Suprasellar cistern tuberculoma presenting as unilateral ocular motility disorder and ptosis: A case report.

BACKGROUND: Intracranial tuberculoma is a rare and serious type of tuberculosis, which mostly occurs in the frontoparietal and cerebellar hemispheres, with predominance in the gray-white matter junction area, while tuberculomas only in the cistern are extremely rare with only a few reported cases in the literature. We describe a unique case of isolated tuberculoma in the suprasellar cistern, with only right ocular motility disorder and upper eyelid ptosis. CASE SUMMARY: A 5-year-old boy without any medical history presented with right ocular motility disorder and upper eyelid ptosis one month ago. He had no history of fever, headache, vomiting, convulsions, or limb weakness. Neurological examination showed right third cranial nerve palsy with restrictions of eye movements and ptosis, pupil dilation and negative light reflex. Imaging suggested a space-occupying lesion in the suprasellar cistern with calcification and ring-enhancement. Moreover, no Mycobacterium tuberculosis was found in cerebrospinal fluid by polymerase chain reaction (PCR). The lesion was initially diagnosed as a tumor, while postoperative pathology combined with PCR indicated tuberculoma. The patient continued postoperative anti-tuberculosis treatment. At present, the patient's condition is stable and the symptoms are partially relieved compared with those before surgery. CONCLUSION: This case confirmed that isolated intracranial tuberculoma can occur in the suprasellar cistern. Therefore, for space-occupying lesions in the suprasellar cistern, tuberculoma should be included in the differential diagnosis even if there is no history or indication of tuberculosis infection.

Enhanced expression of the Erns protein of classical swine fever virus in yeast and its application in an indirect enzyme-linked immunosorbent assay for antibody differentiation of infected from vaccinated animals.

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a devastating disease of swine worldwide. Although a mandatory vaccination with the modified live vaccine C-strain has been implemented in China for decades, CSF remains a serious threat to the swine industry. To facilitate the control and eradication of CSF in China, the E2-based marker vaccine rAdV-SFV-E2, an adenovirus-delivered, alphavirus replicon-vectored vaccine, has been developed. Accordingly, an accompanying discriminatory test that allows differentiating infected from vaccinated animals (DIVA) is required. Here, the enhanced expression of E(rns) protein of CSFV was achieved in the methyltropic yeast Pichia pastoris by codon-optimization of the E(rns) gene, and an indirect enzyme-linked immunosorbent assay (iELISA) based on the yeast-expressed E(rns) (yE(rns)) was developed and evaluated. The optimized iELISA was able to detect CSFV-specific antibodies in the serum samples from the CSFV-infected pigs as early as 6 days post-infection, and discriminate the CSFV-infected pigs from those vaccinated with rAdV-SFV-E2. The iELISA was evaluated using a panel of swine sera, and showed comparable sensitivity (94.6%) and specificity (97.1%), and the consistence rates with the virus neutralization test were 96.8% for CSFV-infected swine sera, 83.3% for C-strain-vaccinated swine sera, and 95.0% for field swine sera. In addition, the iELISA showed higher sensitivity (90.4%) compared with PrioCHECK CSFV E(rns) (59.6%). Taken together, the yE(rns)-based iELISA is specific and sensitive, representing a promising DIVA test for E2-based marker vaccines against CSF.

Effects of the nuclear localization of the N(pro) protein of classical swine fever virus on its virulence in pigs.

The N(pro) protein of classical swine fever virus (CSFV) is localized in the cytoplasm and nucleus. However, it is unknown whether the nuclear localization of N(pro) correlates with the virulence of CSFV in the host. Previously, we showed that the N(pro) protein fused with interferon regulatory factor 3 (IRF3) was present only in the cytoplasm. Here, we generated and evaluated a recombinant CSFV vSM-IRF3 harboring the IRF3 gene inserted into the N(pro) gene of the highly virulent CSFV Shimen strain. Compared to the even nuclear and cytoplasmic distribution of the enhanced green fluorescent protein (EGFP)-N(pro) fusion expressed by the recombinant CSFV EGFP-CSFV, vSM-IRF3 expressed an IRF3-N(pro) fusion protein that only was localized in the cytoplasm. vSM-IRF3 was markedly attenuated in vitro and in vivo, and the inoculated pigs were completely protected from lethal CSFV challenge, whereas the parental virus as well as EGFP-CSFV exhibited a typical virulent phenotype. Taken together, the nuclear localization of N(pro) plays a significant role in the CSFV replication and virulence.

Pathogenicity and genomic characterization of a pseudorabies virus variant isolated from Bartha-K61-vaccinated swine population in China.

Pseudorabies (PR) or Aujeszky's disease (AD), caused by pseudorabies virus (PRV), is an economically important viral disease worldwide. Recently, PR outbreaks occurred in a large number of Bartha-K61-vaccinated swine herds in many regions of China. Here, we isolated a PRV variant, named TJ strain, from a Bartha-K61-vaccinated pig farm in China, evaluated the pathogenicity of the TJ strain in susceptible animals and analyzed its complete genomic sequence obtained by 454 pyrosequencing. Vaccination-challenge experiment in sheep showed that the classical Bartha-K61 vaccine could not provide complete protection against the challenge with the PRV TJ strain. In mice, the 50% lethal dose (LD50) of the TJ strain (10(2.3) TCID50) was lower than that of the classical PRV SC strain (10(3.0) TCID50). Furthermore, the TJ strain displayed higher mortality for pigs, as compared with the SC strain. The PRV TJ strain genome was determined to be 143,642 bp in length, encoding 67 open reading frames. The TJ strain was clustered to an independent branch together with some recent PRV isolates in China in the phylogenetic tree, which was relatively distant from previous PRV isolates. The TJ strain showed unique variations in the viral proteins that play key roles in the viral replication cycle. Taken together, the TJ strain is a highly pathogenic PRV variant with unique molecular signatures. Further studies are needed to explore the relevance of the sequence differences to the virulence alteration of the PRV variant.

Visualization of the Npro protein in living cells using biarsenically labeling tetracysteine-tagged classical swine fever virus.

Real-time fluorescence imaging of viral proteins in living cells is a valuable means to study virus-host interactions, and tetracysteine (TC)-biarsenical technology has been used in several viruses but not in classical swine fever virus (CSFV). Here, we generated CSFV mutants vSMTC385 or vSMTC412 bearing the small TC tag (CCPGCC) in the N-terminal region of the N(pro) protein. The mutants showed growth characteristics indistinguishable from that of the wild-type virus, and retained similar N(pro) subcellular localization to that of the parent virus. Furthermore, labeling with membrane-permeable biarsenical dye resulted in the fluorescent N(pro) protein in the context of virus infection. Finally, we showed that N(pro) was localized in the cytoplasm of CSFV-infected cells at 27 h post-infection (hpi) and present in the nucleus at 48 hpi, and the nuclear import and export was clearly observed from 36.5 to 37 hpi. Interestingly, our results demonstrated that N(pro) transported across the nuclear pores by passive diffusion, which might be prevented by exogenous interferon regulatory factor 3 interacting with N(pro). Taken together, biarsenical labeling allows real-time visualization of the nucleus import and export of the fluorescent N(pro) protein in CSFV-infected living cells.

The role of noncoding regions of classical swine fever virus C-strain in its adaptation to the rabbit.

Classical swine fever (CSF) is a devastating disease of swine caused by classical swine fever virus (CSFV). C-strain, a modified live vaccine against CSF, was developed through hundreds of passages of a highly virulent CSFV in the rabbit in China in the mid-1950s. To identify the role of noncoding regions (NCRs) of C-strain in its adaptation to the rabbit, we generated and evaluated a series of chimeric viruses derived from C-strain and the highly virulent Shimen strain. The results demonstrated that the NCRs of the C-strain were essential for its fever induction in rabbits and the coding region but not NCRs was necessary for its replication in the spleen of rabbits.

Beta-actin interacts with the E2 protein and is involved in the early replication of classical swine fever virus.

The E2 glycoprotein of classical swine fever virus (CSFV) is involved in viral infection and induction of neutralizing antibodies. Little is known about how E2 interacts with host cells. To understand the cellular factors involved in viral replication cycle, E2 was used as bait in yeast two-hybrid screens, resulting in the identification of ß-actin as a potential E2-interacting partner. E2-ß-actin interaction was confirmed by co-immunoprecipitation, GST pulldown, laser confocal and bimolecular fluorescence complementation assays. The E2-interacting domain of ß-actin was mapped to amino acids (aa) 95-188 and two ß-actin-interacting regions were identified in E2 (aa 182-261 and aa 262-341). Knockdown of ß-actin by RNA interference and disruption of filamentous ß-actin with cytochalasin D at 4h post-infection caused a significant reduction of viral RNA copies and titers. Collectively, the results indicated that ß-actin is involved in the early replication of CSFV.

[Metagenomics-based detection of swine viruses].

UNLABELLED: Extreme varieties of viruses exist in the environment and animals, some of which are unknown. However, many unknown viruses are barely detected by means of conventional virus isolation and PCR assay. OBJECTIVE: To develop a technology platform for detecting unknown viruses. METHODS: We established the technology based on viral metagenomics in combination with novel molecular diagnostics. The technology is consisted of removal of host nucleic acid, random PCR amplification, large-scale sequencing, and bioinformatics. RESULTS: The technology was applied to detect classical swine fever virus (CSFV)-infected cells and a tissue sample of a pig infected with porcine circovirus type 2 (PCV2). We amplified 13.7% sequences of CSFV genome and 47.2% those of PCV2 genome, respectively. Moreover, we amplified 16.4% sequences of the simian parainfluenza virus type 5 genome from an unknown virus cell culture using the developed method. In addition, using the developed method combined with the high-throughput sequencing, we detected 1.1% virus sequences, including CSFV, PCV2, torque teno sus virus (TTSuV), porcine bocavirus (PBoV) and human adenovirus type 6 (Ad6) from 7 clinical swine samples of unknown causative agents. CONCLUSION: The developed metagenomics-based method showed good sensitivity for detection of both DNA and RNA viruses from diverse swine samples, and has potential for universal detection of known and unknown viruses. It might facilitate the diagnosis of emerging viral diseases.

Comprehensive evaluation of the adenovirus/alphavirus-replicon chimeric vector-based vaccine rAdV-SFV-E2 against classical swine fever.

Classical swine fever (CSF) is an economically important, highly contagious swine disease caused by classical swine fever virus (CSFV). Marker vaccines and companion serological diagnostic tests are thought to be a promising strategy for future control and eradication of CSF. Previously, we have demonstrated that an adenovirus-vectored Semliki forest virus replicon construct expressing the E2 glycoprotein from CSFV, rAdV-SFV-E2, induced sterile immunity against a lethal CSFV challenge. In this study, we further evaluated the vaccine with respect to its safety, number and dose of immunization, and effects of maternally derived antibodies, re-immunization of the vaccine or co-administration with pseudorabies vaccine on the vaccine efficacy. The results showed that: (1) the vaccine was safe for mice, rabbits and pigs; (2) two immunizations with a dose as low as 6.25×10(5) TCID(50) or a single immunization with a dose of 10(7) TCID(50) rAdV-SFV-E2 provided complete protection against a lethal CSFV challenge; (3) maternally derived antibodies had no inhibitory effects on the efficacy of the vaccine; (4) the vaccine did not induce interfering anti-vector immunity; and (5) co-administration of rAdV-SFV-E2 with a live pseudorabies vaccine induced antibodies and protection indistinguishable from immunization with either vaccine administered alone. Taken together, the chimeric vaccine represents a promising marker vaccine candidate for control and eradication of CSF.

Poly(C)-binding protein 1, a novel N(pro)-interacting protein involved in classical swine fever virus growth.

N(pro) is a multifunctional autoprotease unique to pestiviruses. The interacting partners of the N(pro) protein of classical swine fever virus (CSFV), a swine pestivirus, have been insufficiently defined. Using a yeast two-hybrid screen, we identified poly(C)-binding protein 1 (PCBP1) as a novel interacting partner of the CSFV N(pro) protein and confirmed this by coimmunoprecipitation, glutathione S-transferase (GST) pulldown, and confocal assays. Knockdown of PCBP1 by small interfering RNA suppressed CSFV growth, while overexpression of PCBP1 promoted CSFV growth. Furthermore, we showed that type I interferon was downregulated by PCBP1, as well as N(pro). Our results suggest that cellular PCBP1 positively modulates CSFV growth.