoviruses, picornaviruses, parvoviruses, coronaviruses, reoviruses, rotaviruses, and poxviruses. Mixed populations were also readily demonstrable by this technique: the most common mixed infections consisted of coronaviruses and rotaviruses, and picorna- or parvo-viruses with coronaviruses, rotaviruses, herpesviruses, or adenoviruses. Immunoelectron microscopy was also used to serotype viral agents present in the specimens examined. Viruses identified by this technique were bovine rotaviruses, coronaviruses, and herpesviruses, and bovine and equine adenoviruses.

Animals↗

Interferon regulatory factor-1 promoter polymorphism and the outcome of hepatitis C virus infection.

OBJECTIVE: Interferon-alpha (IFN-alpha), an important mediator for the host's innate antiviral defense system, has been approved for the treatment of persistent viral infections. We investigated whether two functional polymorphisms in genes involved in IFN-alpha signaling and effector functions are associated with the natural outcome of hepatitis C virus (HCV) infection and the responsiveness of chronic hepatitis C patients to IFN-alpha therapy. METHODS: Forty-four individuals who had resolved HCV infection spontaneously and 147 patients who developed chronic hepatitis C were analyzed for functional single nucleotide polymorphisms in the promoter regions of the interferon regulatory factor-1 (IRF-1) and myxovirus resistance protein-1 (MxA) genes at positions -300 and -88, respectively. RESULTS: With regard to -300 IRF-1 or -88 MxA genotype distributions or minor allele frequencies, individuals who spontaneously resolved the infection displayed no significant difference compared with those with chronic infections. Among patients with chronic infections, however, the -300AA IRF-1 genotype, associated with a higher IRF-1 transcriptional activity, was absent in patients with chronic HCV genotype 3a infections, with one exception. In contrast to expectations, -300AA IRF-1 individuals with HCV genotype 3a infection were not represented in higher numbers among those with self-limited infections. Regarding IFN-alpha therapy, -300AA IRF-1 chronic hepatitis C genotype 1 patients tend to respond more often than those with the other IRF-1 genotypes. CONCLUSION: Our findings suggest the possibility that the -300AA IRF-1 genotype is associated with outcome in patients with HCV genotype 3 infection. In addition, in HCV genotype-1-infected patients, this genotype appears associated with response to therapy.

Adult↗

ZBITNEW A: UPPER RESPIRATORY DISEASE IN THOUROUGHBRED HORSES: STUDIES OF ITS VIRAL ETIOLOGY IN THE TORONTO AREA, 1960 TO 1963.

From outbreaks of upper respiratory infection of horses in the Toronto area between 1960 and 1963, several viruses have been isolated. The viruses, isolated in tissue cultures or eggs, include an equine strain of Myxovirus parainfluenzae 3; two strains of equine influenza virus, A/equi-1/Prague/56, and A/equi-2/Miami/63; equine rhinopneumonitis virus, and two newly recognized viruses of the horse, equine rhinoviruses. In addition serological evidence suggested a widespread infection with these viruses in the population under study. Because of the identical clinical picture seen and the complex etiology of the disease, immunization against upper respiratory disease of the horse does not appear to be completely feasible at this time.

Animals↗

The biological effects of five feline IFN-alpha subtypes.

IFN-alpha has been shown to induce both antiviral and antiproliferative activities in animals. This report describes the biological activity of five recently identified feline IFN-alpha subtypes expressed in the Chinese hamster ovary (CHO) cell line (rfeIFN-alpha1[CHO], rfeIFN-alpha2[CHO], rfeIFN-alpha3[CHO], rfeIFN-alpha5[CHO] and rfeIFN-alpha6[CHO]) and the feIFN-alpha6 subtype expressed in and purified from Pichia pastoris (rfeIFN-alpha6[P. pastoris]). The rfeIFN-alpha[CHO] subtypes were tested for antiviral activity against either Vesicular stomatitis virus (VSV) or feline calicivirus (FCV) infected feline embryonic fibroblast cell line (AH927) or Crandell feline kidney cell line (CRFK). Antiviral activity was induced against both VSV and FCV infected AH927 cells and VSV infected CRFK cells by all five of the rfeIFN-alpha[CHO] subtypes and rfeIFN-alpha6[P. pastoris]. In addition, the IFN-alpha inducible Mx gene (associated with antiviral activity) was upregulated in vivo 24 h following treatment with rfeIFN-alpha6[P. pastoris], compared to baseline levels seen prior to treatment. All of the rfeIFN-alpha[CHO] subtypes and rfeIFN-alpha6[P. pastoris] exhibited antiproliferative activity in the FeT-J cell line (an IL-2 independent feline T-cell line). Both necrosis and apoptosis were observed in rfeIFN-alpha6[P. pastoris]-treated FeT-J cells. The rfeIFN-alpha3[CHO] subtype consistently exhibited lower antiviral and antiproliferative activity compared to that observed with the other four rfeIFN-alpha[CHO] subtypes. In summary, this paper demonstrates that five previously described feIFN-alpha subtypes induce both antiviral and antiproliferative activities in vitro and are capable of upregulating the feMx gene in vivo.

Animals↗

Association of a new type of cytopathogenic myxovirus with infantile croup.

Viruses producing an unusual "sponge-like" cytopathogenic change in monkey kidney tissue culture were isolated from the pharyngeal swabs of 2 of 12 infants with croup. The infants from whom the viruses were isolated and 3 additional patients developed significant increases in neutralizing or hemagglutination-inhibition and complement-fixing or all 3 varieties of antibody during convalescence. The isolated agents appeared to be similar antigenically. Fluid from infected monkey kidney tissue culture agglutinated chick erythrocytes and in lower titer human "O" red cells. Hemagglutination occurred best at 4 degrees C. and pH 8.0. Agglutination was reversed at 37 degrees C. but resuspension and sedimentation of red cells at 4 degrees C. resulted in a restitution of positive patterns. In addition, the virus was capable of partially removing receptors from the erythrocyte surface, but only when large quantities of virus were incubated with red cells for 24 hours at 37 degrees C. and small doses of hemagglutinin used to test the treated cells. RDE removed both the erythrocyte receptors and the inhibitor for hemagglutinin present in certain normal sera. Low level multiplication occurred at a slow rate in the amniotic cavity of the fertile hen' egg. Gradocol membrane filtration yielded a size of 90 to 135 mmicro. The virus was stable at -70 degrees C. but infectivity was lost after treatment with 20 per cent ether for 15 hours. The properties of the isolated viruses were consistent with those required for classification in the myxovirus group. No antigenic relationship with influenza A, A', B, and C, Newcastle or Sendai viruses was found. The viruses were distinct from mumps virus but the existence of a common antigen was suggested. The high incidence of infection with this new virus in one group of croup patients suggests that it may be at least one of the etiologic agents of this clinical syndrome, but more extensive control studies will be necessary to establish a specific etiologic association. For the present the group will be referred to as CA viruses; i.e., croup-associated viruses.

Antigens↗