Kaiyuan Cao

New Hepatitis E Virus Genotype in Bactrian Camels, Xinjiang, China, 2013.

To the Editor: Hepatitis E virus (HEV) is a member of the family Hepeviridae, genus Orthohepevirus, which comprises 4 species, Orthohepevirus A–D. Orthohepevirus A contains 7 genotypes (HEV1–7) (1,2). HEV1 and HEV2 infect humans only; HEV3, HEV4, and HEV7 can infect humans and other mammals; and HEV5 and HEV6 have been detected in animals only. Worldwide, HEV is the most common cause of acute viral hepatitis in humans. The disease is generally selflimiting, but high death rates have been observed among HEV-infected pregnant women. Chronic HEV infection is a problem in immunocompromised patients, such as solid organ transplant recipients (3). Human HEV3 and HEV4 infections have been associated with consumption of undercooked pork or game meat (4). In 2014, we described the discovery of a novel genotype of HEV in dromedaries (Camelus dromedarius or 1-humped camels), suggesting another possible source of human HEV infection (5). This dromedary HEV was subsequently classified as a novel Orthohepevirus A genotype, HEV7 (1,2). Recently, this HEV7 genotype was also isolated from a liver transplant recipient from the Middle East with chronic HEV infection (6). The patient regularly consumed dromedary camel meat and milk, implying camelto-human transmission of the virus (6). Like the dromedary, the Bactrian camel (Camelus bactrianus or 2-humped camels) is an Old World camelid species. Thus, we hypothesize that Bactrian camels may also be reservoirs of HEV. To test this hypothesis and increase our understanding of the epidemiology of HEV in camels, we performed a molecular epidemiology study using feces samples from camels in China. During November 2012–May 2013, we collected and tested 1 feces sample each from 205 Bactrian camels on a farm in Xinjiang, China. We performed RNA extraction and reverse transcription PCR (RT-PCR) as previously described (7). We screened for HEV by PCR amplification of a 251-bp fragment of open-reading frame (ORF) 2, using primers 5′-GTTGTCTCAGCCAATGGCGA-3′ and 5′-GTAGTTTGGTCATACTCAGCAGC-3′. PCR was performed, using previously described conditions (7), with the annealing temperature set at 50°C. DNA sequencing and quantitative real-time RT-PCR were performed as previously described (7). Three samples were positive for HEV; we performed complete genome sequencing of these samples as described (online Technical Appendix, http:// wwwnc.cdc.gov/EID/article/22/12/16-0979-Techapp1.pdf) (5,7). We also performed comparative genomic analysis as previously described (1,2,8). We constructed a phylogenetic tree using the maximum-likelihood method and MEGA7 (9); bootstrap values were calculated from 1,000 trees. The optimal substitution model for each ORF was selected by MEGA7 (Figure). RT-PCR for a 251-bp fragment in ORF2 of HEV was positive for 3 (1.5%) of the 205 fecal samples; virus loads were 1.6 × 103, 2.1 × 103, and 1.8 × 104 copies/mg, respectively. Whole-genome sequencing of the 3 Bactrian camel HEV (BcHEV) strains (GenBank accession nos. KX387865–7) showed genome sizes of 7,212–7,223 bp and a G + C content of 52.7%–53.1%. Overall, nucleotides in the BcHEV genome differed by >20% compared with those in all other HEVs. Genomes of the 3 BcHEV isolates contained 3 major ORFs; genome organization was typical of and characteristics were similar to those of HEVs from other Orthohepevirus A species. Phylogenetic trees constructed using ORF1, ORF2, ORF3, and concatenated ORF1/ORF2, excluding the hypervariable region, showed that these 3 BcHEV isolates clustered with the 2 dromedary camel HEV7 strains and the HEV7 strain from the liver-transplant recipient with chronic hepatitis (Figure; online Technical Appendix Figure 1)

A non-synonymous single nucleotide polymorphism in IFNAR1 affects susceptibility to chronic hepatitis B virus infection.

Summary.  The type I interferon (IFN‐α/β) receptor 1 (IFNAR1) mediates the potent antiviral and immuno‐regulatory effects of IFN‐α/β that are believed to be pivotal to eradicate hepatitis B virus (HBV) infection. IFNAR1 promoter polymorphisms (at −568/−77) have been shown to be associated with susceptibility to chronic HBV infection; however, whether these markers are genetic determinants of HBV infection remains unknown. The functional significance of promoter −568/−77 polymorphisms was assessed by mutagenesis and luciferase assays. Sequencing and restriction fragment length polymorphisms in 328 chronic HBV patients, 130 spontaneous resolvers and 148 healthy blood donors identified other polymorphism at IFNAR1 open reading frame. IFNAR1 expression levels in peripheral blood cells were detected by flow cytometry. We found that the −568/−77 promoter variants were unlikely to affect transcription levels. A C/G single nucleotide polymorphism, in strong linkage disequilibrium with the promoter polymorphisms, was found in the coding sequence of IFNAR1 (nt19158). This resulted in a nonsynonymous substitution in the extracellular region of IFNAR1 protein and correlated with susceptibility to chronic HBV infection. Bioinformatic analysis suggested decreased stability of the IFNAR1 protein. Chronic HBV patients with the 19158C/C genotype (Leu141) exhibited higher IFNAR1 protein expression levels in peripheral blood monocytes than those with the 19158G/G genotype (Val141). In conclusion, IFNAR1 19158C/G polymorphism is primarily associated with susceptibility to chronic HBV infection.

Epidemiology characteristics of respiratory viruses found in children and adults with respiratory tract infections in southern China

Summary Background The World Health Organization (WHO) ranks respiratory tract infection (RTI) as the second leading cause of death worldwide for children under 5 years of age. The aim of this work was to evaluate the epidemiology characteristics of respiratory viruses found in children and adults with RTI from July 2009 to June 2012 in southern China. Methods In this work, a total of 14 237 nasopharyngeal swabs (14 237 patients from 25 hospitals) were analyzed, and seven respiratory viruses (influenza virus, respiratory syncytial virus, parainfluenza virus, adenovirus, human metapneumovirus, human coronavirus, human bocavirus) were detected using PCR/RT-PCR from nasopharyngeal swabs. Results The demographic characteristics, viral prevalence, age distribution, seasonal distribution, and pathogen spectrum of the patients with RTIs were analyzed. Co-infection was observed in 483 specimens, but it was more common in male patients, inpatients, children, and young adults. It varied by season, being more prevalent in the spring and summer and less so in the winter. Human coronavirus and human bocavirus were the most common pathogens, tending to occur in co-infection with other respiratory viruses. Conclusions This work adds to our knowledge of the epidemiology characteristics of these seven common respiratory viruses among patients with RTI in southern China. The detection of the specific viral causes of infection provides a useful starting point for an understanding of illness attributable to respiratory infection, and might also provide data relevant to the development of prevention strategies.

Surveillance and Genome Analysis of Human Bocavirus in Patients with Respiratory Infection in Guangzhou, China

Human bocavirus (HBoV) is a novel parvovirus associated with respiratory tract diseases and gastrointestinal illness in adult and pediatric patients throughout the world. To investigate the epidemiological and genetic variation of HBoV in Guangzhou, South China, we screened 3460 throat swab samples from 1686 children and 1774 adults with acute respiratory infection symptoms for HBoV between March 2010 and February 2011, and analyzed the complete genome sequence of 2 HBoV strains. Specimens were screened for HBoV by real-time PCR and other 6 common respiratory viruses by RT-PCR or PCR. HBoV was detected in 58 (1.68%) out of 3460 samples, mostly from pediatric patients (52/58) and inpatient children (47/58). Six adult patients were detected as HBoV positive and 5 were emergency cases. Of these HBoV positive cases, 19 (32.76%) had co-pathogens including influenza virus (n = 5), RSV (n = 5), parainfluenza (n = 4), adenovirus (n = 1), coronavirus (n = 7). The complete genome sequences of 2 HBoVs strains (Genbank no. JN794565 and JN794566) were analyzed. Phylogenetic analysis showed that the 2 HBoV strains were HBoV1, and were most genetically close to ST2 (GenBank accession number DQ0000496). Recombination analysis confirmed that HBoV strain GZ9081 was an intra–genotype recombinant strain among HBoV1 variants.

The expression and role of protein kinase C (PKC) epsilon in clear cell renal cell carcinoma

Protein kinase C epsilon (PKCε), an oncogene overexpressed in several human cancers, is involved in cell proliferation, migration, invasion, and survival. However, its roles in clear cell renal cell carcinoma (RCC) are unclear. This study aimed to investigate the functions of PKCε in RCC, especially in clear cell RCC, to determine the possibility of using it as a therapeutic target. By immunohistochemistry, we found that the expression of PKCε was up-regulated in RCCs and was associated with tumor Fuhrman grade and T stage in clear cell RCCs. Clone formation, wound healing, and Borden assays showed that down-regulating PKCε by RNA interference resulted in inhibition of the growth, migration, and invasion of clear cell RCC cell line 769P and, more importantly, sensitized cells to chemotherapeutic drugs as indicated by enhanced activity of caspase-3 in PKCε siRNA-transfected cells. These results indicate that the overexpression of PKCε is associated with an aggressive phenotype of clear cell RCC and may be a potential therapeutic target for this disease.

Amino acid sequence analysis and identification of mutations under positive selection in hemagglutinin of 2009 influenza A (H1N1) isolates

The 2009 flu pandemic is caused by a new strain of influenza A (H1N1) virus, A/H1N1/09. With its high transmissibility, this novel virus has caused a pandemic and infected over 600,000 people globally. By comparing the hemaglutinin (HA) gene and protein sequences among over 700 A/H1N1/09 isolates, mutations in the receptor-binding sites and antigenic epitope regions were identified. Among these mutations, T220 and E/G239 were found to be strongly positively selected over the course of spreading of the A/H1N1/09 virus worldwide. Interestingly, both sites are located in the highly variable epitope regions of HA1, and residue 239 also plays an important role in the receptor-binding process. Further analyses demonstrated that the percentage of T220 mutants among all isolates increased rapidly during the evolution, and that an E/G239 mutation could decrease the binding affinity of the virus with its cellular receptor. Thus, due to a potential functional importance of residues 220 and 239, mutations at these sites, as well as the significant of positive selection on these sites deserves more attention, while new vaccines and therapeutic drugs are developed against this novel virus.

Screening and identification of significant genes related to tumor metastasis and PSMA in prostate cancer using microarray analysis

Tumor metastasis is one of the causes for the high mortality rate of prostate cancer (PCa) patients, yet the molecular mechanisms of PCa metastasis are not fully understood. In our previous studies, we found that PSMA suppresses the metastasis of PCa, yet the underlying mechanism remains unknown. To identify the genes related to tumor metastasis possibly regulated by PSMA, we performed tumor metastasis PCR array assay to analyze the differentially expressed tumor metastasis-related genes. Eighty-four tumor metastasis related genes were screened in si-PSMA LNCap cells (PSMA silenced by siRNA)/LNCap cells and in PC-3/LNcap cells, respectively. Expression levels of possible related genes were verified by real-time PCR in 4 prostate cancer cell lines (LNCap, 22RV1, PC-3 and DU145) and in 85 clinical samples (12 normal, 26 benign prostatic hypertrophy and 47 prostate cancer tissues). The results showed that 10 genes (including CDH6 and CXCL12) were upregulated and 4 genes (CCL7, ITGB3, MDM2 and MMP2) were downregulated in the si-PSMA LNCap cells. There were 41 genes significantly upregulated and 15 genes downregulated in PC-3 cells when compared with LNCap cells. Eight common genes were found in both the si-PSMA and PSMA(-) groups. CDH6, MMP3, MTSS1 were further identified as PSMA-related genes in the prostate cancer cell lines and clinical samples, and their expression showed a negative correlation with the stage of prostate cancer (P<0.0001) and PSMA level (P<0.05) in clinical samples, indicating their possible involvement in PSMA-related PCa metastasis regulation. These findings may provide insights into the mechanism involved in the suppression of PCa metastasis by PSMA and its possible interacting proteins, and may provide clues for further exploration of the molecular mechanism of PCa metastasis.

The expression and role of tyrosine kinase ETK/BMX in renal cell carcinoma.

BackgroundExpression of the non-receptor tyrosine kinase ETK/BMX has been reported in several solid tumors, but the underlying molecular mechanisms and its clinical significance in renal cell carcinoma (RCC) remain to be elucidated.MethodsETK expression in 90 human RCC and 30 human normal renal tissue samples was examined by immunohistochemistry and compared with several clinicopathologic parameters. To further demonstrate the biological function of ETK in RCC, Western blot was used to test the expression level of ETK protein in RCC cell lines. Subsequent to the downregulation of ETK by small interfering RNA, the effects of ETK on RCC cell growth, apoptosis, migration and invasion were assessed by methyl thiazol tetrazolium assay, flow cytometry and transwell assay. And the varying expression of VEGF, STAT3 and phosphorylated STAT3 (p-STAT3) in RCC were evaluated by Western blot.ResultsImmunohistochemistry analysis showed that ETK expression was highly increased in RCC and was positively correlated with clinical stage, grade and metastasis. Simultaneously, the overall survival time in patients with higher ETK expression was obviously shorter than that in patients with lower ETK expression. ETK was also detected in RCC cell lines. Moreover, the down-regulating ETK significantly inhibited RCC cell growth, migration, invasion and promoted apoptosis. The expression of VEGF and p-STAT3 were also decreased.ConclusionsOur study suggests that the overexpression of ETK is associated with the malignancy and disease progression of RCC. Since ETK is also involved in RCC cell biological function and VEGF-ETK-STAT3 loop, ETK may be used as a potential therapeutic target for RCC.

Bax-interacting factor-1 inhibits cell proliferation and promotes apoptosis in prostate cancer cells

Prostate cancer (PCa) is one of the most common malignant tumors and the second leading cause of cancer-related death among males. Bax-interacting factor-1 (Bif-1) is a member of Endophilin family, which binds to and activates the BAX protein in response to the apoptosis signaling pathway. Loss of Bif-1 may suppress the intrinsic pathway of apoptosis and promote tumorigenesis, but there is also converse evidence that Bif-1 could in part be responsible for the tumorigenesis and the role of Bif-1 in PCa development is not clear. In the present study, we aimed to understand the relationships between Bif-1 expression and PCa development. The mRNA and protein expression levels of Bif-1 in PCa cell lines, benign prostatic hyperplasia (BPH) (n=100) and PCa tissues (n=100, including low Gleason-scored PCa n=43 and high Gleason-scored PCa n=57) were detected and the effects of Bif-1 overexpression on the apoptosis, proliferation and migration in LNCaP cells were explored. Bif-1 mRNA levels of PCa cell lines were analyzed by real-time PCR and the protein levels were detected by western blotting. Bif-1 expression in BPH and PCa samples was detected by immunohistochemistry. To build Bif-1 overexpression PCa cells, Bif-1 gene was transfected into LNCaP cells by pcDNA3.1(+)‑Bif-1 vector. Cell apoptosis was detected by flow cytometric analysis, cell proliferation measured by 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide (MTT) assay and cell migration was analyzed by wound‑healing assay. The results proved that Bif-1 is downregulated in both PCa cell lines (P<0.01) and clinical samples (P<0.05), and Bif-1 expression is suppressed with the cancer progression (BPH vs. PCa P<0.01, and low Gleason-scored PCa vs. high Gleason-scored PCa P<0.05). Overexpression of Bif-1 could significantly inhibit cell proliferation (P<0.05) and enhancing PCa cell apoptosis (P<0.05), but it did not affect the migration ability (P>0.05). Our findings give strong evidence that Bif-1 is involved in PCa tumorigenesis and acts as a suppressor in PCa progression, and may have significance in understanding the process of PCa development.

Vasculogenic mimicry plays an important role in adrenocortical carcinoma

To determine the prognostic role of vasculogenic mimicry in adrenocortical carcinoma, and to explore its relationship with vascular endothelial growth factor receptor 2 expression.