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.

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.

Differentiation of Rice Tungro Spherical Virus Variants by RTPCR and RFLP

Differentiation of rice tungro spherical virus variants by RTPCR and RFLP tungro bacilliform virus (RTBV), the other causal agent, which causes the symptoms. RTSV is a single-stranded RNA virus of 12,180 nucleotides (Hull 1996).

Sequence Changes in Six Variants of Rice Tungro Bacilliform Virus and Their Phylogenetic Relationships

The DNA of three biological variants, G1, Ic and G2, which originated from the same greenhouse isolate of rice tungro bacilliform virus (RTBV) at the International Rice Research Institute (IRRI), was cloned and sequenced. Comparison of the sequences revealed small differences in genome sizes. The variants were between 95 and 99% identical at the nucleotide and amino acid levels. Alignment of the three genome sequences with those of three published RTBV sequences (Phi-1, Phi-2 and Phi-3) revealed numerous nucleotide substitutions and some insertions and deletions. The published RTBV sequences originated from the same greenhouse isolate at IRRI 20, 11 and 9 years ago. All open reading frames (ORFs) and known functional domains were conserved across the six variants. The cysteine-rich region of ORF3 showed the greatest variation. When the six DNA sequences from IRRI were compared with that of an isolate from Malaysia (Serdang), similar changes were observed in the cysteine-rich region in addition to other nucleotide substitutions and deletions across the genome. The aligned nucleotide sequences of the IRRI variants and Serdang were used to analyse phylogenetic relationships by the bootstrapped parsimony, distance and maximum-likelihood methods. The isolates clustered in three groups: Serdang alone; Ic and G1; and Phi-1, Phi-2, Phi-3 and G2. The distribution of phylogenetically informative residues in the IRRI sequences shared with the Serdang sequence and the differing tree topologies for segments of the genome suggested that recombination, as well as substitutions and insertions or deletions, has played a role in the evolution of RTBV variants. The significance and implications of these evolutionary forces are discussed in comparison with badnaviruses and caulimoviruses.

Comparison of Nucleotide Sequences between Northern and Southern Philippine Isolates of Rice Grassy Stunt Virus Indicates Occurrence of Natural Genetic Reassortment

Rice grassy stunt virus is a member of the genus Tenuivirus, is persistently transmitted by a brown planthopper, and has occurred in rice plants in South, Southeast, and East Asia (similar to North and South America). We determined the complete nucleotide (nt) sequences of RNAs 1 (9760 nt), 2 (4069 nt), 3 (3127 nt), 4 (2909 nt), 5 (2704 nt), and 6 (2590 nt) of a southern Philippine isolate from South Cotabato and compared them with those of a northern Philippine isolate from Laguna (Toriyama et al., 1997, 1998). The numbers of nucleotides in the terminal untranslated regions and open reading frames were identical between the two isolates except for the 5′ untranslated region of the complementary strand of RNA 4. Overall nucleotide differences between the two isolates were only 0.08% in RNA 1, 0.58% in RNA 4, and 0.26% in RNA 5, whereas they were 2.19% in RNA 2, 8.38% in RNA 3, and 3.63% in RNA 6. In the intergenic regions, the two isolates differed by 9.12% in RNA 2, 11.6% in RNA 3, and 6.86% in RNA 6 with multiple consecutive nucleotide deletion/insertions, whereas they differed by only 0.78% in RNA 4 and 0.34% in RNA 5. The nucleotide variation in the intergenic region of RNA 6 within the South Cotabato isolate was only 0.33%. These differences in accumulation of mutations among individual RNA segments indicate that there was genetic reassortment in the two geographical isolates; RNAs 1, 4, and 5 of the two isolates came from a common ancestor, whereas RNAs 2, 3, and 6 were from two different ancestors.

Complete Nucleotide Sequence of Rice Tungro Spherical Virus Genome of a Highly Virulent Strain Vt6

The complete nucleotide sequence of rice tungro spherical virus (RTSV) strain Vt6, originally from Mindanao, the Philippines, with higher virulence to resistant rice cultivars, was determined and compared with the published sequence for the Philippine-type strain A (RTSV-A-Shen). It was reported that RTSV-A was not able to infect a rice resistant cultivar TKM 6 (10). RTSV-Vt6 and RTSV-A-Shen share 90% and 95% homology at nucleotide and amino-acid levels, respectively. The N-terminal leader sequence of RTSV-Vt6 contained a 39-amino acids-region (positions 65 to 103) which was totally different from that of RTSV-A-Shen; the difference resulted from frame shifting by nucleotide insertions and deletions. To confirm the amino-acid sequence differences of the leader polypeptide, the same region was cloned and sequenced using a newly obtained variant of RTSV-type 6, which had been collected in the field of IRRI, and seven field isolates from Mindanao, the Philippines. Since all the sequences of the target region are identical to that of the Vt6 leader polypeptide, the sequence difference in the leader region seems not to correlate with the virulence of Vt6.

Inheritance of Resistance to Rice Tungro Spherical Virus in a Near-Isogenic Line Derived from Utri Merah and in Rice Cultivar TKM6

Resistance to rice virus diseases is an important requirement in many Southeast Asian rice breeding programs. Inheritance of resistance to rice tungro spherical virus (RTSV) in TW5, a near-isogenic line derived from Indonesian rice cultivar Utri Merah, was compared to that in TKM6, an Indian rice cultivar. Both TKM6 and Utri Merah are cultivars resistant to RTSV infections. Crosses were made between TKM6 and TN1, a susceptible cultivar, and between TW5 and TN1, and F3 lines were evaluated for their resistance to RTSV using two RTSV inoculum sources and a serological assay (ELISA). In TKM6, the resistance to the mixture of RTSV-V + RTBV inoculum source was controlled by a single recessive gene, whereas in TW5, the resistance was controlled by two recessive genes. A single recessive gene, however, controlled the resistance in TW5 when another RTSV variant, RTSV-VI, was used, suggesting that the resistance in TW5 depends on the nature of the RTSV inoculum used. RT-PCR, sequence, and phylogenetic analyses confirmed that RTSV-VI inoculum differs from RTSV-V inoculum and accurate phenotyping of the resistance to RTSV requires the use of a genetic marker.

Long term immunity to live attenuated Japanese encephalitis chimeric virus vaccine : randomized, double-blind, 5-year phase II study in healthy adults.

In a randomized, double-blind study, 202 healthy adults were randomized to receive a live, attenuated Japanese encephalitis chimeric virus vaccine (JE-CV) and placebo 28 days apart in a cross-over design. A subgroup of 98 volunteers received a JE-CV booster at month 6. Safety, immunogenicity, and persistence of antibodies to month 60 were evaluated. There were no unexpected adverse events (AEs) and the incidence of AEs between JE-CV and placebo were similar. There were three serious adverse events (SAE) and no deaths. A moderately severe case of acute viral illness commencing 39 days after placebo administration was the only SAE considered possibly related to immunization. 99% of vaccine recipients achieved a seroprotective antibody titer ≥ 10 to JE-CV 28 days following the single dose of JE-CV, and 97% were seroprotected at month 6. Kaplan Meier analysis showed that after a single dose of JE-CV, 87% of the participants who were seroprotected at month 6 were still protected at month 60. This rate was 96% among those who received a booster immunization at month 6. 95% of subjects developed a neutralizing titer ≥ 10 against at least three of the four strains of a panel of wild-type Japanese encephalitis virus (JEV) strains on day 28 after immunization. At month 60, that proportion was 65% for participants who received a single dose of JE-CV and 75% for the booster group. These results suggest that JE-CV is safe, well tolerated and that a single dose provides long-lasting immunity to wild-type strains

Concomitant or sequential administration of live attenuated Japanese encephalitis chimeric virus vaccine and yellow fever 17D vaccine : randomized double-blind phase II evaluation of safety and immunogenicity

A randomized, double-blind, study was conducted to evaluate the safety, tolerability and immunogenicity of a live attenuated Japanese encephalitis chimeric virus vaccine (JE-CV) co-administered with live attenuated yellow fever (YF) vaccine (YF-17D strain; Stamaril(®), Sanofi Pasteur) or administered successively. Participants (n = 108) were randomized to receive: YF followed by JE-CV 30 days later, JE followed by YF 30 days later, or the co-administration of JE and YF followed or preceded by placebo 30 days later or earlier. Placebo was used in a double-dummy fashion to ensure masking. Neutralizing antibody titers against JE-CV, YF-17D and selected wild-type JE virus strains was determined using a 50% serum-dilution plaque reduction neutralization test. Seroconversion was defined as the appearance of a neutralizing antibody titer above the assay cut-off post-immunization when not present pre-injection at day 0, or a least a four-fold rise in neutralizing antibody titer measured before the pre-injection day 0 and later post vaccination samples. There were no serious adverse events. Most adverse events (AEs) after JE vaccination were mild to moderate in intensity, and similar to those reported following YF vaccination. Seroconversion to JE-CV was 100% and 91% in the JE/YF and YF/JE sequential vaccination groups, respectively, compared with 96% in the co-administration group. All participants seroconverted to YF vaccine and retained neutralizing titers above the assay cut-off at month six. Neutralizing antibodies against JE vaccine were detected in 82-100% of participants at month six. These results suggest that both vaccines may be successfully co-administered simultaneously or 30 days apart.

Molecular evolutionary dynamics of Ross River virus and implications for vaccine efficacy

Ross River virus (RRV) is a mosquito-borne member of the genus Alphavirus that causes epidemic polyarthritis in humans, costing the Australian health system at least US$10 million annually. Recent progress in RRV vaccine development requires accurate assessment of RRV genetic diversity and evolution, particularly as they may affect the utility of future vaccination. In this study, we provide novel RRV genome sequences and investigate the evolutionary dynamics of RRV from time-structured E2 gene datasets. Our analysis indicates that, although RRV evolves at a similar rate to other alphaviruses (mean evolutionary rate of approx. 8x10(-4) nucleotide substitutions per site year(-1)), the relative genetic diversity of RRV has been continuously low through time, possibly as a result of purifying selection imposed by replication in a wide range of natural host and vector species. Together, these findings suggest that vaccination against RRV is unlikely to result in the rapid antigenic evolution that could compromise the future efficacy of current RRV vaccines.

Rapid displacement of dengue virus type 1 by type 4, Pacific region, 2007-2009

Since 2000-2001, dengue virus type 1 has circulated in the Pacific region. However, in 2007, type 4 reemerged and has almost completely displaced the strains of type 1. If only 1 serotype circulates at any time and is replaced approximately every 5 years, DENV-3 may reappear in 2012.

Agroinoculation Allows the Screening of Rice for Resistance to Rice Tungro Bacilliform Virus

A system for agroinoculating rice tungro bacilliform virus (RTBV), one of the two viruses of the rice tungro disease complex, has been optimised. A nontumour-inducing strain of Agrobacterium (pGV3850) was used in order to conform with biosafety regulations. Fourteen-day-old seedlings survived the mechanical damage of the technique and were still young enough to support virus replication. The level of the bacterial inoculum was important to obtain maximum infection, with a high inoculum level (0.5 × 1012 cells/ml) resulting in up to 100% infection of a susceptible variety that was comparable with infection by insect transmission. Agroinoculation with RTBV was successful for all three rice cultivarss tested; TN1 (tungro susceptible), Balimau Putih (tungro tolerant), and IR26 (RTSV and vector resistant). Agroinoculation enables resistance to RTBV to be distinguished from resistance to the leafhopper vector of the virus, and should prove useful in screening rice germplasm, breeding materials, and transgenic rice lines.

Expected values for pedometer-determined physical activity in older populations

The purpose of this review is to update expected values for pedometer-determined physical activity in free-living healthy older populations. A search of the literature published since 2001 began with a keyword (pedometer, "step counter," "step activity monitor" or "accelerometer AND steps/day") search of PubMed, Cumulative Index to Nursing & Allied Health Literature (CINAHL), SportDiscus, and PsychInfo. An iterative process was then undertaken to abstract and verify studies of pedometer-determined physical activity (captured in terms of steps taken; distance only was not accepted) in free-living adult populations described as ≥ 50 years of age (studies that included samples which spanned this threshold were not included unless they provided at least some appropriately age-stratified data) and not specifically recruited based on any chronic disease or disability. We identified 28 studies representing at least 1,343 males and 3,098 females ranging in age from 50–94 years. Eighteen (or 64%) of the studies clearly identified using a Yamax pedometer model. Monitoring frames ranged from 3 days to 1 year; the modal length of time was 7 days (17 studies, or 61%). Mean pedometer-determined physical activity ranged from 2,015 steps/day to 8,938 steps/day. In those studies reporting such data, consistent patterns emerged: males generally took more steps/day than similarly aged females, steps/day decreased across study-specific age groupings, and BMI-defined normal weight individuals took more steps/day than overweight/obese older adults. The range of 2,000–9,000 steps/day likely reflects the true variability of physical activity behaviors in older populations. More explicit patterns, for example sex- and age-specific relationships, remain to be informed by future research endeavors.

Expected values for steps/day in special populations

Objective To assemble expected values for free-living steps/day in special populations living with chronic illnesses and disabilities. Method Studies identified since 2000 were categorized into similar illnesses and disabilities, capturing the original reference, sample descriptions, descriptions of instruments used (i.e., pedometers, piezoelectric pedometers, accelerometers), number of days worn, and mean and standard deviation of steps/day. Results Sixty unique studies represented: 1) heart and vascular diseases, 2) chronic obstructive lung disease, 3) diabetes and dialysis, 4) breast cancer, 5) neuromuscular diseases, 6) arthritis, joint replacement, and fibromyalgia, 7) disability (including mental retardation/intellectual difficulties), and 8) other special populations. A median steps/day was calculated for each category. Waist-mounted and ankle-mounted instruments were considered separately due to fundamental differences in assessment properties. For waist-mounted instruments, the lowest median values for steps/day are found in disabled older adults (1214 steps/day) followed by people living with COPD (2237 steps/day). The highest values were seen in individuals with Type 1 diabetes (8008 steps/day), mental retardation/intellectual disability (7787 steps/day), and HIV (7545 steps/day). Conclusion This review will be useful to researchers/practitioners who work with individuals living with chronic illness and disability and require such information for surveillance, screening, intervention, and program evaluation purposes. Keywords: Exercise; Walking; Ambulatory monitoring