The power and the promise of synthetic lethality for clinical application in cancer treatment.

Synthetic lethality is a phenomenon wherein the simultaneous deficiency of two or more genes results in cell death, while the deficiency of any individual gene does not lead to cell death. In recent years, synthetic lethality has emerged as a significant topic in the field of targeted cancer therapy, with certain drugs based on this concept exhibiting promising outcomes in clinical trials. Nevertheless, the presence of tumor heterogeneity and the intricate DNA repair mechanisms pose challenges to the effective implementation of synthetic lethality. This review aims to explore the concepts, development, and ethical quandaries surrounding synthetic lethality. Additionally, it will provide an in-depth analysis of the clinical application and underlying mechanism of synthetic lethality.

Identification and validation of functional roles for three MYC-associated genes in hepatocellular carcinoma.

BACKGROUND: Aberrations in MYC underlie a large proportion of liver hepatocellular carcinoma (LIHC) cases; however, MYC is difficult to target because of its undruggable structure. We aimed to uncover MYC-associated molecular targets to provide new strategies for LIHC treatment. METHODS: LIHC transcriptome datasets and clinical information were obtained from The Cancer Genome Atlas. A series of bioinformatics analyses were performed for 370 patients who were stratified based on the median MYC expression level (high-MYC group and low-MYC group). Correlation analysis was performed to determine relationships between the expression of key MYC-associated genes and prognosis, DNA promotor methylation, and immune cell infiltration. Gene ontology and Kyoto Encyclopedia of Genes and Genomes Pathway enrichment analyses were performed to elucidate the functions of these genes in LIHC. Their expression and functions in LIHC were further verified using transgenic mice overexpressing c-Myc under control of the hepatocyte-specific promoter (Alb-Cre). RESULTS: AURKB, CCNB2, and CDKN3 were overexpressed in LIHC patients with high MYC expression and were associated with poor prognosis. Upregulation of these 3 genes was significantly correlated with hypomethylated promoter status, advanced T stage, metastasis, and immune cell infiltration in LIHC patients. Functional enrichment analyses indicated that these genes participate in the "p53 signaling pathway" and "cell cycle". Furthermore, RT-PCR and IHC analysis revealed that their mRNA and protein expression levels were upregulated in an Alb-Cre;cMYClsl/- mouse model. Drugs that target these 3 MYC-related genes were identified. CONCLUSION: Taken together, our results identify biomarkers of potential utility for managing liver cancer therapy owing to their significance in tumorigenesis, proliferation, and tumor immunity.

Recent trends in the incidence and survival of stage I liver cancer: a surveillance, epidemiology, and end results analysis.

BACKGROUND: Improvements in screening and imaging technologies and treatment of liver disease have influenced the trend in diagnosis for stage I liver cancer. In this article, recent trends in age, incidence, tumour size, and survival of different stages of liver cancer are analysed. METHODS: Surveillance, Epidemiology, and end results data from the National Cancer Institute were used to analyse trends in age-adjusted incidence rate, mean tumour size at diagnosis, age at diagnosis, and 5-year survival probability for stage I liver cancer. RESULTS: Stage I cases of liver cancer increased most tremendously over the study period, with a greater increase from 2004 to 2012 following a smaller increase from 2012 to 2015. Moreover, the mean age of stage I liver cancer increased by 1.72 years from 2004 to 2015. The 5-year-overall survival for stage I liver cases worsened from 97.9% to 83.7% from 2004 to 2011, whereas the 10-year survival probability for stage I cases worsened from 97.3% in 2004 to 79.6% in 2006. Comparing with higher stage cases, stage I liver cancer were more likely to be females, be married, live in metro areas, receive chemotherapy, and carry medical insurance. CONCLUSIONS: The incidence of stage I liver cancer has increased over the study period, with an increase in age of diagnosis, decrease in tumour size, and generally stable overall survival rate with slight decrease. These trends emphasized the importance of early detection of liver cancer and regular screening and better treatment for high-risk populations.RESEARCH HIGHLIGHTSImprovements in screening and imaging technologies and treatment of liver disease have influenced the trend in diagnosis for liver cancer.Stage I cases of liver cancer increased most tremendously over the study period, with a greater increase from 2004 to 2012 following a smaller increase from 2012 to 2015.These trends emphasized the importance of early detection of liver cancer and regular screening and better treatment for high-risk populations.

An N-nitrosation reaction-based fluorescent probe for detecting nitric oxide in living cells and inflammatory zebrafish.

Nitric oxide (NO), an essential biological messenger molecule, participates in various physiological and pathological processes. The sensitive and specific detection of NO is of great significance for understanding the biological function of NO. Here, we synthesized a fluorescent probe (Rho-NO) for highly selective detection of NO both in vitro and in vivo. The high selectivity of Rho-NO is attributed to the fact that NO is easily replaced by electron donor amino group to form N-nitrosation products, causing rhodamine spiro ring open and fluorescence emit. Rho-NO showed a good linear response to NO (0-100 µM) with a low detection limit (0.06 µM). Importantly, it exhibited excellent specificity for NO detection in human serum and was also applied for imaging NO in living cells and inflammatory model of zebrafish. This work proves the potential of Rho-NO in pathological research and disease diagnosis.

Deciphering cell-cell interactions and communication in the tumor microenvironment and unraveling intratumoral genetic heterogeneity via single-cell genomic sequencing.

A tumor's heterogeneity has important implications in terms of its clonal origin, progression, stemness, and drug resistance. Therefore, because of its significance in treatment, it is important to understand the gene expression pattern of a single cell, track gene expression or mutation in heterogeneous cells, evaluate the clonal origin of cancer cells, and determine the selective evolution of different subpopulations of cancer cells. Researchers are able to trace a cell's mutation and identify different types of tumor cells by measuring the whole transcriptome with single-cell sequencing (scRNA-seq). This technology provides a better understanding of the molecular mechanisms driving tumor growth than that offered by traditional RNA sequencing methods. In addition, it has revealed changes in the mutations and functions of somatic cells as a tumor evolves; it has also clarified immune cell infiltration and activation. Research on scRNA-seq technology has recently advanced significantly, suggesting new strategies for the treatment of cancer. In short, cancer researchers have become increasingly dependent on scRNA-seq. This paper reviews the development, detection principles, and processes of scRNA-seq technology and their application in tumor research. It also considers potential clinical applications.

Heterogeneity helps us determine the clonal origin, progression, and drug resistance of cancer cells.The gene expression pattern of a single cell has important biological significance.scRNA-seq enables a better understanding of cancer cells' molecular mechanisms.scRNA-seq provides information about the entirety of a tumor from what we can learn about a single cell.

Effect of Mulberry Leaf powder on reproductive performance, serum indexes and milk amino acid composition in lactating sows.

Experiment was conducted to study the effects of Mulberry Leaf (ML) powder on reproductive performance, serum and milk amino acid composition in sows. Fifty sows (D 85 at gestation) with parity 3 or 4 were randomly divided into 5 groups: C, M100, M200, M300 and M400, receiving 0, 100, 200, 300 and 400 g ML powder per sow per day. Blood and milk of sows at Days 1 and 21 of lactation were collected. Results showed that average daily feed intake (ADFI) during lactation was higher in groups supplemented ML compared with control group (p < 0.01). Litter weight gain during lactation was higher in M400 than in groups M200 and C (p < 0.05), with no significant difference compared with M100 and M300. Serum glucose concentration in groups M400 and M300 was higher than those in the other groups (p < 0.01). Serum HDL-C concentration in group M400 was significantly greater than those in groups M100 and M200 (p < 0.05), with no significant difference between group M400 and groups M300, control. Milk amino acid concentrations such as isoleucine, leucine, lysine and valine were all lower in group M400 than in control (p < 0.01). Serum methionine (Met) concentration was higher in M300 than in other groups (p < 0.01). Milk Met concentration in group C was higher than those of the sows in the group M400, with no significant difference compared with groups M100, M200 and M300 (p < 0.05). Serum Lys and Met concentrations were lower in M400 than in control group (p < 0.05). In summary, our results have revealed the ML supplementation at a high dose such as 300 g/day during later gestation and lactation showed benefit in regulating lipid and amino acid metabolism in sows and then improved growth performance of their offspring.

Isolation and Characterization of Encephalomyocarditis Virus from Dogs in China.

Encephalomyocarditis virus (EMCV) is as a potential zoonotic agent with a wide host range. Here, we describe an EMC virus isolate, identified as EMCV C15, which was successfully obtained from the serum of dogs from animal hospitals. Virus production in cell culture was confirmed by EMCV-specific real-time RT-PCR, indirect immunofluorescence assays and electron microscopy. In addition, the open reading frame sequence (ORF) of the EMCV C15 virus was determined. From sequence comparison and phylogenetic analysis among 24 reference EMCV strains, it appears that the EMCV C15 strain is closely genetically related to strain BEL2887A/91 (>99.0% nucleotide identity). In artificially challenged dogs, the heart and brain were important targets of EMCV C15. This study provides genetic and pathogenic characterization of the EMCV C15 strain isolated in Beijing and calls for sustained surveillance of EMCV infection in China to support better prevention and control of the disease.

Genome mining and motif truncation of glycoside hydrolase family 5 endo-ß-1,4-mannanase encoded by Aspergillus oryzae RIB40 for potential konjac flour hydrolysis or feed additive.

Two novel glycosyl hydrolase family 5 (GH5) ß-mannanases (AoMan5A and AoMan5B) were identified from Aspergillus oryzae RIB40 by genome mining. The AoMan5A contains a predicted family 1 carbohydrate binding module (CBM-1), located at its N-terminal. The AoMan5A, AoMan5B and truncated mutant AoMan5AΔCL (truncating the N-terminal CBM and linker of AoMan5A) were expressed retaining the N-terminus of the native protein in Pichia pastoris GS115 by pPIC9KM. The specific enzyme activity of the purified reAoMan5A, reAoMan5B and reAoMan5AΔCL towards locust bean gum at pH 3.6 and 40°C for 10min, was 8.3, 104.2 and 15.8U/mg, respectively. The temperature properties of the reAoMan5AΔCL were improved by truncating CBM. They can degrade the pretreated konjac flour and produce prebiotics. In addition, they had excellent stability under simulative gastric fluid and simulative prilling process. All these properties make these recombinant ß-mannanases potential additives for use in the food and feed industries.

Full-length genomic characterization and molecular evolution of canine parvovirus in China.

Canine parvovirus type 2 (CPV-2) can cause acute haemorrhagic enteritis in dogs and myocarditis in puppies. This disease has become one of the most serious infectious diseases of dogs. During 2014 in China, there were many cases of acute infectious diarrhoea in dogs. Some faecal samples were negative for the CPV-2 antigen based on a colloidal gold test strip but were positive based on PCR, and a viral strain was isolated from one such sample. The cytopathic effect on susceptible cells and the results of the immunoperoxidase monolayer assay, PCR, and sequencing indicated that the pathogen was CPV-2. The strain was named CPV-NY-14, and the full-length genome was sequenced and analysed. A maximum likelihood tree was constructed using the full-length genome and all available CPV-2 genomes. New strains have replaced the original strain in Taiwan and Italy, although the CPV-2a strain is still predominant there. However, CPV-2a still causes many cases of acute infectious diarrhoea in dogs in China.

A recombinant porcine circovirus type 2 expressing the VP1 epitope of the type O foot-and-mouth disease virus is infectious and induce both PCV2 and VP1 epitope antibodies.

Porcine circovirus type 2 (PCV2) is the etiological agent of postweaning multisystemic wasting syndrome, a disease that causes huge economic damage in swine industry. A recombinant PCV2 expressing the neutralizing VP1 epitope (aa 141-160) of the foot-and-mouth disease virus (FMDV) was rescued using an infectious cloning technique. The PCV2 antigen and FMDV-VP1 antigenic epitope of the cloned strain recPCV2-CL-VP1 were confirmed by an immunoperoxidase monolayer assay (IPMA) and a capture enzyme-linked immunosorbent assay (ELISA). The morphological features of the recPCV2-CL-VP1 were not discernibly different from those of its parental strain (PCV2-CL). However, the recombinant virus could be differentiated from its parental virus by PCR and capture ELISA. The recPCV2-CL-VP1 was demonstrated to replicate stably in PK-15 cells through ten passages. An infection experiment using BALB/c mice showed that both recPCV2-CL-VP1 and PCV2-CL could replicate in the mice, cause various pathological changes, and induce a high level of anti-Cap antibodies. The recombinant virus emulsified with Freund's adjuvant was used to immunize BALB/c mice and induced antibodies against the FMDV-VP1 epitope. Hence, the recombinant PCV2 strain, which expressed the neutralizing FMDV-VP1 epitope, provides a valuable platform to develop novel genetic vaccines.

Development and evaluation of the rVP-ELISA for detection of antibodies against porcine parvovirus.

The gene encoding the VP2 protein of porcine parvovirus (PPV) was expressed in an insect-baculovirus system. The recombinant (r) VP2 was similar antigenically/functionally to the native capsid protein as demonstrated by hemagglutination (HA), Western blotting using PPV positive sera. The purified rVP2 proteins were used as coating antigen to establish a rVP-ELISA method for detection of PPV positive and negative sera from pigs. The optimal operating conditions of the rVP-ELISA were: the concentration of rVP2 proteins coated on the wells was 2 µg/mL; the diluted concentration of serum was 1: 150 and that of the enzyme-labeled antibody was 1: 6000. A total of 596 sera were detected by this assay, and the average positive rate was 87%. Compared with France LSI kit, the result showed that the coincidence rate was 96.7%. In conclusion, the rVP2-ELISA is a sensitive and specific method for detecting antibodies against PPV.

A new nanoPCR molecular assay for detection of porcine bocavirus.

Nanoparticle-assisted polymerase chain reaction (nanoPCR) is a novel method for the rapid amplification of DNA and has been used for the detection of virus. For detection of porcine bocavirus (PBoV), a sensitive and specific nanoPCR assay was developed with a pair of primers that were designed based on NS1 gene sequences available in GenBank. Under the optimized conditions of the PBoV nanoPCR assay, the nanoPCR assay was 100-fold more sensitive than a conventional PCR assay. The lower detection limit of the nanoPCR assay was about 6.70×10(1) copies. The nanoPCR assay amplified the specific 482-bp fragment of the PBoV NS1 recombinant plasmid but did not produce any product with genomic DNA or cDNA of porcine parvovirus, porcine circovirus type II, porcine reproductive and respiratory syndrome virus, pseudorabies virus, classic swine fever virus, Encephalomyocarditis virus, Porcine Teschovirus or African swine fever virus plasmid. Of 65 clinical samples collected from diseased pigs, 73.8% and 86.2% were determined to be PBoV positive by PBoV conventional PCR and PBoV nanoPCR assay, respectively. Of 36 clinical samples from healthy pigs, 27.8% and 44.4% were PBoV positive by PBoV conventional PCR and PBoV nanoPCR assay, respectively. The nanoPCR assay will be useful for diagnosing PBoV and for studying its epidemiology and pathology.

Correlation of the cyclin A expression level with porcine circovirus type 2 propagation efficiency.

Porcine circovirus type 2 (PCV2) is an important pathogen in swine, and it is assumed that PCV2 replication is cell cycle dependent (especially during S phase). However, the cellular molecules that regulate PCV2 replication have not been fully identified. Here, we cloned the porcine cyclin A (CycA) and CDK2 genes, the major regulators of the S phase, and established CycA or CDK2 overexpression and lower-expression cell lines. The propagation efficiency of strains PCV2a/CL or PCV2b/YJ in these cell lines was investigated using a capture enzyme-linked immunosorbent assay (ELISA) or an immunoperoxidase monolayer assay (IPMA), and the cell cycle was analyzed by flow cytometry. The results showed that CycA overexpression suppressed PCV2 replication. In contrast, CycA down-regulation by shRNA induced increases during the S and G2/M phases and resulted in increased PCV2 propagation. In contrast, overexpression or lower expression of CDK2 exhibited no significant influence on PCV2 replication. Furthermore, the subcellular localization of the PCV2 replicase protein (Rep) and capsid protein (Cap), CycA, and CDK2 in PK-15 cells was analyzed by confocal microscopy. The results showed that overexpression of CycA, rather than CDK2, altered normal nuclear localization of PCV2-Rep, which was transferred to the cytoplasm. In conclusion, PCV2 replication is both S- and G2/M-phase dependent and CycA, is an important regulator of the PCV2 life cycle.

Experimental infection and comparative genomic analysis of a highly pathogenic PRRSV-HBR strain at different passage levels.

A highly pathogenic strain of porcine reproductive and respiratory syndrome virus (PRRSV-HBR) was passaged on Marc-145 cells for 125 passages. In order to elucidate the change in virulence of PRRSV-HBR strain during the process of passage in vitro, swine infection experiment was performed with the viruses of low (F5 and F10) and high passage (F125). In addition, to identify the mutations related to the change in virulence of PRRSV-HBR strain, we compared and analyzed the genomic sequences of the F5, F10 and F125 of the strain. The virulence of F125 was significantly lower than that of F5 in the virus-infected pigs. In comparison with F5 and F125, there were 45 amino acids (aa) mutations and a deletion of 2 continuous aa by means of the virus genome sequence analysis. For these mutations, 33 aa (73.3%) occurred in the viral nonstructural proteins and the other 12 aa (26.7%) were contained in the viral structural proteins. Of the mutations, only 15 aa (33.3%) appeared in F10 and 30 aa (66.7%) occurred during passage from F10 to F125. The data showed that the latter 30 aa mutations were probably associated with attenuation of PRRSV-HBR strain, and that the change in virulence of the virus was determined by multiple alterations both in the structural and nonstructural genes. The virulence of PRRSV-HBR strain was remarkably attenuated after serial passages, and it can be used as vaccine candidate for control of the PRRS.

Prevalence of porcine hokovirus and its co-infection with porcine circovirus 2 in China.

Here, we report the frequency of porcine hokovirus (PHoV) infection and its co-infection with porcine circovirus 2 (PCV2) in China. A total of 485 domestic pig samples were tested for both PHoV and PCV2, and NS1 gene sequences from 11 PHoV strains were used for phylogenetic analysis. The prevalence of PHoV and PCV2 was 51.3 % and 36.3 %, respectively, and co-infection occurred in 20.2 %. PHoVs from the Chinese mainland showed a close relationship to those isolated in Hong Kong. Co-infection with both viruses was prevalent, and PHoV may contribute to the induction of postweaning multisystemic wasting syndrome (PMWS).

Amino acid mutations in the capsid protein produce novel porcine circovirus type 2 neutralizing epitopes.

Porcine circovirus type 2 (PCV2) is associated with postweaning multisystemic wasting syndrome in pigs. A monoclonal antibody (mAb) 8E4 against the PCV2 capsid protein has the capacity to neutralize the virus. However, this mAb can only react with some PCV2a strains (LG, CL, and JF2; mAb 8E4-positive strains), but does not cross-react with some PCV2b strains (YJ and JF; mAb 8E4-negative strains). In the present study, site-directed mutagenesis was performed targeting the external amino acids of the capsid proteins, which are different between mAb 8E4-positive and -negative strains. A mutation of arginine to alanine at position 59 in the capsid protein of strain JF allowed the mutant to be recognized and neutralized by mAb 8E4. Likewise, mutations of arginine to alanine at position 59 together with alanine to threonine at position 60 in the capsid protein of the YJ strain resulted in a gain of neutralization and recognition by mAb 8E4. Here, we demonstrated that the amino acids at positions 59 and 60 in the capsid protein of PCV2 participate in the formation of conformational neutralizing epitopes and mutations at positions 59 or 59/60 result in novel neutralizing epitopes of mAb 8E4-negative strains. This study provides valuable information for further in-depth mapping of the conformational neutralizing epitopes, clarification of antigenic differences among PCV2 strains, and development of a useful vaccine candidate for control of PCV2-associated diseases.

Enhanced protective immune response to PCV2 subunit vaccine by co-administration of recombinant porcine IFN-γ in mice.

The capsid (Cap) protein of PCV2 is the major immunogenic protein that is crucial to induce PCV2-specific neutralizing antibodies and protective immunity; thus, it is a suitable target antigen for the research and development of genetically engineered vaccines against PCV2 infection. IFN-γ has exhibited potential efficacy as an immune adjuvant that enhances the immunogenicity of certain vaccines in experimental animal models. In this study, three recombinant proteins: PCV2-Cap protein, porcine IFN-γ (PoIFN-γ), and the fusion protein (Cap-PoIFN-γ) of PCV2-Cap protein and PoIFN-γ were respectively expressed in the baculovirus system, and analyzed by Western blot and indirect ELISA. Additionally, we evaluated the enhancement of the protective immune response to the Cap protein-based PCV2 subunit vaccine elicited by co-administration of PoIFN-γ in mice. Vaccination of mice with the PCV2-Cap+PoIFN-γ vaccine elicited significantly higher levels of PCV2-specific IPMA antibodies, neutralizing antibodies, and lymphocyte proliferative responses compared to the Cap-PoIFN-γ vaccine, the PCV2-Cap vaccine, and LG-strain. Following virulent PCV2 challenge, no viraemia was detected in all immunized groups, and the viral loads in lungs of the PCV2-Cap+PoIFN-γ group were significantly lower compared to the Cap-PoIFN-γ group, the LG-strain group, and the mock group, but slightly lower compared to the PCV2-Cap group. These findings suggested that PoIFN-γ substantially enhanced the protective immune response to the Cap protein-based PCV2 subunit vaccine, and that the PCV2-Cap+PoIFN-γ subunit vaccine potentially serves as an attractive candidate vaccine for the prevention and control of PCV2-associated diseases.

In vitro inhibition of transmissible gastroenteritis coronavirus replication in swine testicular cells by short hairpin RNAs targeting the ORF 7 gene.

BACKGROUND: Transmissible gastroenteritis (TGE) is a highly contagious viral disease of swine, characterized by severe vomiting, diarrhea, and high mortality. Currently, the vaccines for it are only partially effective and no specific drug is available for treatment of TGE virus (TGEV) infection. RNA interference has been confirmed as a new approach for controlling viral infections. In this study, the inhibitory effect of short hairpin RNAs (shRNAs) targeting the ORF 7 gene of TGEV on virus replication was examined. RESULTS: Four theoretically effective sequences of TGEV ORF 7 gene were designed and selected for construction of shRNA expression plasmids. In the reporter assays, three of four shRNA expression plasmids were able to inhibit significantly the expression of ORF 7 gene and replication of TGEV, as shown by real-time quantitative RT-PCR analysis of viral ORF 7 and N genes and detection of virus titers (TCID50/ml). Stable swine testicular (ST) cells expressing the shRNAs were established. Observation of the cytopathic effect and apoptosis, as well as a cell proliferation assay demonstrated that the three shRNAs were capable of protecting ST cells against TGEV destruction, with high specificity and efficiency. CONCLUSIONS: Our results indicated that plasmid-transcribed shRNAs targeting the ORF 7 gene in the TGEV genome effectively inhibited expression of the viral target gene and viral replication in vitro. These findings provide evidence that the shRNAs have potential therapeutic application for treatment of TGE.

A porcine circovirus type 2 (PCV2) mutant with 234 amino acids in capsid protein showed more virulence in vivo, compared with classical PCV2a/b strain.

BACKGROUND: Porcine circovirus type 2 (PCV2) is considered to be the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), which has become a serious economic problem for the swine industry worldwide. The major genotypes, PCV2a and PCV2b, are highly prevalent in the pig population and are present worldwide. However, another newly emerging PCV2b genotype mutant, which has a mutation in its ORF2-encoded capsid protein, has been sporadically present in China, as well as in other countries. It is therefore important to determine the relative virulence of the newly emerging PCV2b genotype mutant, compared with the existing PCV2a and PCV2b genotypes, and to investigate whether the newly emerging mutant virus induces more severe illness. METHODOLOGY/PRINCIPAL FINDINGS: Twenty healthy, 30-day-old, commercial piglets served as controls or were challenged with PCV2a, PCV2b and the newly emerging mutant virus. A series of indexes representing different parameters were adopted to evaluate virulence, including clinical signs, serological detection, viral load and distribution, changes in immune cell subsets in the peripheral blood, and evaluation of pathological lesions. The newly emerging PCV2 mutant demonstrated more severe signs compatible with PMWS, characterized by wasting, coughing, dyspnea, diarrhea, rough hair-coat and depression. Moreover, the pathological lesions and viremia, as well as the viral loads in lymph nodes, tonsils and spleen, were significantly more severe (P<0.05) for piglets challenged with the newly emerging mutant compared with those in the groups challenged with PCV2a and PCV2b. In addition, a significantly lower average daily weight gain (P<0.05) was recorded in the group challenged with the newly emerging PCV2 mutant than in the groups challenged with the prevailing PCV2a and PCV2b. CONCLUSIONS: This is believed to be the first report to confirm the enhanced virulence of the newly emerging PCV2 mutant in vivo.

Identification of two internal signal peptide sequences: critical for classical swine fever virus non-structural protein 2 to trans-localize to the endoplasmic reticulum.

BACKGROUND: The membrane topology and molecular mechanisms for endoplasmic reticulum (ER) localization of classical swine fever virus (CSFV) non-structural 2 (NS2) protien is unclear. We attempted to elucidate the subcellular localization, and the molecular mechanisms responsible for the localization of this protein in our study. The NS2 gene was amplified by reverse transcription polymerase chain reaction, with the transmembrane region and hydrophilicity of the NS2 protein was predicted by bioinformatics analysis. Twelve cDNAs of the NS2 gene were amplified by the PCR deletion method and cloned into a eukaryotic expression vector, which was transfected into a swine umbilical vein endothelial cell line (SUVEC). Subcellular localization of the NS2 protein was characterized by confocal microscopy, and western blots were carried out to analyze protein expression. RESULTS: Our results showed that the -NH2 terminal of the CSFV NS2 protein was highly hydrophobic and the protein localized in the ER. At least four transmembrane regions and two internal signal peptide sequences (amino acids103-138 and 220-262) were identified and thought to be critical for its trans-localization to the ER. CONCLUSIONS: This is the first study to identify the internal signal peptide sequences of the CSFV NS2 protein and its subcellular localization, providing the foundation for further exploration of this protein's function of this protein and its role in CSFV pathogenesis.