Seroprevalence of neutralizing antibodies against adenovirus type 26 and 35 in healthy populations from Guangdong and Shandong provinces,China

Human adenoviruses type 26 (HAdV26) and type 35 (HAdV35) have increasingly become the choice of adenovirus vectors for vaccine application. However, the population pre-existing immunity to these two adenoviruses in China, which may reduce vaccine efficacy, remains largely unknown. Here, we established micro-neutralizing (MN) assays to investigate the seroprevalence of neutralizing antibodies (nAbs) against HAdV26 and HAdV35 in the general population of Guangdong and Shandong provinces, China. A total of 1184 serum samples were collected, 47.0% and 15.8% of which showed HAdV26 and HAdV35 nAb activity, respectively. HAdV26-seropositive individuals tended to have more moderate nAbs titers (201–1000), while HAdV35-seropositive individuals appeared to have more low nAbs titers (72–200). The seropositive rates of HAdV26 and HAdV35 in individuals younger than 20 years old were very low. The seropositive rates of HAdV26 increased with age before 70 years old and decreased thereafter, while HAdV35 seropositive rates did not show similar characteristics. Notably, the seropositive rates and nAb levels of both HAdV26 and HAdV35 were higher in Guangdong Province than in Shandong Province, but did not exert significant differences between males and females. The seroprevalence between HAdV26 and HAdV35 showed little correlation, and no significant cross-neutralizing activity was detected. These results clarified the characteristics of the herd immunity against HAdV26 and HAdV35, and provided information for the rational development and application of HAdV26 and HAdV35 as vaccine vectors in China.     

Analysis of severe human adenovirus infection outbreak in Guangdong Province,southern China in 2019

During 2018-2019, a severe human adenovirus (HAdV) infection outbreak occurred in southern China. Here, we screened 18 respiratory pathogens in 1704 children (≤ 14 years old) hospitalized with acute respiratory illness in Guangzhou, China, in 2019. In total, 151 patients had positive HAdV test results; 34.4% (52/151) of them exhibited severe illness. HAdV infection occurred throughout the year, with a peak in summer. The median patient age was 3.0 (interquartile range:1.1-5.0) years. Patients with severe HAdV infection exhibited increases in 12 clinical indexes (P ≤ 0.019) and decreases in four indexes (P ≤ 0.007), compared with patients exhibiting nonsevere infection. No significant differences were found in age or sex distribution according to HAdV infection severity (P > 0.05); however, the distributions of comorbid disease and HAdV co-infection differed according to HAdV infection severity (P < 0.05). The main epidemic types were HAdV-3 (47.0%, 71/151) and HAdV-7 (46.4%, 70/151). However, the severe illness rate was significantly higher in patients with HAdV-7 (51.4%) than in patients with HAdV-3 (19.7%) and other types of HAdV (20%) (P < 0.001). Sequencing analysis of genomes/capsid genes of 13 HAdV-7 isolates revealed high similarity to previous Chinese isolates. A representative HAdV-7 isolate exhibited a similar proliferation curve to the curve described for the epidemic HAdV-3 strain Guangzhou01 (accession no. DQ099432) (P > 0.05); the HAdV-7 isolate exhibited stronger virulence and infectivity, compared with HAdV-3 (P < 0.001). Overall, comorbid disease, HAdV co-infection, and high virulence and infectivity of HAdV-7 were critical risk factors for severe HAdV infection; these data can facilitate treatment, control, and prevention of HAdV infection.     

A slow spread of adenovirus type 7 infection after its re-emergence in Yamagata, Japan, in 1995

We have continued the epidemiological study on adenovirus type 7 (Ad7), which re-emerged in 1995 in Yamagata, Japan. Between 1999 and 2004, we isolated only four strains from 10,778 throat swab specimens among children with acute respiratory infections. A serological survey of 303 specimens revealed the antibody-positive rate against Ad7 to be 0-7.4% in children under 10 years of age in 2005, although it was 3.3-16.7% in 1997 and 0% in 1993. Our results suggest that a re-emergence does not always provoke a sudden major outbreak, even if the antibody-positive rate against Ad7 is low in the local community.     

Co-infection of human metapneumovirus with adenovirus or respiratory syncytial virus among children in Japan

Human metapneumovirus (hMPV) is one of the etiological agents of acute respiratory tract infections. From June 2005 to May 2006, we collected 185 clinical specimens from children in Osaka City, Japan, and detected 41 hMPV RNA. Of the 41 specimens, four (9.8%) also contained other viruses (3 with adenovirus [AdV] and 1 with respiratory syncytial virus [RSV]). The clinical symptoms of patients coinfected with AdV were indistinct from those of patients mono-infected with hMPV. The symptoms of the one patient co-infected with RSV were clinically severe. Further research is needed to clarify the effect of hMPV on other respiratory viruses or vice versa.     

Complementation of translational defect for growth of human adenovirus type 2 in Simian cells by a Simian virus 40-induced factor

The defective step which leads human adenovirus type 2 infection of African green monkey kidney cells (clone C14) to be abortive and its complementation in simian virus 40-transformed cells (clone T22) were studied by comparing the synthesis and function of macromolecules in these cell lines. Neither a quantitative nor a qualitative difference was detected in virus DNA replication and in virus mRNA synthesis in these cells, while a definite difference was observed in protein synthesis. The capsid proteins, such as hexon or penton, were synthesized in T22 cells but not in C14 cells. Inability of polyribosomes to synthesize the capsid proteins in C14 cells infected with adenovirus type 2 may not be due to a defect in elongation of nascent polypeptides or their release, since nascent polypeptides pulse-labelled with [³H]leucine were completely released from polyribosomes after the chase. The electrophoretic analysis of proteins synthesized in vitro with polyribosomes from either infected T22 or C14 cells using the pH 5 enzyme and S100 fraction from T22 cells revealed that hexon was synthesized with polyribosomes from T22 cells but not from C14 cells, thereby suggesting that the defect is not ascribed to a component in the pH 5 enzyme and S100 fraction, but resides in polyribosomes. The analysis of late adenovirus mRNA associated with polyribosomes in the infected T22 and C14 cells by hybridization competition or by sedimentation revealed that all the species of virus mRNA were present in the cytoplasm of these cells; however, certain species of virus mRNA larger than 20 S were absent in polyribosomes of the infected C14 cells. Sedimentation analysis of late adenovirus mRNA following separation on poly(U)-Sepharose or by membrane filtration gave the same results. These results suggest that the defect of C14 cells to support growth of adenoviruses is due to the inability of ribosomes to associate with certain species of late virus mRNA to form polyribosomes and suggest that a factor complementing this defect is induced by simian virus 40.     

乳酸脱氢酶基因调控对HEK-293细胞代谢和腺病毒生产的影响

减少乳酸积累一直是哺乳动物细胞生物技术产业的一个目标。体外培养动物细胞时,乳酸积累主要是2种代谢途径作用的综合结果:一方面,葡萄糖在乳酸脱氢酶A(lactate dehydrogenase A,LDHA)的作用下生成乳酸;另一方面,乳酸可通过乳酸脱氢酶B(LDHB)或乳酸脱氢酶C(LDHC)氧化为丙酮酸重新进入三羧酸循环。本研究综合评估了乳酸代谢关键基因调控对人胚胎肾细胞(human embryonic kidney 293 cells,HEK-293)细胞生长、代谢和人腺病毒(human adenovirus,HAdV)生产的影响,有效提高了HEK-293细胞的HAdV生产能力,并为哺乳动物细胞的乳酸代谢工程调控提供了理论基础。通过改造乳酸代谢关键调控基因(敲除ldha基因以及过表达ldhb和ldhc基因),有效改善了HEK-293细胞的物质和能量代谢效率,显著提高了HAdV的生产。与对照细胞相比,3个基因改造均能促进细胞生长,降低乳酸和氨的积累,明显增强细胞的物质和能量代谢效率,显著提高了HEK-293细胞的HAdV生产能力。ldhc基因过表达对HEK-293细胞的生长、代谢和HAdV生产调控最显著,最大细胞密度提高了约38.7%,乳酸对葡萄糖得率和氨对谷氨酰胺得率分别下降了33.8%和63.3%,HAdV滴度提高了至少16倍。此外,相比于对照细胞株,改造细胞株的腺苷三磷酸(adenosine triphosphate,ATP)生成速率、ATP/O_(2)比率、ATP与腺苷二磷酸(adenosine diphosphate,ADP)的比值以及还原型辅酶Ⅰ(nicotinamide adenine dinucleotide,NADH)含量均有不同程度的提高,能量代谢效率明显改善。     

Application of Human Adenovirus Genotyping by Phylogenetic Analysis in an Outbreak to Identify Nosocomial Infection

Nosocomial infections are common in pediatric patients and can be fatal in infants and immunocompromised patients. In September 2018, a high positive rate of human adenovirus HAdV was occurred among hospitalized children in the Children's Hospital Affiliated to the Capital Institute of Paediatrics in Beijing. To investigate whether this outbreak of HAdV was related to nosocomial infections or the result of community infections, we collected respiratory specimens from patients with acute respiratory infections in a respiratory ward during June to December 2018, and screened for respiratory viruses. Among 1,840 cases included, 95(5.2%, 95/1840) were positive for HAdV and 81 were genotyped based on phylogenetic analysis, including seven as HAdV-1(8.6%), 30 HAdV-3(37.0%), two HAdV-6(2.5%), and 42 HAdV-7(51.9%). More HAdV-positive samples were collected in August(4.7%, 12/255), September(15.0%, 41/274) and October(6.9%, 17/247), with a peak in September 2018. By combining the results of HAdV phylogenetic analysis with clinical data of patients, there were 77 cases(4.2%, 77/1840; 81.1%, 77/95) excluded from nosocomial infections, eight cases representing possible infections transmitted by visitors or attending parents, three cases without sequences that might have been due to infection transmitted by roommates positive for HAdV, one case of a roommate without an HAdV sequence, and six cases that shared highly homologous sequences with those of their roommates, for which nosocomial infections might be considered. In conclusion, genotyping of HAdVs based on phylogenetic analysis combined with clinical information provides a powerful method to distinguish nosocomial infections from community acquired infection, especially when tracing the origins of nosocomial infections.     

Genetic Analysis of Human Adenovirus Type 7 Strains Circulating in Different Parts of China

To investigate the molecular epidemiology and genetic variation of human adenovirus type 7(HAdV-7) in children with acute respiratory infections(ARI) in China. HAdV-7-positive respiratory samples collected from children with ARI in Beijing, Shijiazhuang, Wenzhou and Guangzhou from 2014–2018 were selected for gene amplification and sequence analysis. Fifty-seven HAdV-7 clinical strains with hexon, penton base and fiber gene sequences were obtained. Meanwhile17 strains were selected randomly from different cities for whole genome sequencing. Phylogenetic and variation analyses were performed based on the obtained sequences, HAdV-7 prototype strain Gomen(AY594255), vaccine strains(AY495969 and AY594256) and representative sequences of strains. The phylogenetic trees constructed based on whole genome sequences, major capsid protein genes(hexon, penton base and fiber) and the early genes(E1, E2, E3 and E4) were not completely consistent. The HAdV-7 strains obtained in this study always clustered with most of the circulating strains worldwide from the 1980 s to the present. Compared with the HAdV-7 prototype strain Gomen(AY594255), some amino acid mutations in loop1 and loop2 of hexon and the RGD loop region of the penton base gene were observed. Recombination analysis showed that partial regions of 55 k Da protein and 100 kDa hexon-assembly associated protein genes among all HAdV-7 strains in this study were from HAdV-16 and HAdV-3, respectively. Our study demonstrated the molecular evolution characteristics of HAdV-7 strains circulating in China and provided basic reference data for the prevention, control and vaccine development of HAdV-7.     

Thermostability/infectivity defect caused by deletion of the core protein V gene in human adenovirus type 5 is rescued by thermo-selectable mutations in the core protein X precursor

Mastadenoviruses represent one of the four major genera of the Adenoviridae family comprising a variety of mammalian pathogens including human adenovirus (Ad), whose genomes encode a gene for minor core protein V (pV), not found in other genera of Adenoviridae. Deletion of other genus-specific genes (gene IX and E3 genes) from the Ad type 5 (Ad5) genome has been studied experimentally in vitro and the results on biological characterization of the mutants support the phylogenetic evidence of those genes being non-essential for Ad viability. On this basis it seemed logical to suggest that a deletion of gene V from the Ad5 genome could also be tolerated. To test this hypothesis we constructed and rescued the first pV-deletion mutant of human Ad5. As compared to Ad5, this mutant formed small plaques, had dramatically reduced thermostability and lower infectivity. A subsequent thermoselection screen of the pV-deleted Ad5 allowed isolation of a suppressor mutant Ad5-dV/TSB with restored biological characteristics. Since replication and viral assembly of Ad5-dV/TSB could still occur in the absence of pV, we conclude that pV is a non-essential component of the virion. The observed rescue of the biological defects appears to be associated with a cluster of point mutations in the gene encoding the precursor for the other core protein, X/Mu. This finding, thus, suggests possible roles of pV and protein X/Mu precursor in viral assembly. It also provides an interesting insight into genetic events that mediate molecular adaptation of viruses to possible changes in the genetic background in the course of their evolutionary divergence. The possible mechanism of the observed genetic suppression is discussed.     

Seroprevalence of Neutralizing Antibodies against Six Human Adenovirus Types Indicates the Low Level of Herd Immunity in Young Children from Guangzhou,China

Human adenoviruses(HAdVs) commonly cause many diseases such as respiratory diseases, gastroenteritis, cystitis worldwide. HAdV-3,-7,-4 and emergent HAdV-55 and HAdV-14 are the most important types causing severe respiratory diseases. There is no effective drug available for clinical treatment, and no vaccine available for the general population.Therefore, it is important to investigate the seroprevalence against HAdV for developing novel vaccines and vectors. In this study, we investigated the seroprevalence and titer levels of neutralizing antibodies(NAb) against HAdV-3,-4,-7,-14,-55,and-11 in total 278 healthy populations between 0 months and 49 years of age(228 children and 50 adults) from Guangzhou. In children under the age of 18 years, the seropositive rates were significantly increased against HAdV-3 at12.07%, 33.96%, and 64.29% and against HAdV-7 at 0%, 18.87%, and 19.05% in age groups of 1–2, 3–5, and 6–17 years,respectively. The seroprevalence was very low(0% * 8.1%) for all other four types. In adults aged between 18 and49 years, HAdV-3,-4, and-7([ 50.00%) were the most common types, followed by HAdV-14(38.00%),-55(34.00%),and-11(24.00%). Adults tended to have high NAb titers against HAdV-4 and-55. HAdV-55-seropositive donors tended to be HAdV-11-and HAdV-14-seropositive. These results indicated the low level of herd immunity against all six HAdV types in young children, and HAdV-14,-55,-11 in adults from Guangzhou City. Our findings demonstrate the importance of monitoring HAdV types and developing vaccines against HAdV for children and adults.     

Prevalence of human T‐lymphotropic virus type 1 carriers among pregnant women in Hokkaido,Japan

As there is a risk of MTCT of HTLV‐1, the HSGP HTLV‐1 MTCT was organized in 2011. To determine how many pregnant women are infected with HTLV‐1 in Hokkaido, which is the northernmost and the second largest island in Japan with a population of 5 467 000 and 39 392 newborns in 2011, the HSGP HTLV‐1 MTCT asked all facilities that may care for pregnant women in Hokkaido in July 2013 to provide information on the number of pregnant women who underwent screening for anti‐HTLV‐1 antibody using particle agglutination or chemiluminescent enzyme immunoassay, and the numbers of those with positive, equivocal, and negative test results in the screening and confirmation tests using western blotting or PCR methods in 2012, respectively. A total of 111 facilities participated in this study and provided information on 33 617 pregnant women who underwent screening in 2012, corresponding to approximately 85% of all pregnant women who gave birth in Hokkaido in 2012. Of 81 candidates for a confirmation test because of positive (n = 77) or equivocal (n = 4) results on screening, 63 (78%) underwent the confirmation test and, finally, 34 (0.1%) and 33 563 (99.8%) women were judged to be HTLV‐1 carriers and non‐carriers, respectively. It was concluded that the prevalence rate of HTLV‐1 carriers was low, one per 1000 pregnant women in Hokkaido. Approximately 40 infants are born yearly to mothers infected with HTLV‐1 in Hokkaido.     

Tricalcium phosphate nanoparticles enable rapid purification, increase transduction kinetics, and modify the tropism of mammalian viruses

Adenoviral, adeno-associated viral, and retroviral particles are chosen as gene delivery shuttles in more than 50% of all gene therapy clinical trials. Bulk availability of clinical-grade viral particles and their efficiency to transduce the therapeutic cargo into specific target cells remain the most critical bottlenecks in gene therapy applications to date. Capitalizing on the flame-spray technology for the reproducible economic large-scale production of amorphous tricalcium phosphate nanoparticulate powders (ATCP), we designed a scalable ready-to-use gravity-flow column set-up for the straightforward concentration and purification of transgenic adenoviral, adeno-associated viral, and lentiviral particles. Specific elution buffers enabled rapid release of viral particles from the ATCP matrix of the column and provided high-titer virus preparations in an unsurpassed period of time. The interaction of ATCP with adenoviral, adeno-associated viral, and lentiviral particles in solution increased the transduction kinetics of several mammalian cell lines in culture. The nanoparticles were also able to modify the tropism of murine leukemia virus (MLV) towards transduction of human cells. Based on these findings, we believe that the use of flame-spray tricalcium phosphate nanoparticles will lead to important progress in the development of future gene therapy initiatives.     

Cloning of human,murine, and marsupial keratin 7 and a survey of K7 expression in the mouse

Keratins are cytoplasmic intermediate filament proteins expressed by epithelial cells. Keratin 7 (K7) is expressed in a wide range of epithelial structures in humans. We have cloned and fully sequenced the human and mouse K7 genes and mRNAs, and the K7 mRNA from the marsupial Potorous tridactylis, from which the widely used simple epithelial cell lines PtK1 and PtK2 are derived. Percentage identity plots comparing the mouse and human genomic sequences revealed a number of conserved CpG islands associated with the K7 gene. There was considerable conservation of introns between the two species, which may indicate the presence of intronic regulatory elements. Only the most proximal 500bp of the promoter was conserved, although an additional conserved sequence island was found 2-3kb upstream. Protein sequence comparisons between the three species allowed identification of conserved regions of the keratin variable domains that may be candidates for protein-protein interactions and/or regulatory modification. From the mouse sequence, we generated a polyclonal rabbit antibody specific for murine K7. This antibody was used to perform a survey of K7 expression in the mouse. The expression pattern was similar to the reported human distribution, with substantial expression observed in lung, bladder, mesothelium, hair follicle, and ductal structures. We also noted previously unreported expression of K7 in the gastrointestinal tract and filiform papillae of the tongue and specific K7 expression in a range of "hard" epithelial tissues. The distribution of K7 in mouse and availability of genomic sequence from the 129/Sv mouse strain will allow the generation and analysis of transgenic mice expressing mutant forms of K7 and to predict the phenotype of human genetic disorders caused by mutations in this keratin.     

Intertypic modular exchanges of genomic segments by homologous recombination at universally conserved segments in human adenovirus species D

Human adenovirus species D (HAdV-D), which is composed of clinically and epidemiologically important pathogens worldwide, contains more taxonomic “types” than any other species of the genus Mastadenovirus, although the mechanisms accounting for the high level of diversity remain to be disclosed. Recent studies of known and new types of HAdV-D have indicated that intertypic recombination between distant types contributes to the increasing diversity of the species. However, such findings raise the question as to how homologous recombination events occur between diversified types since homologous recombination is suppressed as nucleotide sequences diverge. In order to address this question, we investigated the distribution of the recombination boundaries in comparison with the landscape of intergenomic sequence conservation assessed according to the synonymous substitution rate (d_S). The results revealed that specific genomic segments are conserved between even the most distantly related genomes; we call these segments “universally conserved segments” (UCSs). These findings suggest that UCSs facilitate homologous recombination, resulting in intergenomic segmental exchanges of UCS-flanking genomic regions as recombination modules. With the aid of such a mechanism, the haploid genomes of HAdV-Ds may have been reshuffled, resulting in chimeric genomes out of diversified repertoires in the HAdV-D population analogous to the MHC region reshuffled via crossing over in vertebrates. In addition, some HAdVs with chimeric genomes may have had the opportunity to avoid host immune responses thereby causing epidemics.     

Possible relationship of PFGE patterns of Moraxella catarrhalis between hospital- and community-acquired respiratory infections in a community hospital

We describe a prospective study of molecular analysis of Moraxella catarrhalis isolated from a community hospital. Our study was designed to investigate the possible relationship of pulsed-field gel electrophoresis (PFGE) patterns of M. catarrhalis between hospital- and community-acquired respiratory infections. A nosocomial outbreak of M. catarrhalis was observed between September 2000 and September 2001. During the study period, 40 strains of M. catarrhalis were isolated from a total of 32 patients with respiratory infections (26 strains from 18 inpatients, and 14 strains from 14 outpatients). We compared the PFGE patterns in 40 strains of M. catarrhalis isolated from the respiratory tract of the study patients. The genomic types of M. catarrhalis were classified into three PFGE patterns (A, B, and C). Interestingly, the nosocomial outbreak of M. catarrhalis included two patterns (A and B). Of the three patterns, two patterns (A and B) were found in both inpatients and outpatients. More interestingly, two subtypes of pattern B (B1 and B4) were simultaneously found in both inpatients and outpatients. Our results indicated that PFGE with SmaI chromosomal digestion is a suitable technique to establish the inter-strain genetic relatedness of M. catarrhalis, and suggested that the outbreak of M. catarrhalis occasionally included miscellaneous PFGE patterns. The results also showed that PFGE patterns of M. catarrhalis isolates were similar between hospital- and community-acquired respiratory infections. Analysis of the subtypes suggested that there might be some association between hospital- and community-acquired respiratory infections caused by M. catarrhalis.     

细菌内同源重组法制备FMDV聚蛋白编码基因重组腺病毒

采用PCR方法从重组质粒pMD18_T/PP中扩增出FMDV的聚蛋白(PP)编码基因,再亚克隆至腺病毒穿梭质粒中,形成重组穿梭质粒rpAd_CMV/PP;将获得的重组穿梭质粒与腺病毒骨架载体通过在大肠杆菌内质粒间同源重组获得重组腺病毒质粒rpAd/PP。将腺病毒载体线性化后用脂质体介导转染293细胞从而获得含有口蹄疫病毒PP编码基因的重组腺病毒。通过倒置显微镜观测,可见明显的细胞病变,利用荧光显微镜可观测到报告基因绿色荧光蛋白的表达,并在电镜下观察到FMDV的空衣壳。结果证明已成功获得了含有口蹄疫病毒PP编码基因的重组腺病毒rAd/PP,并成功表达组装FMDV空衣壳,为FMDV腺病毒活载体疫苗的研究奠定了基础。     

重组人源SNX7蛋白在大肠杆菌中的表达、纯化及磷脂结合特异性分析

Sorting nexins(SNXs)是一类含有SNX-PX结构域,并在细胞内吞和内体分选运输过程中发挥重要调节作用的蛋白。SNX7是SNXs家族中的一员,含有PX结构域和BAR结构域,属于SNX-PX-BAR亚家族。斑马鱼实验表明,SNX7是在肝脏中大量表达的抗凋亡蛋白,并在胚胎肝脏的发育中发挥关键作用。为了从蛋白水平对SNX7进行研究,首先将编码人源PX-BARSNX7(SNX7的一个片段,包含PX和BAR结构域)的cDNA片段插入到原核表达载体p28a中,再将重组质粒转化到大肠杆菌Rosseta 2(DE3)中诱导表达,并用亲和层析、离子交换和分子筛层析对PX-BARSNX7进行了纯化。Western blotting结果表明,亲和层析、离子交换和分子筛层析纯化后获得了高纯度的PX-BARSNX7蛋白。动态光散射实验显示PX-BARSNX7蛋白均一性良好。磷脂结合实验表明,PX-BARSNX7具有较为广泛的磷脂酰肌醇结合能力,能够与PtdIns(5)P、PtdIns(4,5)P2和PtdIns(3,4,5)P3结合。     

Assessment of human adenovirus removal by qPCR in an advanced water reclamation plant in Georgia,USA

Aims

To assess human adenoviruses (HAdVs) removal in an advanced wastewater treatment facility and compare two parallel tertiary treatment methods for the removal of HAdVs.

Methods and Results

Tangential flow ultrafiltration was used to concentrate the water samples, and HAdVs were precipitated by polyethylene glycol. HAdVs were detected only by TaqMan real‐time PCR, and HAdV genotype was determined by DNA sequence. HAdVs were detected in 100% of primary clarification influent, secondary clarification effluent and granular media (GM) filtration effluent samples but only in 31·2% of membrane filtration (MF) effluent and 41·7% of final effluent (FE) samples, respectively. The average HAdVs loads were significantly reduced along the treatments but HAdVs were still present in FE. Comparison of two parallel treatments (GM vs MF) showed that MF was technically superior to GM for the removal of HAdVs.

Conclusions

These findings indicate that adenoviruses are not completely removed by treatment processes. MF is a better treatment for removal of adenoviruses than GM filtration. Because only qPCR was used, the results only indicate the removal of adenovirus DNA and not the infectivity of viruses.

Significance and Impact of the Study

Presence of HAdVs in FE by qPCR suggests a potential public health risk from exposure to the treated wastewater and using the FE for recreational or water reuse purposes should be cautious.     

shRNA抑制宫颈癌Hela细胞株HPV18 E6、E7基因表达的研究

应用短发夹RNA(Short hairpin RNA,shRNA)表达载体抑制宫颈癌Hela细胞株HPV18 E6、E7基因的表达。应用已鉴定的shRNA表达载体pHPV1、pHPV2转染Hela细胞,G418筛选阳性细胞,建立稳定转染细胞株;倒置荧光显微镜检测转染情况;提取细胞内总RNA,RT-PCR方法检测HPV18 E6、E7 mRNA;WesternBlot检测HPV18 E6、E7蛋白表达的变化;采用灰度分析软件对PCR扩增条带与蛋白质条带进行灰度分析。pHPV1实验组细胞内HPV18 E6、E7 mRNA含量分别为阴性对照组的31%、38%,E6、E7蛋白分别为阴性对照组的37%、31%;pHPV2实验组细胞内HPV18 E6、E7 mRNA含量分别为阴性对照组的54%、77%,E6、E7蛋白分别为阴性对照组的52%、83%。pHPV1、pHPV2表达载体能抑制Hela细胞HPV18 E6、E7的表达,针对外显子区434-452的pHPV1抑制作用更强。     

Anthropogenic and climatic impacts on surface pollen assemblages along a precipitation gradient in north‐eastern China

Aim To understand the scenarios of ‘anthropogenic biomes’ that integrate human and ecological systems, we need to explore the impacts of climate and human disturbance on vegetation in the past and present. Interactions among surface pollen, modern vegetation and human activities along climate and land‐use gradients are tested to evaluate the natural and anthropogenic forces shaping the modern vegetation, and hence to aid the reconstruction of vegetation and climate in the past. This in turn will help with future predictions. Location The North‐east China Transect (NECT) in north‐eastern China. Methods We analysed 33 surface pollen samples and 213 quadrats across four vegetation zones along the moisture/land‐use gradients of the NECT. Detrended correspondence analysis (DCA) and redundancy analysis (RDA) of 52 pollen taxa and three environmental variables were used to distinguish anthropogenic and climatic factors that affect surface pollen assemblages along the NECT. Results The 33 surface samples are divided into four pollen zones (forest, meadow steppe, typical steppe and desert steppe) corresponding to major vegetation types in the NECT. Variations in pollen ratios of fern/herb (F/H), Artemisia/Chenopodiaceae (A/C) and arboreal pollen/non‐arboreal pollen (AP/NAP) represent the vegetation and precipitation gradient along the NECT. DCA and RDA analyses suggest that surface pollen assemblages are significantly influenced by the precipitation gradient. Changes in the abundance of Chenopodiaceae pollen are related to both human activities and precipitation. Main conclusions Surface pollen assemblages, fossil pollen records, archaeological evidence and historical documents in northern China show that a large increase of Chenopodiaceae pollen indicates human‐caused vegetation degradation in sandy habitats. The A/C ratio is a good indicator of climatic aridity, but should be used in conjunction with multiple proxies of human activities and climate change in the pollen‐based reconstruction of anthropogenic biomes.