Phylogenetic analysis of polyomaviruses based on their complete genomes

Perez-Losada et al. [1] analyzed 72 complete genomes corresponding to nine mammalian (67 strains) and 2 avian (5 strains) polyomavirus species using maximum likelihood and Bayesian methods of phylogenetic inference. Because some data of 2 genomes in their work are now not available in GenBank, in this work, we analyze the phylogenetic relationship of the remaining 70 complete genomes corresponding to nine mammalian (65 strains) and two avian (5 strains) polyomavirus species using a dynamical language model approach developed by our group (Yu et al., [26]). This distance method does not require sequence alignment for deriving species phylogeny based on overall similarities of the complete genomes. Our best tree separates the bird polyomaviruses (avian polyomaviruses and goose hemorrhagic polymaviruses) from the mammalian polyomaviruses, which supports the idea of splitting the genus into two subgenera. Such a split is consistent with the different viral life strategies of each group. In the mammalian polyomavirus subgenera, mouse polyomaviruses (MPV), simian viruses 40 (SV40), BK viruses (BKV) and JC viruses (JCV) are grouped as different branches as expected. The topology of our best tree is quite similar to that of the tree constructed by Perez-Losada et al.

Monitoring and analysis of internet traffic targeting unused address spaces

Today’s evolving networks are experiencing a large number of different attacks ranging from system break-ins, infection from automatic attack tools such as worms, viruses, trojan horses and denial of service (DoS). One important aspect of such attacks is that they are often indiscriminate and target Internet addresses without regard to whether they are bona fide allocated or not. Due to the absence of any advertised host services the traffic observed on unused IP addresses is by definition unsolicited and likely to be either opportunistic or malicious. The analysis of large repositories of such traffic can be used to extract useful information about both ongoing and new attack patterns and unearth unusual attack behaviors. However, such an analysis is difficult due to the size and nature of the collected traffic on unused address spaces. In this dissertation, we present a network traffic analysis technique which uses traffic collected from unused address spaces and relies on the statistical properties of the collected traffic, in order to accurately and quickly detect new and ongoing network anomalies. Detection of network anomalies is based on the concept that an anomalous activity usually transforms the network parameters in such a way that their statistical properties no longer remain constant, resulting in abrupt changes. In this dissertation, we use sequential analysis techniques to identify changes in the behavior of network traffic targeting unused address spaces to unveil both ongoing and new attack patterns. Specifically, we have developed a dynamic sliding window based non-parametric cumulative sum change detection techniques for identification of changes in network traffic. Furthermore we have introduced dynamic thresholds to detect changes in network traffic behavior and also detect when a particular change has ended. Experimental results are presented that demonstrate the operational effectiveness and efficiency of the proposed approach, using both synthetically generated datasets and real network traces collected from a dedicated block of unused IP addresses.

Evaluating sewage associated JCV and BKV polyomaviruses for sourcing human fecal pollution in a coastal river in Southeast Queensland Australia

In this study, the host-sensitivity and -specificity of JCV and BKV polyomaviruses were evaluated by testing wastewater/fecal samples from nine host groups in Southeast Queensland, Australia. The JCV and BKV polyomaviruses were detected in 48 human wastewater samples collected from the primary and secondary effluent suggesting high sensitivity of these viruses in human wastewater. Of the 81 animal wastewater/fecal samples tested, 80 were PCR negative for this marker. Only one sample from pig wastewater was positive. Nonetheless, the overall host-specificity of these viruses to differentiate between human and animal wastewater/fecal samples was 0.99. To our knowledge, this is the first study in Australia that reports the high specificity of JCV and BKV polyomaviruses. To evaluate the field application of these viruses to detect human fecal pollution, 20 environmental samples were collected from a coastal river. Of the 20 samples tested, 15% and 70% samples exceeded the regulatory guidelines for E. coli and enterococci levels for marine waters. In all, 5 (25%) samples were PCR positive for JCV and BKV indicated the presence of human fecal pollution in the studied river. The results suggest that JCV and BKV detection using PCR could be a useful tool for the identification of human sourced fecal pollution in coastal waters.

The development of a serological-based diagnostic test for Dasheen mosaic potyvirus (DsMV)

Dasheen mosaic potyvirus (DsMV) is an important virus affecting taro. The virus has been found wherever taro is grown and infects both the edible and ornamental aroids, causing yield losses of up to 60%. The presence of DsMV, and other viruses,prevents the international movement of taro germplasm between countries. This has a significant negative impact on taro production in many countries due to the inability to access improved taro lines produced in breeding programs. To overcome this problem, sensitive and reliable virus diagnostic tests need to be developed to enable the indexing of taro germplasm. The aim of this study was to generate an antiserum against a recombinant DsMV coat protein (CP) and to develop a serological-based diagnostic test that would detect Pacific Island isolates of the virus. The CP-coding region of 16 DsMV isolates from Papua New Guinea, Samoa, Solomon Islands, French Polynesia, New Caledonia and Vietnam were amplified,cloned and sequenced. The size of the CP-coding region ranged from 939 to 1038 nucleotides and encoded putative proteins ranged from 313 to 346 amino acids, with the molecular mass ranging from 34 to 38 kDa. Analysis ofthe amino acid sequences revealed the presence of several amino acid motifs typically found in potyviruses,including DAG, WCIE/DN, RQ and AFDF. When the amino acid sequences were compared with each other and the DsMV sequences on the database, the maximum variability was21.9%. When the core region ofthe CP was analysed, the maximum variability dropped to 6% indicating most variability was present in the N terminus. Within seven PNG isolates ofDsMV, the maximum variability was 16.9% and 3.9% over the entire CP-coding region and core region, respectively. The sequence ofPNG isolate P1 was most similar to all other sequences. Phylogenetic analysis indicated that almost all isolates grouped according to their provenance. Further, the seven PNG isolates were grouped according to the region within PNG from which they were obtained. Due to the extensive variability over the entire CP-coding region, the core region ofthe CP ofPNG isolate Pl was cloned into a protein expression vector and expressed as a recombinant protein. The protein was purified by chromatography and SDS-PAGE and used as an antigen to generate antiserum in a rabbit. In western blots, the antiserum reacted with bands of approximately 45-47 kDa in extracts from purified DsMV and from known DsMV -infected plants from PNG; no bands were observed using healthy plant extracts. The antiserum was subsequently incorporated into an indirect ELISA. This procedure was found to be very sensitive and detected DsMV in sap diluted at least 1:1,000. Using both western blot and ELISA formats,the antiserum was able to detect a wide range ofDsMV isolates including those from Australia, New Zealand, Fiji, French Polynesia, New Caledonia, Papua New Guinea, Samoa, Solomon Islands and Vanuatu. These plants were verified to be infected with DsMV by RT-PCR. In specificity tests, the antiserum was also found to react with sap from plants infected with SCMV, PRSV-P, PRSV-W, but not with PVY or CMV -infected plants.

Rice Tungro Bacilliform

Many well-known specialists have contributed to this book which presents for the first time an in-depth look at the viruses, their satellites and the retrotransposons infecting (or occuring in) one plant family: the Poaceae (Gramineae). After molecular and biological descriptions of the viruses to species level, virus diseases are presented by crop: barley, maize, rice, rye, sorghum, sugarcane, triticales, wheats, forage, ornamental and lawn. A detailed index of the viruses and taxonomic lists will help readers in the search for information.

Broad Bean Stain Virus: Identification, Detectability with ELISA in Faba Bean Leaves and Seeds, Occurrence in West Asia and North Africa and Possible Wild Hosts

Abstract During a survey of faba bean viruses in West Asia and North Africa a virus was identified as broad bean stain virus (BBSV) based on host reactions, electron microscopy, physical properties and serology. An antiserum to a Syrian isolate was prepared. With this antiserum both the direct double antibody sandwich ELISA (DAS-ELISA) and dot-ELISA were very sensitive in detecting BBSV in leaf extracts, ground whole seeds and germi nated embryos. Sens it i vity was not reduced when the two-day procedure was replaced by a one-day procedure. us i ng ELISA the vi rus was detected in 73 out of 589 faba bean samples with virus-like symptoms collected from Egypt (4 out of 70 samples tested), Lebanon (6/44) , Morocco (017), Sudan (19/254), Syria (36/145) and Tunisia (8/69). This is the first report of BBSV infection of faba bean in Lebanon, Sudan, Syria and Tunisia. speci es i ndi genous to Syri a were Fourteen wild legume susceptible to BBSV infection, with only two producing obvious symptoms. The virus was found to be seed transmitted ~n Vicia palaestina.

Broad Bean Mottle Virus: Identification, Serology, Host Range, and Occurrence on Faba Bean (Vicia Faba) in West Asia and Norm Africa

One of the faba bean viruses found in West Asia and North Africa was identified as broad bean mottle virus (BBMV) by host reactions, particle morphology and size, serology, and granular, often vesiculated cytoplasmic inclusions. Detailed research on four isolates, one each from Morocco, Tunisia, Sudan and Syria, provided new information on the virus. The isolates, though indistinguishable in ELISA or gel-diffusion tests, differed slightly in host range and symptoms. Twenty-one species (12 legumes and 9 non-legumes) out of 27 tested were systemically infected, and 14 of these by all four isolates. Infection in several species was symptomless, but major legumes such as chickpea, lentil and especially pea, suffered severely from infection. All 23 genotypes of faba bean, 2 of chickpea, 4 of lentil, 11 out of 21 of Phaseolus bean, and 16 out of 17 of pea were systemically sensitive to the virus. Twelve plant species were found to be new potential hosts and cucumber a new local-lesion test plant of the virus. BBMV particles occurred in faba bean plants in very high concentrations and seed transmission in this species (1.37%) was confirmed. An isolate from Syria was purified and two antisera were produced, one of which was used in ELISA to detect BBMV in faba bean field samples. Two hundred and three out of the 789 samples with symptoms suggestive of virus infection collected in 1985, 1986 and 1987, were found infected with BBMV: 4 out of 70 (4/70) tested samples from Egypt, 0/44 from Lebanon, 1/15 from Morocco, 46/254 from Sudan, 72/269 from Syria and 80/137 from Tunisia. This is the first report on its occurrence in Egypt, Syria and Tunisia. The virus is a potential threat to crop improvement in the region.

Purification and Properties of Closteroviruslike Particles Associated with Grapevine Corky Bark Disease

Closteroviruslike particles, designated as grapevine corky bark-associated virus (GCBaV), were purified from mature leaves and stem phloem tissue of a corky bark-affected grapevine that had indexed negative for other grapevine viruses. Electron microscopy of purified preparations revealed the presence of flexuous rod-shaped viruslike particles that were about 13 nm in diameter and between 1,400 and 2,000 nm long, with a helical pitch of 3.4 nm. In purified preparations, the GCBaV particles degraded within a few weeks, unlike grapevine leafroll associated virus (GLRaV), which was stable for more than 1 mo under the same storage condition. The molecular weight of the coat protein of GCBaV was 24,000. A large dsRNA molecule (about 15.3 kbp), along with lower molecular weight species, was detected in tissues of corky bark-diseased grapevines, but not in healthy grapevines. Polyclonal antisera were produced in rabbits against purified or partially purified virus preparations. In direct enzyme-linked immunosorbent assay (ELISA), antisera to GCBaV did not react to the serologically distinct types (II and III) of the long closteroviruses associated with grapevine leafroll disease and grapevine virus A (GVA), and vice versa. This antiserum also reacted in ELISA with other corky bark-affected grapevines. Our data suggest that closteroviruslike particles, designated as GCBaV, may be the causal agent of corky bark disease. However, definitive proof is still lacking. The inclusion of GCBaV in the group of closteroviruses with citrus tristeza virus is proposed.

Differentiation of Rice Tungro Bacilliform Virus Strains by Restriction Analysis and DNA Hybridization

Rice tungro bacilliform virus (RTBV) is one of the two viruses that cause tungro disease. Four RTBV strains maintained in the greenhouse for 4 years, G1, G2, Ic, and L, were differentiated by restriction fragment length polymorphism (RFLP) analysis of the native viral DNA. Although strains G1 and Ic had identical restriction patterns when cleaved with Pst1, BamHI, EcoRI, and EcoRV, they can be differentiated from strains G2 and L by EcoRI and EcoRV digestion. These same endonucleases also differentiate strain G2 from strain L. When total DNA extracts from infected plants were used instead of viral DNA, and digested with EcoRV, identical restriction patterns for each strain (G2 and L) were obtained from roots, leaves, and leaf sheaths of infected plants. The restriction patterns were consistent from plant to plant, in different varieties, and at different times after inoculation. This technique can be used to differentiate RTBV strains and determine the variability of a large number of field samples.

Evaluating Two Mass Screening Methods for Tungro Disease Resistance

Tungro is one of the most destructive viral diseases of rice in South and Southeast Asia. It is associated with two viruses---rice tungro bacilliform virus (RTBV) ,and rice tungro spherical virus (RTSV) (Hibino et al 1978). Both viruses are transmitted by the green leafhopper (GLH) Nephotettix virescens (Ling 1979), However, prior acquisition of RTSV is required for Ihe transmission of RTBV alone (Hibino 1983). Plants infected with both viruses show severe stunting and yellowing. Those infected with RTBV alone show mild stunting but no leaf discoloration whereas those infected with RTSV alone do not show any apparent symptoms (Hibino el al 1978). Since the late 1960s, tungro has been mainly managed through varietal resistance (Khush 1989). The instability of resistant varieties in the field (Dahal et .a1 1990) led to a reexamination of the nature of the incorporated sources of resistance and to the adoption of more precise and more accurate screening methods.

Mechanism of Resistance to Rice Tungro Spherical Virus (RTSV)

RTSV is one of two viruses that cause tungro disease. RTSV is independently transmitted, whereas the other virus, rice tungro bacilliform virus (RTBV), is dependent on RTSV for its transmission by the green leafhopper (GLH), Nephotettix virescens. The occurrence and spread of tungro disease therefore depend on the presence of RTSV in the field. Resistance to RTSV infection would slow down the spread of the disease.

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