i-Juan Ma

Discovery of DNA Viruses in Wild-Caught Mosquitoes Using Small RNA High throughput Sequencing

Background Mosquito-borne infectious diseases pose a severe threat to public health in many areas of the world. Current methods for pathogen detection and surveillance are usually dependent on prior knowledge of the etiologic agents involved. Hence, efficient approaches are required for screening wild mosquito populations to detect known and unknown pathogens. Methodology/principal findings In this study, we explored the use of Next Generation Sequencing to identify viral agents in wild-caught mosquitoes. We extracted total RNA from different mosquito species from South China. Small 18–30 bp length RNA molecules were purified, reverse-transcribed into cDNA and sequenced using Illumina GAIIx instrumentation. Bioinformatic analyses to identify putative viral agents were conducted and the results confirmed by PCR. We identified a non-enveloped single-stranded DNA densovirus in the wild-caught Culex pipiens molestus mosquitoes. The majority of the viral transcripts (.>80% of the region) were covered by the small viral RNAs, with a few peaks of very high coverage obtained. The +/− strand sequence ratio of the small RNAs was approximately 7∶1, indicating that the molecules were mainly derived from the viral RNA transcripts. The small viral RNAs overlapped, enabling contig assembly of the viral genome sequence. We identified some small RNAs in the reverse repeat regions of the viral 5′- and 3′ -untranslated regions where no transcripts were expected. Conclusions/significance Our results demonstrate for the first time that high throughput sequencing of small RNA is feasible for identifying viral agents in wild-caught mosquitoes. Our results show that it is possible to detect DNA viruses by sequencing the small RNAs obtained from insects, although the underlying mechanism of small viral RNA biogenesis is unclear. Our data and those of other researchers show that high throughput small RNA sequencing can be used for pathogen surveillance in wild mosquito vectors.

Toll-like receptors, tumor necrosis factor-α, and interleukin-10 gene polymorphisms in risk of pulmonary tuberculosis and disease severity.

Toll-like receptors (TLRs) and cytokines play key roles in innate and adaptive immunity against Mycobacterium tuberculosis (M.TB). The aim of this study was to investigate whether the functional genetic variations at position 1805 G/T in TLR1, 2258 A/G in TLR2, -857 C/T, and -863 A/C in tumor necrosis factor-α (TNF-α), as well as -819 C/T in interleukin-10 (IL-10) confer susceptibility to pulmonary tuberculosis (PTB). We performed a hospital-based case-control study using 543 case patients and 544 controls. Multivariate logistic regression analysis revealed that the TT genotype of -857 C/T in TNF-α gene was significantly associated with lower risk of PTB, in comparison with other genotypes (odds ratios [OR] = 0.68, 95% confidence interval [CI] = 0.53-0.86, p = 0.001). Conversely, the genetic variants of -863 A/C in TNF-α gene was associated with susceptibility to PTB (OR = 2.42%, 95% CI = 1.28-4.59, p = 0.007) and clinical severity of disease (OR = 3.59%, 95% CI = 1.41-9.11, p = 0.007). Our results indicated that the variants in TNF-α gene were associated with susceptibility to PTB and clinical severity of disease, whereas no significance could be inferred from TLRs and IL-10 genes polymorphisms.

Increased Thyroid Function and Elevated Thyroid Autoantibodies in Pediatric Patients With Moyamoya Disease: A Case-Control Study

Background and Purpose— The purpose of this study was to investigate whether thyroid function and thyroid autoantibodies were associated with the risk of moyamoya disease in pediatric subjects. Methods— Thyroid function and thyroid autoantibodies were evaluated in patients with moyamoya disease and control subjects, and their associations with moyamoya disease were estimated using multivariate analysis. Results— We included 114 pediatric patients and 114 healthy control subjects. The patients displayed higher prevalence of increased thyroid function and elevated thyroid autoantibodies in comparison with control subjects. These remained significant after multivariate adjustment; the ORs (95% CI) for increased thyroid function and evaluated thyroid autoantibodies were evaluated as 12.47 (1.55 to 100.51) and 4.33 (1.29 to 14.59), respectively. Conclusions— Increased thyroid function and elevated thyroid autoantibodies are associated with moyamoya disease and therefore monitoring of thyroid function and thyroid autoantibodies in patients with moyamoya disease is suggested, which might help to guide subsequent clinical management.

Mixed infections of pandemic H1N1 and seasonal H3N2 viruses in 1 outbreak.

BACKGROUND In early September 2009, an outbreak of influenza occurred at a college campus in Beijing, China, in which both pandemic H1N1 and seasonal H3N2 viruses were detected. METHODS Outbreak investigation was performed in the campus. Epidemiologic, clinical data were collected by interviewing patients and retrieving medical records. Individual contact tracing was performed for detailed contact information. Viruses were identified by reverse-transcription polymerase chain reaction assays followed by sequence analysis. The hemagglutination inhibition test was used to detect antibodies to both viruses for paired serum samples. RESULTS Forty of 45 people with influenza-like illness had laboratory-confirmed influenza A infection; 22 of these 40 people were infected with pandemic H1N1 virus, 12 were infected with seasonal H3N2 virus, and 6 were coinfected with both viruses. In the subsequent generation of cases with mixed infection, we detected pandemic H1N1 virus infection more often than seasonal H3N2 virus infection. The clinical patterns were essentially similar for patients with different virus infections. No substantial differences in sequences were observed in either pandemic H1N1 or seasonal H3N2 virus between patients with mixed and single infection. Sequence analyses revealed that all of the detected viruses were susceptible to oseltamivir but resistant to adamantane. Hemagglutination inhibition tests of paired serum samples confirmed mixed infection in the outbreak. CONCLUSIONS Cocirculation of pandemic H1N1 virus and seasonal H3N2 virus led to a mixed infection in patients. Pandemic H1N1 virus, however, took prevalence over seasonal influenza virus in the course of transmission. Therefore, competitive circulation of seasonal influenza A virus with the pandemic H1N1 virus seems less likely.

Multidrug-resistant and extensively drug-resistant tuberculosis in multi-ethnic region, Xinjiang Uygur Autonomous Region, China.

Background The multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) has emerged as a global threat. Xinjiang is a multi-ethnic region and suffered second highest incidence of TB in China. However, epidemiological information on MDR and XDR TB is scarcely investigated. Methodology/Principal Findings A prospective study was conducted to analyze the prevalence of MDR and XDR TB and the differences of drug resistance TB between Chinese Han and other nationalities population at Chest Hospital of Xinjiang Uygur Autonomous Region, China. We performed in vitro drug susceptibility testing of Mycobacterium tuberculosis to first- and second-line anti-tuberculosis drugs for all 1893 culture confirmed positive TB cases that were diagnosed between June 2009 and June 2011. Totally 1117 (59.0%, 95% CI, 56.8%–61.2%) clinical isolates were resistant to ≥1 first-line drugs; the prevalence of MDR TB was 13.2% (95% CI, 11.7%–14.7%), of which, 77 (30.8%; 95% CI, 25.0%–36.6%) and 31 (12.8%; 95% CI, 8.6%–17.0%) isolates were pre-XDR and XDR TB respectively. Among the MDR/XDR TB, Chinese Han patients were significantly less likely to be younger with an odds ratio 0.42 for age 20–29 years and 0.52 for age 40–49 years; P trend = 0.004), and Chinese Han patients has a lower prevalence of XDR TB (9.6%) than all the other nationality (14.9%). Conclusions/Significance The burden of drug resistance TB cases is sizeable, which highlights an urgent need to reinforce the control, detection and treatment strategies for drug resistance TB. However, the difference of MDR and XDR TB between Chinese Han and other nationalities was not observed.

Lack of Peripheral Memory B Cell Responses in Recovered Patients with Severe Acute Respiratory Syndrome: A Six-Year Follow-Up Study

Six years have passed since the outbreak of severe acute respiratory syndrome (SARS). Previous studies indicated that specific Abs to SARS-related coronavirus (SARS-CoV) waned over time in recovered SARS patients. It is critical to find out whether a potential anamnestic response, as seen with other viral infections, exists to protect a person from reinfection in case of another SARS outbreak. Recovered SARS patients were followed up to 6 y to estimate the longevity of specific Ab. The specific memory B cell and T cell responses to SARS-CoV Ags were measured by means of ELISPOT assay. Factors in relation to humoral and cellular immunity were investigated. Six years postinfection, specific IgG Ab to SARS-CoV became undetectable in 21 of the 23 former patients. No SARS-CoV Ag-specific memory B cell response was detected in either 23 former SARS patients or 22 close contacts of SARS patients. Memory T cell responses to a pool of SARS-CoV S peptides were identified in 14 of 23 (60.9%) recovered SARS patients, whereas there was no such specific response in either close contacts or healthy controls. Patients with more severe clinical manifestations seemed to present a higher level of Ag-specific memory T cell response. SARS-specific IgG Ab may eventually vanish and peripheral memory B cell responses are undetectable in recovered SARS patients. In contrast, specific T cell anamnestic responses can be maintained for at least 6 y. These findings have applications in preparation for the possible reemergence of SARS.

Association of a functional polymorphism in the MMP-3 gene with Moyamoya Disease in the Chinese Han population.

Background: Moyamoya disease (MMD) is an uncommon cerebrovascular disease characterized by progressive stenosis of the intracranial internal carotid arteries and their proximal branches. The important role of genetic factors in the etiology and pathogenesis of MMD is being increasingly recognized. The study was designed to examine the association of single nucleotide polymorphisms in matrix metalloproteinase (MMP) and tissue inhibitors of metalloproteinase (TIMP) genes with MMD occurrence. Methods: A case-control study was performed. Five functional promoter polymorphisms in the MMP-2, MMP-3, MMP-9 and MMP-13 genes and a potentially functional promoter polymorphism in the TIMP-2 gene were determined by polymerase chain reaction-restriction fragment length polymorphism. Their associations with MMD were analyzed by multivariate logistic regression. Results: In total, 208 definite patients with MMD (including 31 familial MMD, FMMD, patients) and 224 healthy subjects were recruited. The frequency of the MMP-3 5A/6A and 5A/5A genotypes was significantly lower in MMD patients (OR = 0.57, 95% CI 0.38–0.86, pcorr = 0.042) compared with healthy controls in a dominant genetic model. Significant differences of the MMP-3 5A/6A polymorphism were also detected between FMMD patients and controls both in the dominant genetic model (OR = 0.23, 95% CI 0.08–0.68, pcorr = 0.048) and the additive genetic model (OR = 0.24, 95% CI 0.08–0.69, pcorr = 0.048). Conclusion: The functional polymorphism in the MMP-3 promoter might be associated with susceptibility to both MMD and FMMD in the Chinese Han population. The findings need to be validated in further studies including more subjects from different populations.

Genetic variants in MARCO are associated with the susceptibility to pulmonary tuberculosis in Chinese Han population.

Background Susceptibility to tuberculosis is not only determined by Mycobacterium tuberculosis infection, but also by the genetic component of the host. Macrophage receptor with a collagenous structure (MARCO) is essential components required for toll like receptor-signaling in macrophage response to Mycobacterium tuberculosis, which may contribute to tuberculosis risk. Principal Findings To specifically investigated whether single nucleotide polymorphisms (SNPs) in MARCO gene are associated with pulmonary tuberculosis in Chinese Han population. By selecting tagging SNPs in MARCO gene, 17 tag SNPs were identified and genotyped in 923 pulmonary tuberculosis patients and 1033 healthy control subjects using a hospital based case-control association study. Single-point and haplotype analysis revealed an association in intron and exon region of MARCO gene. One SNP (rs17009726) was associated with susceptibility to pulmonary tuberculosis, where the carriers of the G allele had a 1.65 fold (95% CI = 1.32–2.05, p corrected = 9.27E–5) increased risk of pulmonary tuberculosis. Haplotype analysis revealed that haplotype GC containing G allele of 17009726 and haplotype TGCC (rs17795618T/A, rs1371562G/T, rs6761637T/C, rs2011839C/T) were also associated with susceptibility to pulmonary tuberculosis (p corrected = 0.0001 and 0.029, respectively). Conclusions Our study suggested that genetic variants in MARCO gene were associated with pulmonary tuberculosis susceptibility in Chinese Han population, and the findings emphasize the importance of MARCO mediated immune responses in the pathogenesis of tuberculosis.

Presence of entomobirnaviruses in Chinese mosquitoes in the absence of Dengue virus co-infection.

Birnaviruses, including the genus Entomobirnavirus, are socio-economically important viruses. Currently, only Drosophila X virus has been formally assigned to the genus Entomobirnavirus, but two more viruses were recently isolated, Espirito Santo virus (ESV) and Culex Y virus. The host mosquito has been reported to carry many viruses, but seldom entomobirnaviruses. To discover potential pathogens in mosquitoes, we exploited small-RNAs high-throughput sequencing of three mosquito species caught in South China. A virus that genetically likes entomobirnavirus, Mosquito X virus (MXV), was identified from Anopheles sinensis and was 97% identical to ESV, which co-infects with Dengue virus (DENV). However, the absence of DENV in the A. sinensis suggested the independence of MXV infection from dengue co-infection. Our discovery complements prior research on entomobirnaviruses and proved that MXV may be widespread in mosquitoes on different continents. This work also highlights the applying of high-throughput sequencing of small RNAs to survey viruses carried by insect vectors.

Clinical and Immunological Characteristics of Patients with 2009 Pandemic Influenza A (H1N1) Virus Infection after Vaccination

BACKGROUND We followed a cohort of 773 individuals who received a monovalent vaccine against 2009 pandemic influenza A (H1N1). Approximately 6 weeks after vaccination, 12 persons developed the disease. METHODS Three groups of subjects were studied (12 patients who had or had not received previous monovalent vaccine and 1 group of 49 control subjects who had previously been immunized with the same vaccine). For all patients, clinical features were characterized and the causative viruses sequenced for possible mutations. Nasopharyngeal swabs, serum specimens, and peripheral blood monocyte cells (PBMCs) were collected at different time points up to 11 weeks after symptom onset to measure the virus load and humoral and cellular immune responses. Serum samples and PBMCs were also collected from 49 and 16 vaccinated control subjects, respectively. RESULTS Both patient groups had similar clinical manifestations. No substantial viral mutations were detected. Compared with unvaccinated patients, viral loads in vaccinated patients were initially higher, but the levels decreased faster to undetectable levels. However, the virus became detectable again for 6 of them. Two weeks after infection, vaccinated and unvaccinated patients had similar neutralizing antibody levels as the vaccinated control subjects. Thereafter, the neutralizing antibody levels decreased markedly in vaccinated patients. During the acute phase, memory T cell counts and tumor necrosis factor-α levels were significantly higher in vaccinated than in unvaccinated patients. CONCLUSIONS Although the clinical consequences of infection are comparable between vaccinated and unvaccinated patients, humoral and cellular immune responses in vaccinated patients are boosted for some weeks, indicating an additional benefit of vaccination against 2009 pandemic influenza A (H1N1) virus.