Purification,electron microscopy and serology of virus isolated fromEuonymus europaea

“Euonymus mosaic virus”, purified from cucumber cotyledons by the differential and density-gradient centrifugation, shows typical nucleoprotein absorption spectrum. Electron microscopy reveals isometric virus particles of about 37 nm diameter. No reaction of purified “Euonymus mosaic virus” was observed with antisera against a raspberry ringspot virus, tobacco ringspot virus, cherry leaf roll virus, strawberry latent ringspot virus, tomato ringspot virus, elm mosaic virus, arabis mosaic virus, tomato bushy stunt virus and watermelon mosaic virus.     

Four Categories of Viral Infection Describe the Health Status of Honey Bee Colonies

Honey bee virus prevalence data are an essential prerequisite for managing epidemic events in a population. A survey study was carried out for seven viruses in colonies representing a healthy Danish honey bee population. In addition, colonies from apiaries with high level Varroa infestation or high level of winter mortality were also surveyed. Results from RT-qPCR showed a considerable difference of virus levels between healthy and sick colonies. In the group of healthy colonies, no virus was detected in 36% of cases, while at least one virus was found in each of the sick colonies. Virus titers varied among the samples, and multiple virus infections were common in both groups with a high prevalence of Sacbrood virus (SBV), Black queen cell virus (BQCV) and Deformed wing virus (DWV). Based on the distribution of virus titers, we established four categories of infection: samples free of virus (C = 0), samples with low virus titer (estimated number of virus copies 0 < C < 10³), samples with medium virus titer (10³ ≤ C < 10⁷) and samples with high virus titer (C ≥ 10⁷). This allowed us to statistically compare virus levels in healthy and sick colonies. Using categories to communicate virus diagnosis results to beekeepers may help them to reach an informed decision on management strategies to prevent further spread of viruses among colonies.     

Arabidopsis thaliana as a model for the study of plant–virus co-evolution

Understanding plant–virus coevolution requires wild systems in which there is no human manipulation of either host or virus. To develop such a system, we analysed virus infection in six wild populations of Arabidopsis thaliana in Central Spain. The incidence of five virus species with different life-styles was monitored during four years, and this was analysed in relation to the demography of the host populations. Total virus incidence reached 70 per cent, which suggests a role of virus infection in the population structure and dynamics of the host, under the assumption of a host fitness cost caused by the infection. Maximum incidence occurred at early growth stages, and co-infection with different viruses was frequent, two factors often resulting in increased virulence. Experimental infections under controlled conditions with two isolates of the most prevalent viruses, cauliflower mosaic virus and cucumber mosaic virus, showed that there is genetic variation for virus accumulation, although this depended on the interaction between host and virus genotypes. Comparison of Q_ST-based genetic differentiations between both host populations with F_ST genetic differentiation based on putatively neutral markers suggests different selection dynamics for resistance against different virus species or genotypes. Together, these results are compatible with a hypothesis of plant–virus coevolution.     

Effect of the Deletion of Genes Encoding Proteins of the Extracellular Virion Form of Vaccinia Virus on Vaccine Immunogenicity and Protective Effectiveness in the Mouse Model

Antibodies to both infectious forms of vaccinia virus, the mature virion (MV) and the enveloped virion (EV), as well as cell-mediated immune response appear to be important for protection against smallpox. EV virus particles, although more labile and less numerous than MV, are important for dissemination and spread of virus in infected hosts and thus important in virus pathogenesis. The importance of the EV A33 and B5 proteins for vaccine induced immunity and protection in a murine intranasal challenge model was evaluated by deletion of both the A33R and B5R genes in a vaccine-derived strain of vaccinia virus. Deletion of either A33R or B5R resulted in viruses with a small plaque phenotype and reduced virus yields, as reported previously, whereas deletion of both EV protein-encoding genes resulted in a virus that formed small infection foci that were detectable and quantifiable only by immunostaining and an even more dramatic decrease in total virus yield in cell culture. Deletion of B5R, either as a single gene knockout or in the double EV gene knockout virus, resulted in a loss of EV neutralizing activity, but all EV gene knockout viruses still induced a robust neutralizing activity against the vaccinia MV form of the virus. The effect of elimination of A33 and/or B5 on the protection afforded by vaccination was evaluated by intranasal challenge with a lethal dose of either vaccinia virus WR or IHD-J, a strain of vaccinia virus that produces relatively higher amounts of EV virus. The results from multiple experiments, using a range of vaccination doses and virus challenge doses, and using mortality, morbidity, and virus dissemination as endpoints, indicate that the absence of A33 and B5 have little effect on the ability of a vaccinia vaccine virus to provide protection against a lethal intranasal challenge in a mouse model.     

Beet curly top virus transmission by artificially fed and injected beet leafhoppers (Circulfer tenellus)

The transmission of beet curly top virus (BCTV) by leafhoppers, Circulifer tenellus, fed virus through Parafilm® membranes was compared with their transmission when injected with virus from phloem exudates of Amsinckia douglasiana. Virus uptake from ³²P-labelled test solutions and the resulting virus transmission, as measured by an infectivity index, varied widely. By contrast, insects injected with virus transmitted with similar efficiencies. If insects were fasted for 3, 5, or 7 h before a 6 h acquisition access period on test solutions, their ³²P, and presumably virus uptake, was greater than that of nonfasted insects and their variability in virus transmission decreased. The proportion of insects transmitting curly top virus, after fasting and given a 6 h acquisition access period, was similar to that of insects injected with virus. Maximum liquid uptake by the beet leafhopper occurred with a 12% sucrose solution.     

Effects of single and mixed infections with wild type and genetically modified Helicoverpa armigera nucleopolyhedrovirus on movement behaviour of cotton bollworm larvae

Naturally occurring insect viruses can modify the behaviour of infected insects and thereby modulate virus transmission. Modifications of the virus genome could alter these behavioural effects. We studied the distance moved and the position of virus‐killed cadavers of fourth instars of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) infected with a wild‐type genotype of H. armigera nucleopolyhedrovirus (HaSNPV) or with one of two recombinant genotypes of this virus on cotton plants. The behavioural effects of virus infection were examined both in larvae infected with a single virus genotype, and in larvae challenged with mixtures of the wild‐type and one of the recombinant viruses. An egt‐negative virus variant caused more rapid death and lower virus yield in fourth instars, but egt‐deletion did not produce consistent behavioural effects over three experiments, two under controlled glasshouse conditions and one in field cages. A recombinant virus containing the AaIT‐(Androctonus australis Hector) insect‐selective toxin gene, which expresses a neurotoxin derived from a scorpion, caused faster death and cadavers were found lower down the plant than insects infected with unmodified virus. Larvae that died from mixed infections of the AaIT‐expressing recombinant and the wild‐type virus died at positions significantly lower, compared to infection with the pure wild‐type viral strain. The results indicate that transmission of egt‐negative variants of HaSNPV are likely to be affected by lower virus yield, but not by behavioural effects of egt gene deletion. By contrast, the AaIT recombinant will produce lower virus yields as well as modified behaviour, which together can contribute to reduced virus transmission under field conditions. In addition, larvae infected with both the wild‐type virus and the toxin recombinant behaved as larvae infected with the toxin recombinant only, which might be a positive factor for the risk assessment of such toxin recombinants in the environment.     

Attenuation of Tick-Borne Encephalitis Virus Using Large-Scale Random Codon Re-encoding

Large-scale codon re-encoding (i.e. introduction of a large number of synonymous mutations) is a novel method of generating attenuated viruses. Here, it was applied to the pathogenic flavivirus, tick-borne encephalitis virus (TBEV) which causes febrile illness and encephalitis in humans in forested regions of Europe and Asia. Using an infectious clone of the Oshima 5–10 strain ("wild-type virus"), a cassette of 1.4kb located in the NS5 coding region, was modified by randomly introducing 273 synonymous mutations ("re-encoded virus"). Whilst the in cellulo replicative fitness of the re-encoded virus was only slightly reduced, the re-encoded virus displayed an attenuated phenotype in a laboratory mouse model of non-lethal encephalitis. Following intra-peritoneal inoculation of either 2.10⁵ or 2.10⁶ TCID50 of virus, the frequency of viraemia, neurovirulence (measured using weight loss and appearance of symptoms) and neuroinvasiveness (detection of virus in the brain) were significantly decreased when compared with the wild-type virus. Mice infected by wild-type or re-encoded viruses produced comparable amounts of neutralising antibodies and results of challenge experiments demonstrated that mice previously infected with the re-encoded virus were protected against subsequent infection by the wild-type virus. This constitutes evidence that a mammalian species can be protected against infection by a virulent wild-type positive-stranded RNA virus following immunisation with a derived randomly re-encoded strain. Our results demonstrate that random codon re-encoding is potentially a simple and effective method of generating live-attenuated vaccine candidates against pathogenic flaviviruses.     

A comparison of the effectiveness of the four British virus vector species of Longidorus and Xiphinema

The frequency with which the four virus-vector species of longidoroid nematodes occurring in Britain transmitted their associated plant viruses were compared in a series of experiments using a standard procedure. In these tests Xiphinema diversicaudatum proved an effective vector of British isolates of arabis mosaic virus and strawberry latent ringspot virus and Longidorus attenuatus of an isolate of tomato black ring virus from England. In comparison, isolates of raspberry ringspot virus and tomato blackring virus from Scotland and of raspberry ringspot virus from England were transmitted much less readily by their respective vectors, L. elongatus and L. macrosoma. These differences in ability to transmit virus were not related to differences in feeding access on the virus source- or bait-plants, in the extent to which virus was retained within the nematode feeding apparatus or in the frequency with which virus was recovered from Longidorus in concurrent slash tests. Three Scottish isolates of raspberry ringspot and tomato black ring viruses were transmitted equally infrequently by two populations of L. elongatus and the frequency with which virus was transmitted was not greatly increased when the species of source or bait plants was changed.     

Isolation, characterisation and identification of a potyvirus from Dioscorea alata L. (water yam) in Nigeria

A yam potyvirus was isolated from Dioscorea alata samples collected in Nigeria. The virus was not transmissible mechanically but was transmitted by Aphis craccivora to four cowpea cultivars (Ife Brown, IT84S-2114, IT82E-10 and TVu2657), and from which it could be mechanically transmitted between the cowpea cultivars. In infectivity- tests using cowpea extracts, the virus had a dilution end point of 10^-4, a thermal inactivation point of 60–65°C and longevity in vitro of 2 days at room temperature. The virus coat protein had an estimated molecular weight of 32 100 daltons. The virus was identified as an isolate of Dioscorea alata virus (DAV; syn. yam virus 1) due to its biological characteristics and its serological reaction with antiserum raised against DAV. The virus is not related to yam mosaic virus, but distantly related to blackeye cowpea mosaic virus and cowpea aphid-borne mosaic virus.     

Cryptoporus volvatus Extract Inhibits Influenza Virus Replication In Vitro and In Vivo

Influenza virus is the cause of significant morbidity and mortality, posing a serious health threat worldwide. Here, we evaluated the antiviral activities of Cryptoporus volvatus extract on influenza virus infection. Our results demonstrated that the Cryptoporus volvatus extract inhibited different influenza virus strain replication in MDCK cells. Time course analysis indicated that the extract exerted its inhibition at earlier and late stages in the replication cycle of influenza virus. Subsequently, we confirmed that the extract suppressed virus internalization into and released from cells. Moreover, the extract significantly reduced H1N1/09 influenza virus load in lungs and dramatically decreased lung lesions in mice. And most importantly, the extract protected mice from lethal challenge with H1N1/09 influenza virus. Our results suggest that the Cryptoporus volvatus extract could be a potential candidate for the development of a new anti-influenza virus therapy.     

cis-acting genomic elements and trans-acting proteins involved in the assembly of RNA viruses

There is now considerable evidence that a specific site (or sites) in the genome of an RNA virus interacts with a viral protein to initiate the assembly of the virus ribonucleoprotein or nucleocapsid. We describe the progress that has been made in defining these elements for a number of different viruses: the togavirus, Sindbis virus; the coronavirus, mouse hepatitis virus; influenza A virus; several retroviruses; and the hepadnavirus, hepatitis B virus. The importance of cis-acting elements in packaging has been established for all of these viruses. For Sindbis virus, specificity in the binding of the RNA element to a region of the viral capsid protein in vitro has also been demonstrated.     

A serum-free Vero production platform for a chimeric virus vaccine candidate

MedImmune Vaccines has engineered a live, attenuated chimeric virus that could prevent infections caused by parainfluenza virus type 3 (PIV3) and respiratory syncytial virus (RSV), causative agents of acute respiratory diseases in infants and young children. The work here details the development of a serum-free Vero cell culture production platform for this virus vaccine candidate. Efforts to identify critical process parameters and optimize culture conditions increased infectious virus titers by approximately 2 log₁₀ TCID₅₀/ml over the original serum-free process. In particular, the addition of a chemically defined lipid concentrate to the pre-infection medium along with the shift to a lower post-infection cultivation temperature increased virus titers by almost 100-fold. This improved serum-free process achieved comparable virus titers to the serum-supplemented process, and demonstrated consistent results upon scale-up: Vero cultures in roller bottles, spinner flasks and bioreactors reproducibly generated maximum infectious virus titers of 8 log₁₀ TCID₅₀/ml.     

Mise en évidence et étude d'un complexe de maladies aParvovirus,baculovirus etIridovirus

Résumé Une maladie à virus complexe due à un virus de densonucléose, à une polyèdrie nucléaire et à un virus irisant a été observée chezGalleria mellonella et étudiée en histologie et en microscopie électronique. Les 3 virus sont capables de se multiplier simultanément dans la même chenille, le même tissu et la même cellule. Les altérations cytopathologiques sont analogues à celles provoquées par chacun de ces virus.

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Summary A virus disease complex induced by a densonucleosis virus, a nuclear polyhedrosis and an iridescent virus was observed inGalleria mellonella and studied by histology and electron microscopy. The 3 viruses are liable to multiply simultaneously in the same individual, the same tissue, and the same cell. The observed cytological injuries are similar to those noticed in each of these virus diseases.

     

Physicochemical properties of tipula iridescent virus

The molecular weight of Tipula iridescent virus, based on sedimentation and diffusion coefficients, was 5.51 × 10⁸, with hydration of 0.57 g of water per g of virus. Deoxyribonucleic acid content, based on total inorganic phosphorus liberated, was 19 ± 0.2%. At 260 mμ, the virus gave an uncorrected absorbance of 18.2 cm²/mg of virus and a light-scattering corrected absorbance of 9.8 cm²/mg of virus. Amino acid analyses of the virus protein revealed a remarkable similarity to Sericesthis iridescent virus. The possibility is discussed that the four iridescent insect viruses reported to date bear a strain relationship.     

Beziehungen zwischen dem Wirkungsgrad antiphytoviraler Substanzen und dem Niveau der quantitativen Sortenresistenz sowie der Virulenz von Virusisolaten an Virus/Wirt-Systemen der Kartoffel

Relationship between the efficiency of antiphytoviral substances, the degree of quantitative resistance of cultivars, and the virulence of virus isolates on virus/host-systems of potato. In vitro systematically infected with potato virus S, potato virus M or potato virus X stem cuttings of different potato varieties were treated with 2,4-dioxohexahydro-1,3,5-triazine and Ribavirin. The reduction of virus replication by chemotherapy differed between varieties depending on their level of quantitative resistance and the virulence of virus isolates. An increasing resistance level of cultivars and a decreasing virulence of the virus isolates resulted in a relative enhancement of the inhibitory of the antiphytoviral substances.     

Purification and Identification of a South African Isolate of Watermelon Mosaic Virus – Morocco

Cucurbit crops in South Africa are seriously affected by a flexuous rod-shaped virus 706 to 770 nm long which causes the plant to be stunted, the leaves to display symptoms of chlorotic mosaic, dark green blisters and malformation, and fruit to be malformed. The virus was purified from infected Cucurbita pepo by extraction in 0.5 M borate buffer, pH 8, containing ethylenediaminotetra-acetic acid and mercapto-ethanol, clarification with chloroform, addition of Triton X-100, sedimentation by ultracentrifugation for which a sucrose cushion was used and centrifugation in 10 to 40 % sucrose gradients. The virus was mechanically transmitted to a limited host range with Chenopodium album, C. amaranticolor, C. quinoa and Gomphrena globosa being the only hosts infected outside the Cucurbitaceae. Luffa cylindrica, Cucumis metuliferus, Coccinia sessilifolia and Citrullus ecirrhosus all members of the Cucurbitaceae, were not infected by the virus. The virus was non-persistently transmitted by Myzus persicae, produced pinwheel and bundle inclusions in the plant cell cytoplasm and has a single coat protein with a molecular weight of 36,000 daltons and a degraded lighter component of 26,000 daltons. Serological comparisons with antiserum to watermelon mosaic virus 2, Papaya ringspot virus strain W and watermelon mosaic virus Morocco (WMV-Mor.) identified the virus as an isolate of WMV-Mor. It was found that WMV-Mor. is the dominant virus in all the main cucurbit producing areas of South Africa which were surveyed.     

Elevated viral antibody titres in spontaneous abortion

Abstract To assess the possible role of virus infections in spontaneous abortion we undertook a prospective study of three groups of patients: women who had a spontaneous abortion; women seen in the ante-natal booking clinic; and those undergoing therapeutic termination of pregnancy. Venous blood was taken for antibody detection, cervical swabs for virus isolation, and in the cases of spontaneous and therapeutic abortion products of conception were sent for virus culture. The infectious agents studied were influenza A and B, adenovirus, respiratory syncytial virus, measles, psittacosis, Varicella zoster , and mumps virus. Statistical analysis revealed that women in the spontaneous abortion group has a higher incidence of antibodies against respiratory syncytial virus ( P <0.01), and mumps virus ( P <0.01). Among women seropositive for each virus, higher titres were found only for respiratory syncytial virus ( P <0.05) in cases rather than controls. These results could be explained if viruses such as respiratory syncytial virus caused abortion or if women having a spontaneous abortion mount exaggerated immune responses to fetal adn viral infections, and the former altered immune response is associated with spontaneous abortion.     

Nora Virus Persistent Infections Are Not Affected by the RNAi Machinery

Drosophila melanogaster is widely used to decipher the innate immune system in response to various pathogens. The innate immune response towards persistent virus infections is among the least studied in this model system. We recently discovered a picorna-like virus, the Nora virus which gives rise to persistent and essentially symptom-free infections in Drosophila melanogaster. Here, we have used this virus to study the interaction with its host and with some of the known Drosophila antiviral immune pathways. First, we find a striking variability in the course of the infection, even between flies of the same inbred stock. Some flies are able to clear the Nora virus but not others. This phenomenon seems to be threshold-dependent; flies with a high-titer infection establish stable persistent infections, whereas flies with a lower level of infection are able to clear the virus. Surprisingly, we find that both the clearance of low-level Nora virus infections and the stability of persistent infections are unaffected by mutations in the RNAi pathways. Nora virus infections are also unaffected by mutations in the Toll and Jak-Stat pathways. In these respects, the Nora virus differs from other studied Drosophila RNA viruses.