Subculturing cells have no effect on CRISPR/Cas9-mediated cleavage of UL30 gene in pseudorabies virus.

CRISPR/Cas9-mediated genome editing can inhibit virus infection by targeting the conserved regions of the viral genomic DNA. Unexpectedly, we found previously that pseudorabies virus (PRV) could escape from CRISPR/Cas9-mediated inhibition. In order to elucidate whether the escape of PRV from Cas9-mediated inhibition was due to cell deficiencies, such as genetic instability of sgRNA or Cas9 protein, the positive cells were passaged ten times, and PRV infection in the sgRNA-expressing cells was evaluated in the present study. The results showed that subculturing cells has no effect on Cas9-mediated cleavage of PRV. Different passages of PX459-PRV cells can stably express sgRNA to facilitate Cas9/sgRNA cleavage on the UL30 gene of PRV, resulting in a pronounced inhibition of PRV infection. Studies to elucidate the mechanism of PRV escape are currently in progress.

Detection of Kobe-type and Otsu-type Babesia microti in wild rodents in China's Yunnan province.

Babesiosis is an emerging tick-transmitted zoonosis prevalent in large parts of the world. This study was designed to determine the rates of Babesia microti infection among small rodents in Yunnan province, where human cases of babesiosis have been reported. Currently, distribution of Babesia in its endemic regions is largely unknown. In this study, we cataloged 1672 small wild rodents, comprising 4 orders, from nine areas in western Yunnan province between 2009 and 2011. Babesia microti DNA was detected by polymerase chain reaction in 4·3% (72/1672) of the rodents analyzed. The most frequently infected rodent species included Apodemus chevrieri and Niviventer fulvescens. Rodents from forests and shrublands had significantly higher Babesia infection rates. Genetic comparisons revealed that Babesia was most similar to the Kobe- and Otsu-type strains identified in Japan. A variety of rodent species might be involved in the enzootic maintenance and transmission of B. microti, supporting the need for further serological investigations in humans.

Identification of MHC I class genes in two Platyrrhini species.

The major histocompatibility complex is a diverse gene family that plays a crucial role in the adaptive immune system. In humans, the MHC class I genes consist of the classical loci of HLA-A, -B, and -C, and the nonclassical loci HLA-E, -F, and -G. In Platyrrhini species, few MHC class I genes have been described so far and were classified as MHC-E, MHC-F, and MHC-G, with MHC-G possibly representing a classical MHC class I locus while there were arguments about the existence of the MHC-B locus in Platyrrhini. In this study, MHC class I genes were identified in eight common marmosets (Callithrix jacchus) and two brown-headed spider monkeys (Ateles fusciceps). For common marmosets, 401 cDNA sequences were sequenced and 18 alleles were detected, including 14 Caja-G alleles and 4 Caja-B alleles. Five to eleven Caja-G alleles and one to three Caja-B alleles were detected in each animal. For brown-headed spider monkeys, 102 cDNA sequences were analyzed, and 9 new alleles were identified, including 5 Atfu-G and 4 Atfu-B alleles. Two or three Atfu-G and two Atfu-B alleles were obtained for each of animal. In phylogenetic analyses, the MHC-G and -B alleles from the two species and other Platyrrhini species show locus-specific clusters with bootstrap values of 86% and 50%. The results of pairwise sequence comparisons and an excess of non-synonymous nucleotide substitutions in the PBR region are consistent with the suggestion that Caja-G and Atfu-G may be classical MHC class I loci in the Platyrrhini species… But it appears that MHC-B locus of the two Platyrrhini species shares features with both classical and nonclasical MHC class I loci. Our results are an important addition to the limited MHC immunogenetic information available for the Platyrrhini species.

[Preparation of polyclonal antibody against sAPRIL and analysis of function in suppressing sAPRIL-mediated lymphocyte proliferation].

This study was aimed to prepare the polyclonal antibody against the soluble proliferation-inducing ligand (sAPRIL) antigen and to investigate its effects in suppressing sAPRIL mediated lymphocyte proliferation. Mutated recombinant sAPRIL protein, which lacks biological activity but maintains immunogenicity, was used as antigen to immunize humanized SCID mice. Sera were obtained at 6 weeks after immunization. Indirect ELISA and Western blot were used to detect the antibody titer and specificity. The inhibition of polyclonal antibodies on Raji and Jurkat cell proliferation stimulated by sAPRIL was assessed by the MTT assay. The results showed that the mutant of sAPRIL could induce the production of polyclonal antibodies against human sAPRIL. Western blot and indirect ELISA analyses indicated that the anti-serum had higher specificity with a titer of 1:640. Functional analysis revealed that these polyclonal antibodies significantly inhibited the proliferation of Raji and Jurkat cell stimulated by sAPRIL (p < 0.05). It is concluded the polyclonal antibody against human sAPRIL is successfully prepared, which can inhibit the proliferation of Raji and Jurkat cells stimulated by sAPRIL in vitro.