Construction of a Sonchus Yellow Net Virus minireplicon: a step toward reverse genetic analysis of plant negative-strand RNA viruses.

Reverse genetic analyses of negative-strand RNA (NSR) viruses have provided enormous advances in our understanding of animal viruses over the past 20 years, but technical difficulties have hampered application to plant NSR viruses. To develop a reverse genetic approach for analysis of plant NSR viruses, we have engineered Sonchus yellow net nucleorhabdovirus (SYNV) minireplicon (MR) reporter cassettes for Agrobacterium tumefaciens expression in Nicotiana benthamiana leaves. Fluorescent reporter genes substituted for the SYNV N and P protein open reading frames (ORFs) exhibited intense single-cell foci throughout regions of infiltrated leaves expressing the SYNV MR derivatives and the SYNV nucleocapsid (N), phosphoprotein (P), and polymerase (L) proteins. Genomic RNA and mRNA transcription was detected for reporter genes substituted for both the SYNV N and P ORFs. These activities required expression of the N, P, and L core proteins in trans and were enhanced by codelivery of viral suppressor proteins that interfere with host RNA silencing. As is the case with other members of the Mononegavirales, we detected polar expression of fluorescent proteins and chloramphenicol acetyltransferase substitutions for the N and P protein ORFs. We also demonstrated the utility of the SYNV MR system for functional analysis of SYNV core proteins in trans and the cis-acting leader and trailer sequence requirements for transcription and replication. This work provides a platform for construction of more complex SYNV reverse genetic derivatives and presents a general strategy for reverse genetic applications with other plant NSR viruses.

WormBase 2012: more genomes, more data, new website.

Since its release in 2000, WormBase (http://www.wormbase.org) has grown from a small resource focusing on a single species and serving a dedicated research community, to one now spanning 15 species essential to the broader biomedical and agricultural research fields. To enhance the rate of curation, we have automated the identification of key data in the scientific literature and use similar methodology for data extraction. To ease access to the data, we are collaborating with journals to link entities in research publications to their report pages at WormBase. To facilitate discovery, we have added new views of the data, integrated large-scale datasets and expanded descriptions of models for human disease. Finally, we have introduced a dramatic overhaul of the WormBase website for public beta testing. Designed to balance complexity and usability, the new site is species-agnostic, highly customizable, and interactive. Casual users and developers alike will be able to leverage the public RESTful application programming interface (API) to generate custom data mining solutions and extensions to the site. We report on the growth of our database and on our work in keeping pace with the growing demand for data, efforts to anticipate the requirements of users and new collaborations with the larger science community.

Brachypodium distachyon line Bd3-1 resistance is elicited by the barley stripe mosaic virus triple gene block 1 movement protein.

Barley stripe mosaic virus North Dakota 18 (ND18), Beijing (BJ), Xinjiang (XJ), Type (TY) and CV21 strains are unable to infect the Brachypodium distachyon Bd3-1 inbred line, which harbours a resistance gene designated Bsr1, but the Norwich (NW) strain is virulent on Bd3-1. Analysis of ND18 and NW genomic RNA reassortants and RNAß mutants demonstrates that two amino acids within the helicase motif of the triple gene block 1 (TGB1) movement protein have major effects on their Bd3-1 phenotypes. Resistance to ND18 correlates with an arginine residue at TGB1 position 390 (R(390)) and a threonine at position 392 (T(392)), whereas the virulent NW strain contains lysines (K) at both positions. ND18 TGB1 R390K ((ND)TGB1(R390K)) and (ND)TGB1(T392K) single substitutions, and an (ND)TGB1(R390K,T392K) double mutation resulted in systemic infections of Bd3-1. Reciprocal (ND)TGB1 substitutions into (NW)TGB1 ((NW)TGB1(K390R) and (NW)TGB1(K392T)) failed to affect virulence, implying that K(390) and K(392) compensate for each other. In contrast, an (NW)TGB1(K390R,K392T) double mutant exhibited limited vascular movement in Bd3-1, but developed prominent necrotic streaks that spread from secondary leaf veins. This phenotype, combined with the appearance of necrotic spots in certain ND18 mutants, and necrosis and rapid wilting of Bd3-1 plants after BJ strain ((BJ)TGB1(K390,T392)) inoculations, show that Bd3-1 Bsr1 resistance is elicited by the TGB1 protein and suggest that it involves a hypersensitive response.

Hordeivirus replication, movement, and pathogenesis.

The last Hordeivirus review appearing in this series 20 years ago focused on the comparative biology, relationships, and genome organization of members of the genus ( 68 ). Prior to the 1989 review, useful findings about the origin, disease occurrence, host ranges, and general biological properties of Barley stripe mosaic virus (BSMV) were summarized in three comprehensive reviews ( 26, 67, 107 ). Several recent reviews emphasizing contemporary molecular genetic findings also may be of interest to various readers ( 15, 37, 42, 69, 70, 88, 113 ). In the current review, we briefly reiterate the biological properties of the four members of the Hordeivirus genus and describe advances in our understanding of organization and expression of the viral genomes. We also discuss the infection processes and pathogenesis of the most extensively characterized Hordeiviruses and frame these advances in the broader context of viruses in other families that have encoded triple gene block proteins. In addition, an overview of recent advances in the use of BSMV for virus-induced gene silencing is presented.

Subcellular localization of the barley stripe mosaic virus triple gene block proteins.

Barley stripe mosaic virus (BSMV) spreads from cell to cell through the coordinated actions of three triple gene block (TGB) proteins (TGB1, TGB2, and TGB3) arranged in overlapping open reading frames (ORFs). Our previous studies (D. M. Lawrence and A. O. Jackson, J. Virol. 75:8712-8723, 2001; D. M. Lawrence and A. O. Jackson, Mol. Plant Pathol. 2:65-75, 2001) have shown that each of these proteins is required for cell-to-cell movement in monocot and dicot hosts. We recently found (H.-S. Lim, J. N. Bragg, U. Ganesan, D. M. Lawrence, J. Yu, M. Isogai, J. Hammond, and A. O. Jackson, J. Virol. 82:4991-5006, 2008) that TGB1 engages in homologous interactions leading to the formation of a ribonucleoprotein complex containing viral genomic and messenger RNAs, and we have also demonstrated that TGB3 functions in heterologous interactions with TGB1 and TGB2. We have now used Agrobacterium tumefaciens-mediated protein expression in Nicotiana benthamiana leaf cells and site-specific mutagenesis to determine how TGB protein interactions influence their subcellular localization and virus spread. Confocal microscopy revealed that the TGB3 protein localizes at the cell wall (CW) in close association with plasmodesmata and that the deletion or mutagenesis of a single amino acid at the immediate C terminus can affect CW targeting. TGB3 also directed the localization of TGB2 from the endoplasmic reticulum to the CW, and this targeting was shown to be dependent on interactions between the TGB2 and TGB3 proteins. The optimal localization of the TGB1 protein at the CW also required TGB2 and TGB3 interactions, but in this context, site-specific TGB1 helicase motif mutants varied in their localization patterns. The results suggest that the ability of TGB1 to engage in homologous binding interactions is not essential for targeting to the CW. However, the relative expression levels of TGB2 and TGB3 influenced the cytosolic and CW distributions of TGB1 and TGB2. Moreover, in both cases, localization at the CW was optimal at the 10:1 TGB2-to-TGB3 ratios occurring in virus infections, and mutations reducing CW localization had corresponding effects on BSMV movement phenotypes. These data support a model whereby TGB protein interactions function in the subcellular targeting of movement protein complexes and the ability of BSMV to move from cell to cell.

Clinical Profile of Elderly Patients (Over 90 Years) with Type 2 Diabetes Seen at a Diabetes Center in South India.

Background: The current life expectancy in India is <70 years. Type 2 diabetes mellitus (T2DM) is known to reduce life expectancy by 6-8 years. Hence elderly people with T2DM in India would be rare. We report on the clinical profile of Asian Indian patients with T2DM who lived beyond 90 years of age and compared them with T2DM patients aged 50 to 60 years. Methods: From the diabetes electronic medical records of >470,000 diabetes patients, we identified T2DM patients who had lived ≥90 years and compared them with those in the 50-60 years age group, matched for gender and duration of diabetes. Clinical data included age at last visit, age at diagnosis, duration of diabetes, family history, smoking and alcohol, details of medications, body mass index (BMI), and blood pressure. Biochemical data included fasting and postprandial plasma glucose, glycated hemoglobin, fasting and stimulated C-peptide levels, lipid profile, and renal function studies. Assessment of retinopathy, nephropathy, neuropathy, coronary artery disease (CAD), and peripheral vascular disease (PVD) was also done. Results: A total of 325 T2DM patients aged ≥90 years and 278 T2DM patients aged between 50 and 60 years were selected for the study. Patients aged ≥90 years had higher systolic blood pressure (P < 0.001) and lower BMI (P < 0.001) than those between 50 and 60 years. Prevalence of retinopathy (29.7% vs. 53.5%) and macroalbuminuria (3.7% vs. 16.0%) was lower in the ≥90 years T2DM patients than in the 50-60 years age group. However, prevalence of neuropathy (89.8% vs. 50.8%), PVD (13.5% vs. 2.0%), and CAD (60.3% vs. 32.0%) was higher among the ≥90 years patients. Eighty-five percent of the T2DM aged ≥90 years were on oral hypoglycemic agents (OHAs), (of whom 64.9% were on sulfonylurea), 12% were on insulin, and 3% on diet alone. Among the 50-60 years old, 87.8% were on OHAs and 12.2% on insulin. Conclusions: This is the first report on the clinical profile of Asian Indians with T2DM aged ≥90 years, and significant differences are seen in their clinical profile compared with younger T2DM patients.

Triple gene block protein interactions involved in movement of Barley stripe mosaic virus.

Barley stripe mosaic virus (BSMV) encodes three movement proteins in an overlapping triple gene block (TGB), but little is known about the physical interactions of these proteins. We have characterized a ribonucleoprotein (RNP) complex consisting of the TGB1 protein and plus-sense BSMV RNAs from infected barley plants and have identified TGB1 complexes in planta and in vitro. Homologous TGB1 binding was disrupted by site-specific mutations in each of the first two N-terminal helicase motifs but not by mutations in two C-terminal helicase motifs. The TGB2 and TGB3 proteins were not detected in the RNP, but affinity chromatography and yeast two-hybrid experiments demonstrated that TGB1 binds to TGB3 and that TGB2 and TGB3 form heterologous interactions. These interactions required the TGB2 glycine 40 and the TGB3 isoleucine 108 residues, and BSMV mutants containing these amino acid substitution were unable to move from cell to cell. Infectivity experiments indicated that TGB1 separated on a different genomic RNA from TGB2 and TGB3 could function in limited cell-to-cell movement but that the rates of movement depended on the levels of expression of the proteins and the contexts in which they are expressed. Moreover, elevated expression of the wild-type TGB3 protein interfered with cell-to-cell movement but movement was not affected by the similar expression of a TGB3 mutant that fails to interact with TGB2. These experiments suggest that BSMV movement requires physical interactions of TGB2 and TGB3 and that substantial deviation from the TGB protein ratios expressed by the wild-type virus compromises movement.

Transgenic expression of coat protein gene of Rice tungro bacilliform virus in rice reduces the accumulation of viral DNA in inoculated plants.

Rice tungro, a devastating disease of rice in south and southeast Asia, is caused by the joint infection of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV). In order to obtain transgenic resistance against RTBV, indica rice cultivar Pusa Basmati-1 was transformed to express the coat protein (CP) gene of an Indian isolate of RTBV. Rice plants containing the transgene integrated in low copy numbers were obtained, in which the CP was shown to accumulate in the leaf tissue. The progenies representing three independent transformation events were challenged with Indian isolates of RTBV using viruliferous Green leafhoppers, and the viral titers in the inoculated plants were monitored using DNA dot-blot hybridization. As compared to non-transgenic controls, two independent transgenic lines showed significantly low levels of RTBV DNA, especially towards later stages of infection and a concomitant reduction of tungro symptoms.

Postpartum development of type 1 diabetes in Asian Indian women with gestational diabetes.

AIM: To study the postpartum conversion of gestational diabetes mellitus (GDM) to different types of diabetes among Asian Indian women. MATERIALS AND METHODS: Using data from electronic medical records, 418 women with GDM seen at a tertiary diabetes care center for diabetes in Chennai in South India between 1991 and 2014 were evaluated for development of diabetes postpartum. RESULTS: Of the 418 GDM women followed up postpartum, 388 progressed to diabetes. Of these 359 (92.5%) developed type 2 diabetes (T2DM) and 29 women (7.5%) developed type 1 diabetes (T1DM). The median time to development of T1DM was 2 years (interquartile range 2 [IQR]) while for T2DM it was 5 years (IQR 6). Women who developed T1DM had significantly lower mean body mass index (BMI) (20.4 ± 2.8 vs. 27.5 ± 4.4 kg/m2, P = 0.001), and higher fasting plasma glucose (222 ± 105 vs. 165 ± 62 mg/dl P = 0.008) and glycated hemoglobin levels (10.2 ± 2.7 vs. 8.5 ± 2.1% P < 0.001) compared to those who developed T2DM. Glutamic acid decarboxylase (GAD) autoantibodies were present in 24/29 (82.7%) of women who developed T1DM. CONCLUSION: A small but significant proportion of women with GDM progress to T1DM postpartum. Measurement of GAD antibodies in leaner women with more severe diabetes could help to identify women who are likely to develop T1DM and thus prevent their presentation with acute hyperglycemic emergencies after delivery.