Simon Rayner

ORF-FINDER: a vector for high-throughput gene identification.

We have developed a simple and efficient system (ORF-FINDER) for selecting open reading frames (ORFs) from randomly fragmented genomic DNA fragments. The ORF-FINDER vectors are plasmids that contain a translational start site out of frame with respect to the gene for green fluorescent protein (GFP). Insertion of DNA fragments that bring the initiating ATG in frame with GFP and that contain no stop codons (that is, ORFs) results in the expression of ORF-GFP fusion proteins. In addition, we have developed software (GeneWorks and GenomeAnalyzer) to predict the optimal insert size for maximizing the number of gene-coding ORFs and minimizing unintentionally selected non-coding ORFs. To demonstrate the feasibility of using the ORF-FINDER system to screen genomes for ORFs, we cloned yeast genomic DNA and succeeded in enriching for ORFs by 25-fold. Furthermore, we have shown that the vector can effectively isolate ORFs from the more complex genomes of eukaryotic parasites. We envision that ORF-FINDER will have several applications including genome sequencing projects, gene building from oligonucleotides and construction of expression libraries enriched for ORFs.

Emergence of Genotype I of Japanese Encephalitis Virus as the Dominant Genotype in Asia

ABSTRACT Japanese encephalitis virus (JEV), a mosquito-borne zoonotic pathogen, is one of the major causes of viral encephalitis worldwide. Previous phylogenetic studies based on the envelope protein indicated that there are four genotypes, and surveillance data suggest that genotype I is gradually replacing genotype III as the dominant strain. Here we report an evolutionary analysis based on 98 full-length genome sequences of JEV, including 67 new samples isolated from humans, pigs, mosquitoes, midges. and bats in affected areas. To investigate the relationships between the genotypes and the significance of genotype I in recent epidemics, we estimated evolutionary rates, ages of common ancestors, and population demographics. Our results indicate that the genotypes diverged in the order IV, III, II, and I and that the genetic diversity of genotype III has decreased rapidly while that of genotype I has increased gradually, consistent with its emergence as the dominant genotype.

Comparative Proteomics Reveal Fundamental Structural and Functional Differences between the Two Progeny Phenotypes of a Baculovirus

ABSTRACT The replication of lepidopteran baculoviruses is characterized by the production of two progeny phenotypes: the occlusion-derived virus (ODV), which establishes infection in midgut cells, and the budded virus (BV), which disseminates infection to different tissues within a susceptible host. To understand the structural, and hence functional, differences between BV and ODV, we employed multiple proteomic methods to reveal the protein compositions and posttranslational modifications of the two phenotypes of Helicoverpa armigera nucleopolyhedrovirus. In addition, Western blotting and quantitative mass spectrometry were used to identify the localization of proteins in the envelope or nucleocapsid fractions. Comparative protein portfolios of BV and ODV showing the distribution of 54 proteins, encompassing the 21 proteins shared by BV and ODV, the 12 BV-specific proteins, and the 21 ODV-specific proteins, were obtained. Among the 11 ODV-specific envelope proteins, 8 either are essential for or contribute to oral infection. Twenty-three phosphorylated and 6 N-glycosylated viral proteins were also identified. While the proteins that are shared by the two phenotypes appear to be important for nucleocapsid assembly and trafficking, the structural and functional differences between the two phenotypes are evidently characterized by the envelope proteins and posttranslational modifications. This comparative proteomics study provides new insight into how BV and ODV are formed and why they function differently.

The epidemic origin and molecular properties of B': a founder strain of the HIV-1 transmission in Asia.

Objective:To clarify the epidemic origin and molecular properties of the B′ subtype that is an important strain in the HIV-1 epidemic in Asia. Design:The genealogical relationship between the B′ and B subtype was investigated with two globally representative datasets covering the gag and env regions. B′ sequences were identified, from which the epidemic origin, population genetics and the signature mutation sites of the B′ subtype were inferred. Methods:Two globally representative datasets were compiled, using phylogenetic methods. Through coalescent-based analysis, the genealogical relationship between the B′ and B subtypes was investigated. The divergence times and population genetic parameters of B′ were estimated in a Bayesian framework using Markov Chains Monte Carlo sampling under a relaxed molecular clock method. Additionally, molecular properties of the B′ were identified by performing comparative sequence analysis with the HIV-1 M group. Results:About 15 years later after the B subtype began to spread, the B′ diverged from the B subtype. The demographic history of B′ was reconstructed, and the epidemic of B′ was estimated to originate around 1985. Eight and nine distinct signature mutation sites, unique to B′, were found around the p17 and V3 regions, respectively. Conclusion:Our research is the first large-scale investigation on HIV-1 B′ at a global level and provides a deep insight into one of the founder strains of HIV-1 epidemic in Asia. Our results provide an important reference for HIV scientists, public health officials and HIV vaccine designers.

Seroprevalence of Kaposi's Sarcoma-Associated Herpesvirus and Risk Factors in Xinjiang, China

Xinjiang, China is an endemic area for Kaposis sarcoma (KS) but the seroprevalence of Kaposis sarcoma‐associated herpesvirus (KSHV) and risk factors remain undefined. In this study, antibodies to one KSHV latent protein (ORF73) and two KSHV lytic proteins (ORF65 and ORF‐K8.1) were examined in 2,228 subjects from the general population and 37 subjects infected with HIV‐1 in Xinjiang, and 560 subjects from the general population in Hubei, a low KS incidence region. The serostatus of a serum sample was defined based on positive results in any one of the three serologic assays. The seroprevalence of KSHV in the general population was higher in Xinjiang than in Hubei (19.2% vs. 9.5%; odds ratios [OR], 2.28; 95% confidence interval [CI], 1.68–3.08; P < 0.001). Among the ethnic groups in Xinjiang, 68 (15.8%) Han, 182 (20.7%) Uygur, 140 (19.9%) Hazakh, 9 (33.3%) Xibo, and 29 (16.8%) Hui were KSHV‐seropositive, respectively. Compared to the Han, the latter groups had an increase in the risk of KSHV of 62.2%, 63.8%, 180.1%, and 30.2% (P = 0.003, 0.004, 0.018, and 0.286, respectively). Subjects aged <20, 20–50, and >50 had a seroprevalence of KSHV of 11.8%, 17.9%, and 24.6%, respectively. Compared to subjects aged <20, the latter groups had an increase in the risk of KSHV of 63.3% and 144.5% (P = 0.009 and <0.001, respectively). Subjects infected with HIV‐1 in Xinjiang had a seroprevalence of KSHV of 43.2%, and a 220% increase in the risk of KSHV compared to the general population (P < 0.001). Similar results were obtained when the seroprevalence of KSHV was analyzed with any single or two of the three serologic assays alone. Genotyping identified three unique sequences clustered in the A clade. This study indicates that Xinjiang has a high seroprevalence of KSHV. Geographic location, ethnicity, age and HIV‐1 infection are risk factors. Serologic and genotyping results suggest the introduction of KSHV into Xinjiang by specific ethnic groups. J. Med. Virol. 81:1422–1431, 2009.

Functional characterization of lipase in the pathogenesis of Staphylococcus aureus

During infection, Staphylococcus aureus produces multiple enzymes that enable it to invade and destroy host tissues and metastasize to other sites. One such enzyme, lipase, has been recognized for its relationship in the virulence of S. aureus. However, a direct involvement of lipase in the pathogenesis of S. aureus remains to be demonstrated. Our prior study indicated that anti-lipase serum inhibits biofilm formation in S. aureus clinical strains. The aim of this study was to further characterize the roles of lipase in the pathogenesis in S. aureus. We found that deletion of the lipase-coding gene reduced biofilm formation relative to the wild-type strain. This was shown by culture in 96-well plates coated with collagen to resemble the in vivo infection process. Intraperitoneal inoculation of mice with a lipase mutant strain showed defective formation of peritoneal abscesses, and bacterial loads in different organs were much lower compared with the wild-type. Importantly, active immunization with recombinant lipase protected mice against a lethal challenge with S. aureus. Altogether, our data provide evidence that S. aureus lipase plays important roles in the pathogenesis of S. aureus.

MicroRNA miR-21 Attenuates Human Cytomegalovirus Replication in Neural Cells by Targeting Cdc25a

ABSTRACT Congenital human cytomegalovirus (HCMV) infection is a leading cause of birth defects, primarily manifesting as neurological disorders. HCMV infection alters expression of cellular microRNAs (miRs) and induces cell cycle arrest, which in turn modifies the cellular environment to favor virus replication. Previous observations found that HCMV infection reduces miR-21 expression in neural progenitor/stem cells (NPCs). Here, we show that infection of NPCs and U-251MG cells represses miR-21 while increasing the levels of Cdc25a, a cell cycle regulator and known target of miR-21. These opposing responses to infection prompted an investigation of the relationship between miR-21, Cdc25a, and viral replication. Overexpression of miR-21 in NPCs and U-251MG cells inhibited viral gene expression, genome replication, and production of infectious progeny, while shRNA-knockdown of miR-21 in U-251MG cells increased viral gene expression. In contrast, overexpression of Cdc25a in U-251MG cells increased viral gene expression and production of infectious progeny and overcame the inhibitory effects of miR-21 overexpression. Three viral gene products—IE1, pp71, and UL26—were shown to inhibit miR-21 expression at the transcriptional level. These results suggest that Cdc25a promotes HCMV replication and elevation of Cdc25a levels after HCMV infection are due in part to HCMV-mediated repression of miR-21. Thus, miR-21 is an intrinsic antiviral factor that is modulated by HCMV infection. This suggests a role for miR-21 downregulation in the neuropathogenesis of HCMV infection of the developing CNS. IMPORTANCE Human cytomegalovirus (HCMV) is a ubiquitous pathogen and has very high prevalence among population, especially in China, and congenital HCMV infection is a major cause for birth defects. Elucidating virus-host interactions that govern HCMV replication in neuronal cells is critical to understanding the neuropathogenesis of birth defects resulting from congenital infection. In this study, we confirm that HCMV infection downregulates miR-21 but upregulates Cdc25a. Further determined the negative effects of cellular miRNA miR-21 on HCMV replication in neural progenitor/stem cells and U-251MG glioblastoma/astrocytoma cells. More importantly, our results provide the first evidence that miR-21 negatively regulates HCMV replication by targeting Cdc25a, a vital cell cycle regulator. We further found that viral gene products of IE1, pp71, and UL26 play roles in inhibiting miR-21 expression, which in turn causes increases in Cdc25a and benefits HCMV replication. Thus, miR-21 appears to be an intrinsic antiviral factor that represents a potential target for therapeutic intervention.

The amino acid substitutions rtP177G and rtF249A in the reverse transcriptase domain of hepatitis B virus polymerase reduce the susceptibility to tenofovir

Long term antiviral therapy with nucleoside/nucleotide analogs have been routinely used to treat chronic hepatitis B virus (HBV) infection but may lead to the emergence of drug-resistant viral mutants. However, the HBV resistance mutations for tenofovir (TDF) remain controversial. It is speculated that the genetic barrier for TDF resistance may be high for HBV. We asked whether selected amino acid substitutions in HBV polymerase may reduce susceptibility to TDF. A series of amino acids in HBV polymerase were selected based on bioinformatics analysis for mutagenesis. The replication competence and susceptibility to TDF of the mutated HBV clones were determined both in vitro and in vivo. nineteen mutations in HBV polymerase were included and impaired the replication competence of HBV genome in different degrees. The mutations at rtL77F (sS69C), rtF88L (sF80Y), and rtP177G (sR169G) also significantly affected HBsAg expression. The HBV mutants with rtP177G and rtF249A were found to have reduced susceptibility to TDF in vitro with a resistance index of 2.53 and 12.16, respectively. The testing in in vivo model based on the hydrodynamic injection revealed the antiviral effect of TDF against wild type and mutated HBV genomes and confirmed the reduced the susceptibility of mutant HBV to TDF.

The spatial and temporal dynamics of rabies in China

Background and Objectives Recent years have seen a rapid increase in the number of rabies cases in China and an expansion in the geographic distribution of the virus. In spite of the seriousness of the outbreak and increasing number of fatalities, little is known about the phylogeography of the disease in China. In this study, we report an analysis of a set of Nucleocapsid sequences consisting of samples collected through the trial Chinese National Surveillance System as well as publicly available sequences. This sequence set represents the most comprehensive dataset from China to date, comprising 210 sequences (including 57 new samples) from 15 provinces and covering all epidemic regions. Using this dataset we investigated genetic diversity, patterns of distribution, and evolutionary history. Results Our analysis indicates that the rabies virus in China is primarily defined by two clades that exhibit distinct population subdivision and translocation patterns and that contributed to the epidemic in different ways. The younger clade originated around 1992 and has properties that closely match the observed spread of the recent epidemic. The older clade originated around 1960 and has a dispersion pattern that suggests it represents a strain associated with a previous outbreak that remained at low levels throughout the country and reemerged in the current epidemic. Conclusions Our findings provide new insight into factors associated with the recent epidemic and are relevant to determining an effective policy for controlling the virus.

Analysis on Factors Related to Rabies Epidemic in China from 2007-2011*

To analyze features of the rabies epidemic in China between 2007 and 2011, identify factors influencing the epidemic and to provide a scientific basis for further control and prevention of rabies, Descriptive epidemiological methods and statistical analysis was used on data collected from the National Disease Reporting Information System between 2007 to 2011 and the National Active Surveillance System between 2007 and 2010. Our analysis shows that while the number of human rabies cases decreased year by year, the number of districts reporting cases did not show significant change. The situations in Guangdong, Guangxi, Guizhou and Hunan provinces clearly improved over the period but they remain provinces with high-incidence, and consequently influence the epidemic situation of surrounding provinces and possibly the whole country. Summer and autumn were high-incidence seasons. Farmers, students and pre-school children represent the high-risk populations, and rates of cases in farmers increased, those for students decreased, and pre-school children remained unchanged. Provinces with active surveillance programs reported a total of 2346 individual cases, of which 88.53% were associated with canines. Postexposure prophylaxis (PEP) of rabies cases was not significantly improved, whereas PEP in post-exposure population was good. In rural regions of China, canine density was reduced somewhat, and the immunization rate increased slightly. Finally we show that while the epidemic decreased 2007 to 2011 in China, cases continued to be diffused in certain regions. Lack of standardization of PEP on rabies cases was the main reason of morbidity. The high density and low immunization of dog in rural areas and the defective situation of PEP are still continuous occurrences in China and remain a cause for concern.