A New Begomovirus Species Causing Tomato Leaf Curl Disease in Varanasi, India.

In November 2001, a leaf curl disease of tomato, manifested by yellowing of leaf lamina, upward leaf curling, leaf distortion, shrinking of leaf surface, and stunted plant growth was observed in tomato-growing areas in the Varanasi and Mirzapur districts of eastern Uttar Pradesh, India, which caused yield losses up to 100%. The causal agent was infective to tomato cv. Punjab Chuhara by whiteflies and grafting. Inoculated plants developed symptoms observed in naturally infected tomatoes. Viral DNA was isolated from artificially inoculated tomato plants using 1% CTAB (2) followed by a concentration of supercoiled DNA by alkaline denaturation (1). A geminivirus was confirmed by polymerase chain reaction using DNA-A degenerate primers (3), and a 550-bp amplified product was obtained from artificially and naturally infected plants. Full-length viral genomes of DNA-A and DNA-B were cloned in plasmid pUC18 at HindIII and XbaI sites, respectively. Partial tandem dimers of the viral clones were infective to Nicotiana benthamiana and tomato cv. Organ Spring through particle bombardment. Infected N. benthamiana plants exhibited downward and upward leaf curling, big veins, leaf puckering with interveinal chlorosis, and stunting. On tomato, symptoms were the same as those seen on naturally infected plants. Cloned DNA also infected Capsicum annuum cv. California Wonder (upward leaf curling and stunting) and tobacco cv. Xanthi (leaf curling and crinkling) but failed to infect Phaseolus vulgaris, okra, cotton, and N. glutinosa. The Varanasi isolate was sap transmissible (0.1 M potassium phosphate buffer, pH 7.0) from the bombarded plants to N. benthamiana and tomato cv. Organ Spring. DNA-A alone infected N. benthamiana (upward leaf curling and big veins) and tomato cv. Organ Spring (mild leaf curl), but symptoms were delayed and milder. Full-length genome sequencing revealed DNA-A (AY190290) contained 2,757 nt and DNA-B (AY190291) contained 2,688 nt. DNA-A of the Varanasi isolate shares 98.4% identity with a DNA-A sequence (AF449999) obtained from a tomato showing leaf curl symptoms from the same region and 97.1% identity with an isolate from Gujarat (900 km from Varanasi). All three sequences represent isolates of the same species, herein called Tomato leaf curl Gujarat virus, based on the priority of submission of the DNA sequence for the Gujarat region (ToLCGV; AF 413671). All isolates noted were obtained from GenBank. However, except for the DNA-A sequence, no other information is available for these ToLCGV isolates. DNA-A of the ToLCGV-Varanasi isolate shares 66.8% identity with Tomato leaf curl New Delhi virus, severe strain (ToLCNdV-Svr) (U15015), and 84.1% with Tomato leaf curl Karnataka virus (U38239). No DNA-B has been reported for these two ToLCGV isolates, and no infectious clone proving the etiology of the disease has been constructed, except for ToLCGV-Varanasi. DNA-B of ToLCGV-Varanasi shares 79.2% homology with ToLCNdV-Svr and 84.1% with ToLCNdV-Luc (X89653). These results suggest that the isolate from Varanasi belongs to ToLCGV, a previously undescribed geminivirus species causing a devastating tomato leaf curl disease in Gujarat and Uttar Pradesh. References: (1) H. C. Birnboim and J. Doly. Nucleic Acids Res. 7:1513, 1979. (2) K. M. Srivastava et al. J. Virol. Methods 51:297, 1995. (3) S. D. Wyatt and J. K. Brown. Phytopathology 86:1288, 1996.

Tomato leaf curl Gujarat virus, a New Begomovirus Species Causing a Severe Leaf Curl Disease of Tomato in Varanasi, India.

ABSTRACT The biological and molecular properties of Tomato leaf curl Gujarat virus from Varanasi, India (ToLCGV-[Var]) were characterized. ToLCGV-[Var] could be transmitted by grafting and through whitefly transmission in a persistent manner. The full-length genome of DNA-A and DNA-B of ToLCGV-[Var] was cloned in pUC18. Sequence analysis revealed that DNA-A (AY190290) is 2,757 bp and DNA-B (AY190291) is 2,688 bp in length. ToLCGV-[Var] could infect and cause symptoms in tomato, pepper, Nicotiana benthamiana, and N. tabacum when partial tandem dimeric constructs of DNA-A and DNA-B were co-inoculated by particle bombardment. DNA-A alone also is infectious, but symptoms were milder and took longer to develop. ToLCGV-Var virus can be transmitted through sap inoculation from infected tomato plants to the above-mentioned hosts causing the same symptoms. Open reading frames (ORFs) in both DNA-A and DNA-B are organized similarly to those in other begomoviruses. DNA-A and DNA-B share a common region of 155 bp with only 60% sequence identity. DNA-B of ToLCGV-[Var] shares overall 80% identity with DNA-B of Tomato leaf curl New Delhi virus-Severe (ToLCNDV-Svr) and 75% with ToLCNDV-[Lucknow] (ToLCNDV-[Luc]). Comparison of DNA-A sequence with different begomoviruses indicates that ToLCGV-[Var] shares 84% identity with Tomato leaf curl Karnataka virus (ToLCKV) and 66% with ToLCNDV-Svr. ToLCGV-[Var] shares a maximum of 98% identity with another isolate of the same region (ToLCGV-[Mir]; AF449999) and 97% identity with one isolate from Gujarat (ToLCGV-[Vad]; AF413671). All three viruses belong to the same species that is distinct from all the other geminivirus species described so far in the genus Begomovirus of the family Geminiviridae. The name Tomato leaf curl Gujarat virus is proposed because the first sequence was taken from an isolate of Gujarat, India.

Efficacy of single-dose SA 14-14-2 vaccine against Japanese encephalitis: a case control study.

BACKGROUND: In China, since 1989, an estimated 120 million children have been immunised with the SA 14-14-2 live-attenuated Japanese encephalitis (JE) vaccine at ages 1, 2, and 6 years. A case-control study of licensed vaccine found two doses to be 98% effective. Subsequently, researchers found that single-dose vaccine efficacy was high; we aimed to confirm this result. METHODS: During July 11-24, 1999, 160000 doses of JE vaccine were given to children aged 1-15 years, resident in three districts of Nepal. Several cases of JE were admitted to hospital from early August. We obtained names and addresses of cases with serological evidence of a recent infection from Bheri Zonal Hospital, Nepalgunj. We did a matched case-control study and calculated the odds ratio of vaccination among JE cases and age-sex matched village controls. FINDINGS: 20 children, aged 1-15 years, were identified whose illness conformed with the JE case definition and were resident in villages receiving the vaccine. None of 20 JE cases had received JE vaccine compared with 326 of 557 age-sex matched village controls. The efficacy of a single dose of JE vaccine was 99.3% (CI 94.9-100%). INTERPRETATION: A single dose of JE vaccine is highly efficacious in preventing Japanese encephalitis when administered only days or weeks before exposure to infection.

Distribution of beta-thalassemia mutations in the Indian population referred to a diagnostic center.

Hemoglobinopathies are the most commonly inherited genetic disorders in India. Population screening has identified certain communities in India with high risk of beta-thalassemia, the prevalence of carrier status in some being as high as 17%. Over a period of 6 years we have conducted DNA analysis on 1,233 carriers of 23 beta-thalassemia mutations and Hb E, using the amplification refractory mutation system. The studies included analyses of five common mutations for Asian Indians, namely IVS-I-5 (G-->C), 619 bp deletion, IVS-I-1 (G-->T), and the frameshifts at codons 8/9 (+G) and 41/42 (-TTCT). The occurrence of these was seen in 1,066 (86.45%) of the carriers referred to us, the percentage of mutations varying between 5.03-42.58%. We found codon 15 (TGG-->TAG) in 47 (3.81%) samples which was also considered a common mutation in the Indian population. Rare beta-thalassemia mutations were found in 87 (7.06%) individuals. We have designed five new primers and modified four primers for nine rare mutations. These were seen in nine (0.73%) samples. The beta-thalassemia anomaly in 33 (2.68%) carriers remained uncharacterized. State-wide and community-wide distribution patterns of mutations indicated that IVS-I-5 (G-->C) is the most common beta-thalassemia allele in the Indian population. Sindhis settled in Gujrat, and Maharashtra and Lohanas from Gujrat showed a prevalence of the 619 bp deletion mutation in 49.2 and 45.5% carriers, respectively. High frequency of the IVS-I-1 (G-->T) mutation was also found in Sindhis (25.5%), Punjabi Hindus (34.7%), and Lohanas (31.2%). These studies of mutation patterns in different communities have helped us in the quick identification of beta-thalassemia mutations for prenatal diagnosis.

Sources and inheritance of resistance to leaf curl virus in Lycopersicon.

One hundred and twenty-two varieties, lines and wild accessions of Lycopersicon were screened under three different regimes during the autumn/winter season of 1982-83 and 1983-84 for resistance to tomato leaf curl virus (TLCV). L. hirsutum f. glabratum ('B6013') and L. hirsutum f. typicum ('A1904') proved to be highly resistant to TLCV in all three environments. Various accessions of L. peruvianum were also highly resistant. L. pimpinellifolium ('A1921') exhibited no TLCV symptoms within 90 days. Of the cultivated varieties, 'Acc 99' exhibited the minimim score for susceptibility; 'AC 142', 'Collection No. 2', 'Kalyanpur Angurlata' and 'HS 101' had a low rating for virus incidence. The inheritance of resistance was studied in the interspecific crosses between a TLCV resistant line of L. pimpinellifolium ('A1921') and five ('HS 101', 'HS 102', 'HS 110', 'Pusa Ruby' and 'Punjab Chhuhara') susceptible cultivars of L. esculentum. Parents, F1, F2 and backcross progenies were artificially inoculated with local strains of TLCV using vector the viruliferious whitefly, Bemisia tabaci (Genn.). Data indicated that the resistance of L. pimpinellifolium ('A 1921') is monogenic and incompletely dominant over susceptibility.