The second transcribed spacer rDNA sequence: an effective genetic marker for inter-species phylogenetic analysis of trematodes in the order Strigeata.

In the present study, the second nuclear internal transcribed spacer (ITS-2) rDNA of Schistosoma japonicum isolates in mainland China was amplified, sequenced, and assessed for inferring the intra- and inter-species phylogenetic relationships of trematodes in the order Strigeata. The fragment containing ITS-2 rDNA was obtained from 24 S. japonicum isolates from eight epidemic provinces in mainland China. The length polymorphisms were observed among these ITS-2 rDNA sequences, ranging from 343 to 346 bp, and the intra- and inter-population variations in ITS-2 sequence were 0.0-2.1% among S. japonicum isolates in China. Phylogenetic analyses using the maximum parsimony and maximum likelihood methods revealed that the ITS-2 rDNA sequence is not a suitable marker for studying inter- and intra-population variation in S. japonicum. However, phylogenetic analysis of trematodes in the order Strigeata indicated that the ITS-2 rDNA sequence provides an effective molecular marker for studying inter-species phylogenetic relationships among trematodes in this order.

Combined mitochondrial 16S and 12S rDNA sequences: an effective genetic marker for inter-species phylogenetic analysis of zoonotic trematodes.

The present study studied the genetic variation among Schistosoma japonicum isolates from different endemic regions in mainland China and examined the phylogenetic relationships of zoonotic trematodes using the combined mitochondrial 16S and 12S ribosomal DNA sequences. The fragments of 16S and 12S rDNA were amplified from 22 S. japonicum isolates, and sequenced, and the relevant sequences of other nine trematode species belonging to six genera in four families were downloaded from GenBank, and their phylogenetic relationships were re-constructed by unweighted pair-group method with arithmetic averages analyses using the combined 16S and 12S rDNA sequences, with Trichinella spiralis as outgroup. The results showed that the partial sequences of mitochondrial 16S and 12S rDNA of S. japonicum were 757 and 797 bp, respectively, and they were quite conserved among the S. japonicum isolates. Phylogenetic analysis revealed that the combined 16S and 12S rDNA sequences were not able to distinguish S. japonicum isolates in mountainous areas from those in lake/marshland areas in mainland China. However, the combined sequences could distinguish different species of zoonotic trematodes. Therefore, the combined mitochondrial 16S and 12S rDNA sequences provide an effective molecular marker for the inter-species phylogenetic analysis and differential identification of zoonotic trematodes.

Heterogeneity of class I and class II MHC sequences in Schistosoma japonicum from different endemic regions in mainland China.

The present study examined sequence variation in class I and class II major histocompatibility complex (MHC) genes among Schistosoma japonicum isolates from different endemic regions in mainland China and assessed the level of horizontal gene transfer and sequence similarity between parasites and their hosts. S. japonicum cercariae were used to infect male adult rabbits to obtain adult S. japonicum samples. A portion of the class I MHC gene (pMHC I) and class II MHC genes (pMHC II) were amplified separately from individual adult trematodes by polymerase chain reaction and sequenced. Among all the examined isolates of S. japonicum, sequence differences between male and female parasites were 0.0-26.6% for pMHC I and 0.0-7.0% for pMHC II. Sequence variations between male and female parasites among different geographical locations from the mountainous areas were 1.1-26.6% for pMHC I and 1.5-3.0% for pMHC II. Sequence variations between samples from Yunnan and those from Sichuan were 2.7-23.5% for pMHC I and 1.1-3.7% for pMHC II. In the lake/marshland areas, sequence variations between male and female parasites among different geographical locations were 0.0-25.0% for pMHC I and 0.0-7.0% for pMHC II. Sequence variations between S. japonicum isolates from mountainous areas, and those from lake/marshland areas were 0.0-26.1% for pMHC I and 0.4-6.1% for pMHC II. BLASTN analysis indicated that all the pMHC II sequences showed high homology to a portion of exon 3 in rabbit MHC class II DP beta gene with more than 89% similarity, and all the pMHC I sequences except isolates in Yunnan (Eryuan) revealed high homology to the portion of exon 2 in rabbit MHC I gene with more than 81% similarity. Phylogenetic analysis showed no specific clustering comprising parasites from single geographical or endemic regions, and the paired parasites were even found in different clusters. These results demonstrated that pMHC I and II of S. japonicum isolates in mainland China existed heterogeneity, but the pMHC I, II, or combined sequences were not suitable markers for examining genetic relationship among different isolates from endemic regions in mainland China.

Genetic variability among Schistosoma japonicum isolates from different endemic regions in China revealed by sequences of three mitochondrial DNA genes.

The present study examined sequence variation in three mitochondrial DNA (mtDNA) regions, namely cytochrome c oxidase subunit 3 (cox3), NADH dehydrogenase subunits 4 and 5 (nad4 and nad5), among Schistosoma japonicum isolates from different endemic regions in China, and their phylogenetic relationships were re-constructed. A portion of the cox3 gene (pcox3), a portion of the nad4 and nad5 genes (pnad4 and pnad5) were amplified separately from individual trematodes by polymerase chain reaction (PCR) and the amplicons were subjected to direct sequencing. In the mountainous areas, sequence variations between parasites from Yunnan and those from Sichuan were 0.3% for pcox3, 0.0-0.1% for pnad4, and 0.0-0.2% for pnad5. In the lake/marshland areas, sequence variations between male and female parasites among different geographical locations were 0.0-0.3% for pcox3, 0.0-0.7% for pnad4, and 0.0-1.6% for pnad5. Sequence variations between S. japonicum from mountainous areas and those from lake/marshland areas were 0.0-0.5% for pcox3, 0.0-0.7% for pnad4, and 0.0-1.6% for pnad5. Phylogenetic analyses based on the combined sequences of pcox3, pnad4 and pnad5 revealed that S. japonicum isolates from mountainous areas (Yunnan and Sichuan provinces) clustered together. For isolates from the lake/marshland areas, isolates from Anhui and Jiangsu provinces clustered together and was sister to samples from Jiangxi province, while isolates from Hubei and Zhejiang province clustered together. However, isolates from different geographical locations in Hunan province were in different clades. These findings demonstrated the usefulness and attributes of the three mtDNA sequences for population genetic studies of S. japonicum, and have implications for studying population biology, molecular epidemiology, and genetic structure of S. japonicum, as well as for the effective control of schistosomiasis.

Complete nucleotide sequence and genome organization of a Chinese isolate of tobacco bushy top virus.

The complete nucleotide sequence of a Chinese isolate of tobacco bushy top virus (TBTV), designated TBTV-Ch, was determined from cDNA generated from double-stranded RNA extracted from diseased tobacco. The genome is 4152 nucleotides (nt) in size, contains four putative open reading frames (ORFs) and untranslated regions of 10 nt and 645 nt at the 5' and 3' ends, respectively. In genome organization and in the amino acid sequence of its potential products, the RNA of TBTV-Ch is similar to other umbraviruses sequenced to date. The results suggested that TBTV should be regarded as a definitive species of the genus Umbravirus.

First Report of Tobacco Bushy Top Disease in China.

In 1993, a severe epidemic of a new disease of flue-cured tobacco (Nicotiana tabacum) occurred in western Yunnan Province, People's Republic of China. Since then, over 40,000 ha of tobacco have been affected, with an average incidence of ≈15%. Infected plants were stunted, and leaves showed symptoms of vein distortion, vein clearing, mottling, and rounding. Axillary buds sprouted from the main stem of infected plants early and formed lateral shoots, on which other shoots were produced. As a result, the infected plants presented the characteristic "bushy" appearance. Aphid- (Myzus persicae (Sulzer)) and sap-transmission experiments were conducted. In aphid-transmission tests, after an acquisition feeding period of 24 h on diseased tobacco, the shortest test feeding that resulted in infection was 5 min. The causal agent(s) was readily sap-transmissible, but it could not be transmitted from sap-inoculated plants to healthy plants by aphids. Thirty-two species of plants in eleven families were tested by sap-inoculation for infectivity to alternative hosts by the casual agent(s). All the species of Nicotiana tested were infected. All the hosts were restricted to the solanaceae. The symptoms, transmission, and host range of this disease were identical to those of tobacco bushy top disease in Zimbabwe (1). Using umbravirus-specific primers (3), a 550-bp DNA fragment was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from diseased tobacco, whose sequence (GenBank Accession No. AF402620) indicated the occurrence of an umbravirus. The results coincided with the taxonomic status of Tobacco bushy top virus, one of the causal agents of tobacco bushy top disease (1), a tentative species of the genus Umbravirus (2). To our knowledge, this is the first report of tobacco bushy top disease in China and the first report of a large outbreak of the disease outside sub-Saharan Africa. References: (1) L. F. Gates. Ann. Appl. Biol. 50:169, 1962. (2) F. A. Murphy. et al. Page 388 in: Virus Taxonomy: Sixth Report of the International Committee on Taxonomy of Viruses, Page 388, 1995. (3) P. Vercruysse et al. J. Virol. Methods 88:153, 2000.