In this study, suspected classical swine fever (CSF) samples from the Guangxi Province of China were obtained from pigs with acute CSF, aborted fetuses, newborn pigs that died at 1-2 days of age, tonsils of healthy pigs, and leukocytes of immunized sows during 2001-2009. About 92 of 775 samples were found to be positive by RT-PCR, and 41 isolates were obtained. Phylogenetic analysis was performed on the 31 isolates by sequencing the E2 gene, and the isolates were found to cluster into two groups: (1) isolates from aborted fetuses (except GXGZ02), deceased newborn baby pigs, tonsils of healthy pigs, and leukocytes of immunized sows belonged to group 1.1, along with vaccine strain, HCLV, and standard virulent strain, Shimen, of China, and (2) 20 isolates from pigs with acute CSF belonged to group 2.1, 13 of which were clustered into subgroup 2.1b with isolates from other provinces of China, and 7 of which were clustered into subgroup 2.1a with isolates from Italy and Germany.

Subtilisin-like proteases are widely distributed and reported to be required for virulence in pathogenic fungi. In chestnut blight fungus Cryphonectria parasitica, prb1, encoding a putative subtilisin-like protease, was expressed and recombinant Prb1 protein was shown to have a protease activity in vitro. prb1-deleted mutants exhibited reduced total protease activity by 60%. The Δprb1 mutants showed a phenotype of reduced aerial hyphae, lower level of sporulation, and a significant reduction in virulence. Additionally, site-directed mutagenesis of Prb1 protein revealed that D195, H227, and S393 are critical for C. parasitica Prb1 function in vivo. Transcriptional analysis showed that deletion of prb1 also reduced the transcript accumulation levels for genes encoding key components of the heterotrimeric G-protein signaling pathway, including cpga1, cpgb1, cpgc1, and ste12. Furthermore, deletion of prb1 results in the accumulation of autophagic bodies in the fungus. Taken together, our results showed that prb1-encoded protease functions in the regulation of virulence, phenotypical traits, and autophagy in C. parasitica.

S-adenosylhomocysteine (SAH), formed after donation of the methyl group of S-adenosylmethionine (SAM) to a methyl acceptor, is reversibly hydrolyzed to adenosine (ADO) and homocysteine (HCY) by S-adenosylhomocysteine hydrolase (SAHH). In chestnut blight fungus (Cryphonectria parasitica), sahh, a hypovirus-regulated gene that encodes a deduced SAHH protein was shown to have an SAHH enzymatic activity in vitro. Deletion of sahh resulted in the increased accumulation of intracellular SAH and SAM but decreased ADO, and a remarkably increased accumulation of transcripts that encode adenosine kinase, methionine adenosyltransferase, and an O-methyltransferase, key components of the methylation pathway. The Δsahh knockout mutants showed a phenotype of slower growth rate, fewer aerial hyphae, loss of orange pigment, absence of asexual fruiting bodies and conidia, and a significant reduction in virulence. Deletion of sahh significantly reduced the accumulation level of transcripts of the cyp1 that encodes cyclophilin A as well as genes of the heterotrimeric G-protein signaling pathways including cpga1, cpgb1, and cpgc1 and ste12, a target activated by the MAP kinase cascade. Taken together, we demonstrated that SAHH is required for virulence and multiple traits of phenotype in C. parasitica, by regulation of the expression of genes involved in key process of the cell.

No abstract

In this study, a street rabies virus isolate, GXHXN, was obtained from the brain of one rabid cattle in Guangxi province of southern China. To characterize the biological properties of GXHXN, we first evaluated its pathogenicity using 4-week-old adult mice. GXHXN was highly pathogenic with a short incubation period and course of disease. Its LD50 of 10(-6.86)/mL is significantly higher than the LD50 of 10(-5.19)/mL of GXN119, a dog-derived rabies virus isolate. It also displayed a higher neurotropism index than the rRC-HL strain. However, the relative neurotropism index of GXHXN was slightly lower than that of GXN119. Analyzing antigenicity using anti-N and anti-G monoclonal antibodies (MAbs), all tested anti-N MAbs reacted similarly to GXHXN, CVS, and rRC-HL, but the reaction of anti-N MAbs to GXHXN was slightly different from GXN119. Moreover, 2/11 tested anti-G mAbs showed weaker reactivity to GXHXN than rRC-HL, whereas 4/11 showed stronger reactivity to GXHXN than CVS and GXN119, indicating that the structures of G might differ. In order to understand its genetic variation and evolution, the complete GXHXN genome sequence was determined and compared with the known 12 isolates from other mammals. A total of 42 nucleotide substitutions were found in the full-length genome, including 15 non-synonymous mutations. The G gene accounts for the highest nucleotide substitution rate of 0.70 % in ORF and an amino acid substitution rate of 0.95 %. Phylogenetic trees based on the complete genome sequence as well as the N and G gene sequences from 37 known rabies isolates from various mammals demonstrated that the GXHXN is closely related to the BJ2011E isolate from a horse in Beijing, the WH11 isolate from a donkey in Hubei, and isolates from dogs in the Fujian and Zhejiang provinces. These findings will be helpful in exploring the molecular mechanisms underlying interspecies transmission and the genetic variation of the rabies virus in different mammal species.