Isolation and characterization of a tobacco mosaic virus-inducible myb oncogene homolog from tobacco

Salicylic acid (SA) plays an important role in signaling the activation of plant defense responses against pathogen attack including induction of pathogenesis-related (PR) proteins. To gain further insight into the SA-mediated signal transduction pathway, we have isolated and characterized a tobacco mosaic virus (TMV)-inducible myb oncogene homolog (myb1) from tobacco. The myb1 gene was induced upon TMV infection during both the hypersensitive response and development of systemic acquired resistance in the resistant tobacco cultivar following the rise of endogenous SA, but was not activated in the susceptible cultivar that fails to accumulate SA. The myb1 gene was also induced by incompatible bacterial pathogen Pseudomonas syringae pv. syringae during the hypersensitive response. Exogenous SA treatment rapidly (within 15 min) activated the expression of myb1 in both resistant and susceptible tobacco cultivars with the subsequent induction of PR genes occurring several hours later. Biologically active analogs of SA and 2,6-dichloroisonicotinic acid (a synthetic functional analog of SA), which induce PR genes and enhanced resistance, also activated the myb1 gene. In contrast, biologically inactive analogs were poor inducers of myb1 gene expression. Furthermore, the recombinant Myb1 protein was shown to specifically bind to a Myb-binding consensus sequence found in the promoter of the PR-1a gene. Taken together, these results suggest that the tobacco myb1 gene encodes a signaling component downstream of SA that may participate in transcriptional activation of PR genes and plant disease resistance.

Isolation of virus-neutralizing RNAs from a large pool of random sequences.

RNA and ribonuclease-resistant RNA analogs that bound and neutralized Rous sarcoma virus (RSV) were isolated from a large pool of random sequences by multiple cycles of in vitro selection using infectious viral particles. The selected RNA pool of RSV-binding sequences at a concentration of 0.16 microM completely neutralized the virus. Of 19 sequences cloned from the selected pool, 5 inhibited RSV infection. The selected RNA and RNA analogs were shown to neutralize RSV by interacting with the virus, rather than by adversely affecting the host cells. The selection of the anti-RSV RNA and RNA analogs by intact virions immediately suggests the potential application of this approach to develop RNA and RNA analogs as inhibitors of other viruses such as human immunodeficiency virus.

Isolation and molecular characterization of a human T-cell lymphotropic virus type II (HTLV-II), subtype B, from a healthy Pygmy living in a remote area of Cameroon: an ancient origin for HTLV-II in Africa.

We report characterization of a human T-cell lymphotropic virus type II (HTLV-II) isolated from an interleukin 2-dependent CD8 T-cell line derived from peripheral blood mononuclear cells of a healthy, HTLV-II-seropositive female Bakola Pygmy, aged 59, living in a remote equatorial forest area in south Cameroon. This HTLLV-II isolate, designated PYGCAM-1, reacted in an indirect immunofluorescence assay with HTLV-II and HTLV-I polyclonal antibodies and with an HTLV-I/II gp46 monoclonal antibody but not with HTLV-I gag p19 or p24 monoclonal antibodies. The cell line produced HTLV-I/II p24 core antigen and retroviral particles. The entire env gene (1462 bp) and most of the long terminal repeat (715 bp) of the PYGCAM-1 provirus were amplified by the polymerase chain reaction using HTLV-II-specific primers. Comparison with the long terminal repeat and envelope sequences of prototype HTLV-II strains indicated that PYGCAM-1 belongs to the subtype B group, as it has only 0.5-2% nucleotide divergence from HTLV-II B strains. The finding of antibodies to HTLV-II in sera taken from the father of the woman in 1984 and from three unrelated members of the same population strongly suggests that PYGCAM-1 is a genuine HTLV-II that has been present in this isolated population for a long time. The low genetic divergence of this African isolate from American isolates raises questions about the genetic variability over time and the origin and dissemination of HTLV-II, hitherto considered to be predominantly a New World virus.

Protocole d'isolation des virions périnucléaires et extracellulaires chez HSV-1 pour l'étude par une approche protéomique du mécanisme de transport intracellulaire emprunté par le virus

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Protocole d'isolation des virions périnucléaires et extracellulaires chez HSV-1 pour l'étude par une approche protéomique du mécanisme de transport intracellulaire emprunté par le virus

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Genetic Diversity of Rice Tungro Spherical Virus in Tungro-Endemic Provinces of the Philippines and Indonesia

The two adjacent genes of coat protein 1 and 2 of rice tungro spherical virus (RTSV) were amplified from total RNA extracts of serologically indistinguishable field isolates from the Philippines and Indonesia, using reverse transcriptase polymerase chain reaction (RT-PCR). Digestion with HindIII and BstYI restriction endonucleases differentiated the amplified DNA products into eight distinct coat protein genotypes. These genotypes were then used as indicators of virus diversity in the field. Inter- and intra-site diversities were determined over three cropping seasons. At each of the sites surveyed, one or two main genotypes prevailed together with other related minor or mixed genotypes that did not replace the main genotype over the sampling time. The cluster of genotypes found at the Philippines sites was significantly different from the one at the Indonesia sites, suggesting geographic isolation for virus populations. Phylogenetic studies based on the nucleotide sequences of 38 selected isolates confirm the spatial distribution of RTSV virus populations but show that gene flow may occur between populations. Under the present conditions, rice varieties do not seem to exert selective pressure on the virus populations. Based on the selective constraints in the coat protein amino acid sequences and the virus genetic composition per site, a negative selection model followed by random-sampling events due to vector transmissions is proposed to explain the inter-site diversity observed

Inter-and Intra-Site Genetic Diversity of Natural Field Populations of Rice Tungro Bacilliform Virus in the Philippines

The genetic structure of rice tungro bacilliform virus (RTBV) populations within and between growing sites was analyzed in a collection of natural field isolates from different rice varieties grown in eight tungro-endemic sites of the Philippines. Total DNA extracts from 345 isolates were digested with EcoRV restriction enzyme and hybridized with a full-length probe of RTBV, a procedure shown in preliminary experiments capable of revealing high levels of polymorphism in RTBV field isolates. In the total population, 17 distinct EcoRV-based genome profiles (genotypes) were identified and used as indicators for virus diversity. Distinct sets of genotypes occurred in Isabela and North Cotabato provinces suggesting a geographic isolation of virus populations. However, among the sites in each province, there were few significant differences in the genotype compositions of virus populations. The number of genotypes detected at a site varied from two to nine with a few genotypes dominating. In general the isolates at a site persisted from season to season indicating a genetic stability for the local virus population. Over the sampling time, IRRI rice varieties, which have green leafhopper resistance genes, supported similar virus populations to those supported by other varieties, indicating that the variety of the host exerted no apparent selection pressures. Insect transmission experiments on selected RTBV field isolates showed that dramatic shifts in genotype and phenotype distributions can occur in response to host /environmental shifts.

Genetic Composition and Complexity of Virus Populations at Tungro-Endemic and Outbreak Rice Sites

We have recently demonstrated the geographic isolation of rice tungro bacilliform virus (RTBV) populations in the tungro-endemic provinces of Isabela and North Cotabato, Philippines. In this study, we examined the genetic structure of the virus populations at the tungro-outbreak sites of Lanao del Norte, a province adjacent to North Cotabato. We also analyzed the virus populations at the tungro-endemic sites of Subang, Indonesia, and Dien Khanh, Vietnam. Total DNA extracts from 274 isolates were digested with EcoRV restriction enzyme and hybridized with a full-length probe of RTBV. In the total population, 22 EcoRV-restricted genome profiles (genotypes) were identified. Although overlapping genotypes could be observed, the outbreak sites of Lanao del Norte had a genotype combination distinct from that of Subang or Dien Khanh but a genotype combination similar to that identified earlier from North Cotabato, the adjacent endemic province. Sequence analysis of the intergenic region and part of the ORF1 RTBV genome from randomly selected genotypes confirms the geographic clustering of RTBV genotypes and, combined with restriction analysis, the results suggest a fragmented spatial distribution of RTBV local populations in the three countries. Because RTBV depends on rice tungro spherical virus (RTSV) for transmission, the population dynamics of both tungro viruses were then examined at the endemic and outbreak sites within the Philippines. The RTBV genotypes and the coat protein RTSV genotypes were used as indicators for virus diversity. A shift in population structure of both viruses was observed at the outbreak sites with a reduced RTBV but increased RTSV gene diversity

A protocol for the rapid isolation of full geminivirus genomes from dried plant tissue

A high-throughput method of isolating and cloning geminivirus genomes from dried plant material, by combining an Extract-n-Amp™-based DNA isolation technique with rolling circle amplification (RCA) of viral DNA, is presented. Using this method an attempt was made to isolate and clone full geminivirus genomes/genome components from 102 plant samples, including dried leaves stored at room temperature for between 6 months and 10 years, with an average hands-on-time to RCA-ready DNA of 15 min per 20 samples. While storage of dried leaves for up to 6 months did not appreciably decrease cloning success rates relative to those achieved with fresh samples, efficiency of the method decreased with increasing storage time. However, it was still possible to clone virus genomes from 47% of 10-year-old samples. To illustrate the utility of this simple method for high-throughput geminivirus diversity studies, six Maize streak virus genomes, an Abutilon mosaic virus DNA-B component and the DNA-A component of a previously unidentified New Word begomovirus species were fully sequenced. Genomic clones of the 69 other viruses were verified as such by end sequencing. This method should be extremely useful for the study of any circular DNA plant viruses with genome component lengths smaller than the maximum size amplifiable by RCA. © 2008 Elsevier B.V. All rights reserved.

Evidence of bat origin for Menangle virus, a zoonotic paramyxovirus first isolated from diseased pigs

Menangle virus (MenPV) is a zoonotic paramyxovirus capable of causing disease in pigs and humans. It was first isolated in 1997 from stillborn piglets at a commercial piggery in New South Wales, Australia, where an outbreak of reproductive disease occurred. Neutralizing antibodies to MenPV were detected in various pteropid bat species in Australia and fruit bats were suspected to be the source of the virus responsible for the outbreak in pigs. However, previous attempts to isolate MenPV from various fruit bat species proved fruitless. Here, we report the isolation of MenPV from urine samples of the black flying fox, Pteropus alecto, using a combination of improved procedures and newly established bat cell lines. The nucleotide sequence of the bat isolate is 94% identical to the pig isolate. This finding provides strong evidence supporting the hypothesis that the MenPV outbreak in pigs originated from viruses in bats roosting near the piggery. © 2012 Printed in Great Britain.

Survival of Hendra Virus in the Environment: Modelling the Effect of Temperature

Hendra virus (HeV), a highly pathogenic zoonotic paramyxovirus recently emerged from bats, is a major concern to the horse industry in Australia. Previous research has shown that higher temperatures led to lower virus survival rates in the laboratory. We develop a model of survival of HeV in the environment as influenced by temperature. We used 20 years of daily temperature at six locations spanning the geographic range of reported HeV incidents to simulate the temporal and spatial impacts of temperature on HeV survival. At any location, simulated virus survival was greater in winter than in summer, and in any month of the year, survival was higher in higher latitudes. At any location, year-to-year variation in virus survival 24 h post-excretion was substantial and was as large as the difference between locations. Survival was higher in microhabitats with lower than ambient temperature, and when environmental exposure was shorter. The within-year pattern of virus survival mirrored the cumulative within-year occurrence of reported HeV cases, although there were no overall differences in survival in HeV case years and non-case years. The model examines the effect of temperature in isolation; actual virus survivability will reflect the effect of additional environmental factors

Detection, epidemiology and host spectrum of cowpox and Borna disease virus infections

Several orthopoxviruses (OPV) and Borna disease virus (BDV) are enveloped, zoonotic viruses with a wide geographical distribution. OPV antibodies cross-react, and former smallpox vaccination has therefore protected human populations from another OPV infection, rodent-borne cowpox virus (CPXV). Cowpox in humans and cats usually manifests as a mild, self-limiting dermatitis and constitutional symptoms, but it can be severe and even life-threatening in the immunocompromised. Classical Borna disease is a progressive meningoencephalomyelitis in horses and sheep known in central Europe for centuries. Nowadays the virus or its close relative infects humans and also several other species in central Europe and elsewhere, but the existence of human Borna disease with its suspected neuropsychiatric symptoms is controversial. The epidemiology of BDV is largely unknown, and the present situation is even more intriguing following the recent detection of several-million-year-old, endogenized BDV genes in primate and various other vertebrate genomes. The aims of this study were to elucidate the importance of CPXV and BDV in Finland and in possible host species, and particularly to 1) establish relevant methods for the detection of CPXV and other OPVs as well as BDV in Finland, 2) determine whether CPXV and BDV exist in Finland, 3) discover how common OPV immunity is in different age groups in Finland, 4) characterize possible disease cases and clarify their epidemiological context, 5) establish the hosts and possible reservoir species of these viruses and their geographical distribution in wild rodents, and 6) elucidate the infection kinetics of BDV in the bank vole. An indirect immunofluorescence assay and avidity measurement were established for the detection, timing and verification of OPV or BDV antibodies in thousands of blood samples from humans, horses, ruminants, lynxes, gallinaceous birds, dogs, cats and rodents. The mostly vaccine-derived OPV seroprevalence was found to decrease gradually according to the year of birth of the sampled human subjects from 100% to 10% in those born after 1977. On the other hand, OPV antibodies indicating natural contact with CPXV or other OPVs were commonly found in domestic and wild animals: the horse, cow, lynx, dog, cat and, with a prevalence occasionally even as high as 92%, in wild rodents, including some previously undetected species and new regions. Antibodies to BDV were detected in humans, horses, a dog, cats, and for the first time in wild rodents, such as bank voles (Myodes glareolus). Because of the controversy within the human Borna disease field, extra verification methods were established for BDV antibody findings: recombinant nucleocapsid and phosphoproteins were produced in Escherichia coli and in a baculovirus system, and peptide arrays were additionally applied. With these verification assays, Finnish human, equine, feline and rodent BDV infections were confirmed. Taken together, wide host spectra were evident for both OPV and BDV infections based on the antibody findings, and OPV infections were found to be geographically broadly distributed. PCR amplification methods were utilised for hundreds of blood and tissue samples. The methods included conventional, nested and real-time PCRs with or without the reverse transcription step and detecting four or two genes of OPVs and BDV, respectively. OPV DNA could be amplified from two human patients and three bank voles, whereas no BDV RNA was detected in naturally infected individuals. Based on the phylogenetic analyses, the Finnish OPV sequences were closely related although not identical to a Russian CPXV isolate, and clearly different from other CPXV strains. Moreover, the Finnish sequences only equalled each other, but the short amplicons obtained from German rodents were identical to monkeypox virus, in addition to German CPXV variants. This reflects the close relationship of all OPVs. In summary, RNA of the Finnish BDV variant could not be detected with the available PCR methods, but OPV DNA infrequently could. The OPV species infecting the patients of this study was proven to be CPXV, which is most probably also responsible for the rodent infections. Multiple cell lines and some newborn rodents were utilised in the isolation of CPXV and BDV from patient and wildlife samples. CPXV could be isolated from a child with severe, generalised cowpox. BDV isolation attempts from rodents were unsuccessful in this study. However, in parallel studies, a transient BDV infection of cells inoculated with equine brain material was detected, and BDV antigens discovered in archival animal brains using established immunohistology. Thus, based on several independent methods, both CPXV and BDV (or a closely related agent) were shown to be present in Finland. Bank voles could be productively infected with BDV. This experimental infection did not result in notable pathological findings or symptoms, despite the intense spread of the virus in the central and peripheral nervous system. Infected voles commonly excreted the virus in urine and faeces, which emphasises their possible role as a BDV reservoir. Moreover, BDV RNA was regularly reverse transcribed into DNA in bank voles, which was detected by amplifying DNA by PCR without reverse transcription, and verified with nuclease treatments. This finding indicates that BDV genes could be endogenized during an acute infection. Although further transmission studies are needed, this experimental infection demonstrated that the bank vole can function as a potential BDV reservoir. In summary, multiple methods were established and applied in large panels to detect two zoonoses novel to Finland: cowpox virus and Borna disease virus. Moreover, new information was obtained on their geographical distribution, host spectrum, epidemiology and infection kinetics.

Humanized antibody neutralizing 2009 pandemic H1N1 virus

The 2009 pandemic H1N1 S-OIV (swine origin influenza A virus) caused noticeable morbidity and mortality worldwide. In addition to vaccine and antiviral drug therapy, the use of influenza virus neutralizing monoclonal antibodies (MAbs) for treatment purposes is a viable alternative. We previously reported the isolation of a high affinity, potently neutralizing murine MAb MA2077 against 2009 pandemic H1N1 virus. We describe here the humanization of MA2077 and its expression in a mammalian cell line. Six complementarity-determining regions (CDRs) of MA2077 were grafted onto the human germline variable regions; along with six and eight back mutations in the framework of heavy and light chains, respectively, pertaining to the vernier zone and interchain packing residues to promote favorable CDR conformation and facilitate antigen binding. The full length humanized antibody, 2077Hu2, expressed in CHO-K1 cells, showed high affinity to hemagglutinin protein (K-D = 0.75 +/- 0.32 nM) and potent neutralization of pandemic H1N1 virus (IC50 = 0.17 mu g/mL), with marginally higher IC50 as compared to MA2077 (0.08 mu g/mL). In addition, 2077Hu2 also retained the epitope specificity for the ``Sa'' antigenic site on pandemic HA. To the best of our knowledge, this is the first report of a humanized neutralizing antibody against pandemic H1N1 virus.

Studies of temperature sensitive mutants of polyoma virus

Polyoma virus can undergo two different types of interactions with susceptible cells; one type of interaction leads to the production of new infectious virus and eventual cell death while the other leads to a neoplastically transformed cell which is able to continue to divide under conditions that inhibit the multiplication of uninfected normal cells. In order to study the viral genes involved in both of these virus-cell interactions the isolation of temperature sensitive mutants of polyoma virus was undertaken.

Two strains (TS-a, TS-b) which were temperature sensitive in their plaque forming ability at 38.5˚C, but not at 31.5˚C, were isolated from a mutagenized stock of the polyoma wild type virus (PY). TS-a was studied in further detail.

TS-a grown at 31.5˚C was found to be indistinguishable from PY in a number of physical characteristics including the heat sensitivity of the completed viral components. TS-a was inhibited in its ability to produce infectious virus in mouse cells when incubated at 38.5˚C; this inhibition could be overcome by infection with high multiplicities.

The nature of the intracellular temperature sensitive step of TS-a was analysed to some degree. It was found that this step occurs after uncoating of the infecting virus particles and about the time of new viral DNA synthesis. New infectious viral DNA does not appear to be made at the nonpermissive temperature; in contrast noninfectious capsids are made at 38.5˚C, but in amounts smaller than a full yield, such as made by TS-a at 31.5˚C or by PY at both the high and low temperature.

TS-a has also been found to be temperature sensitive in its transforming ability in vitro. Cells transformed at 31.5˚C by TS-a retain their transformed characteristics upon cultivation at 38.5˚C. Thus the temperature sensitive function seems to be important for the initiation of transformation, but not essential for the maintenance of the transformed state. TS-a also appears to be temperature sensitive in the production of tumors in newborn hamsters.