Selection of the 16S rRNA gene target region can substantially influence inferred respiratory microbiome profiles, particularly in low-biomass samples. In this study, we compared full-length V1-V9 (Pacific Biosciences) and V3-V4 (Illumina) amplicon sequencing using exhaled breath condensate collected from 50 healthy adults. Samples that yielded successful amplification for both approaches were included in paired, within-subject comparisons of sequencing depth, alpha diversity, and taxonomic composition. Sequencing of the V3-V4 region yielded significantly higher read counts and greater alpha diversity (richness and Shannon index) than full-length V1-V9 sequencing (all p < 0.001). Both approaches consistently identified a shared set of dominant respiratory-associated genera, including Acinetobacter, Streptococcus, Psychrobacter, Gemella, Neisseria, and Granulicatella. However, relative abundance estimates differed by target region, with Acinetobacter enriched in V1-V9 profiles, whereas Streptococcus and Neisseria were more abundant in V3-V4 data. In contrast, several genera showed comparable abundances across regions. These findings indicate that while overall community membership is broadly conserved, quantitative diversity metrics and genus-level abundance estimates are strongly influenced by primer and target-region selection. Overall, these results provide methodological guidance for interpreting respiratory microbiome data derived from exhaled breath condensate and highlight the importance of aligning sequencing strategies with specific study objectives.

Objective The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been engendering enormous hazards to the world. We obtained the complete genome sequences of SARSCoV-2 from imported cases admitted to the Guangzhou Eighth People's Hospital, which was appointed by the Guangdong provincial government to treat coronavirus disease 2019 (COVID-19). The SARS-CoV-2 diversity was analyzed, and the mutation characteristics, time, and regional trend of variant emergence were evaluated. Methods In total, 177 throat swab samples were obtained from COVID-19 patients (from October 2020 to May 2021). High-throughput sequencing technology was used to detect the viral sequences of patients infected with SARS-CoV-2. Phylogenetic and molecular evolutionary analyses were used to evaluate the mutation characteristics and the time and regional trends of variants. Results We observed that the imported cases mainly occurred after January 2021, peaking in May 2021, with the highest proportion observed from cases originating from the United States. The main lineages were found in Europe, Africa, and North America, and B.1.1.7 and B.1.351 were the two major sublineages. Sublineage B.1.618 was the Asian lineage (Indian) found in this study, and B.1.1.228 was not included in the lineage list of the Pangolin web. A reasonably high homology was observed among all samples. The total frequency of mutations showed that the open reading frame 1a (ORF1a) protein had the highest mutation density at the nucleotide level, and the D614G mutation in the spike protein was the commonest at the amino acid level. Most importantly, we identified some amino acid mutations in positions S, ORF7b, and ORF9b, and they have neither been reported on the Global Initiative of Sharing All Influenza Data nor published in PubMed among all missense mutations. Conclusion These results suggested the diversity of lineages and sublineages and the high homology at the amino acid level among imported cases infected with SARS-CoV-2 in Guangdong Province, China.

The Asian musk shrew (shrew) is a new reservoir of a rat hepatitis E virus (HEV) that has been classified into genotype HEV-C1 in the species Orthohepevirus C. However, there is no information regarding classification of the new rat HEV based on the entire genome sequences, and it remains unclear whether rat HEV transmits from shrews to humans. We herein inoculated nude rats (Long-Evans rnu/rnu) with a serum sample from a shrew trapped in China, which was positive for rat HEV RNA, to isolate and characterize the rat HEV distributed in shrews. A rat HEV strain, S1129, was recovered from feces of the infected nude rat, indicating that rat HEV was capable of replicating in rats. S1129 adapted and grew well in PLC/PRF/5 cells, and the recovered virus (S1129c1) infected Wistar rats. The entire genomes of S1129 and S1129c1 contain four open reading frames and share 78.3–81.8% of the nucleotide sequence identities with known rat HEV isolates, demonstrating that rat HEVs are genetically diverse. We proposed that genotype HEV-C1 be further classified into subtypes HEV-C1a to HEV-C1d and that the S1129 strain circulating in the shrew belonged to the new subtype HEV-C1d. Further studies should focus on whether the S1129 strain infects humans.

目的:观察老年人高尿酸血症的患病特征并对相关因素进行分析.方法:选择2015年9月至2016年12月至广州市番禺区大石街社区卫生服务中心进行健康体检的621例老年人作为研究对象,所有参与研究人员进行血压、身高、体质量以及相关生化检查,据参与研究人员的血尿酸情况将患者分为观察组和对照组.观察组人员血尿酸值≥420 μmol·L-1,共计282例,对照组人员血尿酸值＜420 μmol·L-1,共计339例.观察两组参与研究人员高血压、肥胖、以及各项生化指标异常的人员情况,然后使用非条件Logistic回归进行相关因素分析观察影响老年人高尿酸血症患者的相关因素.结果:观察组患者男性较多,且有高血压、糖尿病病史,肥胖、胆固醇(TC)、三酰甘油(TG)、高密度脂蛋白胆固醇(HDL)、低密度脂蛋白胆固醇(LDL)、血肌酐(Cr)异常人数均明显高于对照组,差异具有统计学意义(P＜0.05),进行多因素Logistic回归分析,结果发现肥胖、高血压、高血糖、血脂异常以及肾功能异常是导致老年人高尿酸血症的相关因素.结论:老年人高尿酸血症与肥胖、高血压、高血糖、血脂异常以及肾功能异常等因素密切相关.

BACKGROUND:Zika virus (ZIKV) is a mosquito-borne virus spreading rapidly in the Americas, Africa, and Asia. No indigenous ZIKV infection had been seen in China. We monitored ZIKV infection among travelers returning to Enping county from ZIKV transmitting countries from 1 March to 10 April 2016.METHODS:We analyzed data including interviews; conducted laboratory test on blood, urine, saliva, conjunctival swab or semen specimens for evidence of ZIKV infection; evaluated household for presence of Aedes mosquitoes or larvae.RESULTS:A total of 925 individuals were screened, 507 (54.8%) were interviewed, 400 (43.2%) provided samples, of which 13 (3.3%) tested positive for ZIKV including 3 asymptomatic. Rash, conjunctivitis, sore throat, fever were the common symptoms; rash was more pronounced in adults than in children. ZIKV RNA was detected for 1-4 days in blood, but longer in urine and saliva (3-32 days and 2-10 days). Among interviewed, 57.0% had good knowledge about ZIKV, 45.8% were worried about ZIKV, 99.2% would go to hospital if they had infection. Aedes mosquitoes or larvae were detected in townships of infected returners.CONCLUSIONS:ZIKV was imported to China. Screening by symptoms alone is inadequate for detecting ZIKV infection. ZIKV surveillance, health-education, and vector control are necessary to decrease risk of ZIKV transmission.

In 2015, an unexpected multiple outbreak of dengue occurred in Guangdong, China. In total, 1,699 cases were reported, of which 1,627 cases were verified to have DENV infections by nucleic acid or NS1 protein, including 44 DENV-1, 1126 DENV-2, 18 DENV-3 and 6 DENV-4, and the other cases were confirmed by NS1 ELISA. Phylogenetic analyses of DENV-1 isolates identified two genotypes (I and V). The predominant DENV-2 outbreak isolates were the Cosmopolitan genotypes, which likely originated from Malaysia. The DENV-3 isolates were assigned into genotype I and genotype III. All 6 DENV-4 isolates from imported cases were likely originally from Cambodia, Thailand and the Philippines. The entomological surveillance showed a moderate risk for the BI index in Chaozhou and Foshan and a low risk in Guangzhou. The imported cases were mostly detected in Guangzhou and Foshan. Surprisingly, the most serious outbreak occurred in Chaozhou, but not in Guangzhou or Foshan. A combined analyses demonstrated the multiple geographical origins of this outbreak, and highlight the detection of suspected cases after the alerting of imported cases, early implementation of control policies and reinforce the vector surveillance strategies were the key points in the chain of prevention and control of dengue epidemics.

Objective To provide scientific evidence for prevention and control of Zika virus diseases in China.Methods The clinical characteristics of 11 cases,specimen detection results of 11 cases and 3 latent infected patients were analyzed.Results Theoretically the incubation of 11 cases was predicted as 1 to 9 days,with median 4 days.The result showed that 11 cases performed rash,following 6 cases performed conjunctival congestion and 4 cases performed pharyngeal hyperemia.Whereas only 2 cases performed fever (body temperature ＞ 37.3 ℃),without muscle pain or arthralgia.Rash was the first symptom of 9 cases,while 2 cases performed fever as their first sign of Zika.The symptom of rash lasted about 2 to 8 days,with median 6 days.The onset of symptom was mainly on chest and backside.The state of rash showed a significant difference between adults and children.Among all specimens of 11 cases,the positive detection rate was 45.45％ (5/11) in serum,90.91％ (10/11) in saliva and 100％ (11/11) in urine.Moreover,there was 1 latent case detected positive in serum and saliva respectively,and 3 latent cases were detected positive in urine.Conclusion Symptom onset of Zika is mild,which impacts the early diagnosis.We suggest using nucleic acid detection on serum,salvia and urine as diagnosis basis.Besides,vector density control should be well conducted for reducing the risks of local transmissions of Zika.

OBJECTIVES:Fast expansion and linkage to microcephaly and Guillain Barre syndrome have made Zika virus (ZIKV) track attention of global health authority concerns. The epidemiology, virological characteristics and genetic evolution of introduced ZIKV to Guangdong, China, were investigated.METHODS:Analyses of the epidemiological characteristics and genetic diversity of ZIKV isolates were performed.RESULTS:A total of twenty-eight confirmed ZIKV infection cases were imported into China in 2016, of which 19 were imported into Guangdong, China from Venezuela (16), the Samoa Islands (1), Suriname (1) and Guatemala (1). Serial sampling studies of the cases indicated longer shedding times of ZIKV particles from urine and saliva samples than from serum and conjunctiva swab samples. Seven ZIKV strains were successfully isolated from serum, urine and conjunctiva swab samples using cell culture and neonatal mouse injection methods. Genomic analysis indicated that all viruses belonged to the Asian lineage but had different evolutionary transmission routes with different geographic origins. The molecular clock phylogenetic analysis of the ZIKV genomes indicated independent local transmission that appeared to have been previously established in Venezuela and Samoa. Additionally, we found 7 unique non-synonymous mutations in the genomes of ZIKV that were imported to China. The mutations may indicate that ZIKV has undergone independent evolutionary history not caused by sudden adaptation to Chinese hosts.CONCLUSION:The increasing number of ex-patriot Chinese returning from ZIKV hyper-endemic areas to Guangdong combined with the presence of a variety of Aedes species indicate the potential for autochthonous transmission of ZIKV in Guangdong.

Background We examined associations between single-nucleotide polymorphisms (SNPs) of IFITM3, TLR3, and CD55 genes and influenza clinical outcomes in Chinese. Methods A multicenter study was conducted on 275 adult cases of avian (H7N9) and pandemic (H1N1pdm09) influenza. Host DNA was extracted from diagnostic respiratory samples; IFITM3 rs12252, TLR3 rs5743313, CD55 rs2564978, and TLR4 rs4986790/4986791 were targeted for genotyping (Sanger sequencing). The primary outcome analyzed was death. Results IFITM3 and TLR3 SNPs were in Hardy-Weinberg equilibrium; their allele frequencies (IFITM3/C-allele 0.56, TLR3/C-allele 0.88) were comparable to 1000 Genomes Han Chinese data. We found over-representation of homozygous IFITM3 CC (54.5% vs 33.2%; P = .02) and TLR3 CC (93.3% vs 76.9%; P = .04) genotypes among fatal cases. Recessive genetic models showed their significant independent associations with higher death risks (adjusted hazard ratio [aHR] 2.78, 95% confidence interval [CI] 1.29-6.02, and aHR 4.85, 95% CI 1.11-21.06, respectively). Cumulative effects were found (aHR 3.53, 95% CI 1.64-7.59 per risk genotype; aHR 9.99, 95% CI 1.27-78.59 with both). Results were consistent for each influenza subtype and other severity indicators. The CD55 TT genotype was linked to severity. TLR4 was nonpolymorphic. Conclusions Host genetic factors may influence clinical outcomes of avian and pandemic influenza infections. Such findings have important implications on disease burden and patient care in at-risk populations.

End-point assays of in vitro cell proliferation and death have been employed to study the mechanisms of fungal pathogenesis and have shown the responses of host cells at individual time points. A new cell analysis technology has been developed that allows for the continuous measurement and quantification of cell activities, thus enabling the dynamic assessment of electrical impedance when various pathogens are cultured in vitro. In this study, this system was evaluated to determine the response of the cell line RAW264.7 to infection by several clinically relevant fungi in vitro, including Aspergillus fumigatus, Candida albicans, and melanized and albino mutant strains of Fonsecaea monophora. The results showed that infection resulted in rounding of the host cells with a loss of contact between individual cells and a decline in the electrical impedance of all test groups. However, changes in the electrical impedance were variable. Aspergillus fumigatus caused initial increases and later significant decreases in the electrical impedance, while for C. albicans and F. monophora, the effect was reduced. The melanized strain of F. monophora caused a faster change in the electrical impedance than the albino strain. Our data proved that this system can be used as an efficient tool for monitoring cellular responses to fungal infection.

BackgroundWhether host genetic factors determine clinical severity of influenza virus infections is unclear. This study examined the effects of single nucleotide polymorphisms (SNPs) of IFITM3, TLR3, CD55, and TLR4 genes on outcomes of avian influenza A (H7N9) and pandemic influenza A (H1N1)pdm09 in Chinese patients.MethodsThis multicentre cohort study took place in four centres in Hong Kong, Beijing, Guangdong, and Shanghai and included adults prospectively diagnosed with H7N9 and H1N1pdm09 infections during three respective seasonal outbreaks (H7N9, 2013–15; H1N1pdm09, 2011–14). The inclusion criteria were PCR-confirmed H7N9 or H1N1pdm09 virus infection, age 18 years or older, and Chinese ethnicity. Host DNA was extracted from diagnostic respiratory samples, and IFITM3(rs12252), TLR3(rs5743313), CD55(rs2564978), and TLR4(rs4986790/4986791) were genotyped by Sanger sequencing. The data were tested to see whether they deviated from the Hardy-Weinberg equilibrium, and were compared with the 1000 Genomes data. The primary outcome for analysis was mortality. The effects of the SNPs were examined under different genetic models (recessive, additive, and dominant). The joint effect of significant loci was tested by computing the genetic risk score. All identifying patient information was removed from the dataset, and only anonymous data were used for analysis; individual consent was not required. Ethics approvals were obtained from the institutional review boards of all participating institutes.FindingsOur cohort consisted of 275 adults with H7N9 and H1N1pdm09 infections, of whom 33 died. In patients who died, we found over-representation of the homozygous IFITM3 CC genotype (18 [54%] of 33 patients with available data who died vs 70 [33%] of 211 with available data who survived; p=0·017) and the TLR3 CC genotype (28 [93%] of 30 vs 160 [77%] of 208; p=0·039). Recessive genetic models, adjusted for age, comorbidity, and antiviral treatment, showed that these genotypes had a significant association with increased risk of death (IFITM3 CC genotype, adjusted hazard ratio [HR] 2·78 [95% CI 1·29–6·02]; and TLR3 CC genotype, adjusted HR 4·85 [1·11–21·06]). Cumulative effects were found (adjusted HR 3·53 [95% CI 1·64–7·59] per risk genotype; adjusted HR 9·99 [1·27–78·59] with both genotypes). The case-fatality rate in patients with a genetic risk score of 2, 1, and 0 was 23%, 11%, and 3%, respectively. Results were similar for each influenza subtype and other severity indicators such as development of acute respiratory failure. The population-attributable risk for mortality was 35·5% (95% CI 9·9–69·8) for IFITM3 CC and 74·6% (21·7–100) for TLR3 CC (combined effect, 83·6% [52·3–100]), owing to their high prevalence among Chinese people. A SNP of CD55 (genotype TT) was linked to severity of influenza as indicated by an increased risk for hospital admission (adjusted odds ratio [OR] 2·77, 95% CI 1·21–6·36; p=0·02); TLR4 was shown to be non-polymorphic in this cohort.InterpretationHost genetic factors might influence clinical outcomes of avian and pandemic influenza. Our findings could have important implications on public health and health-care planning, patient care, and design of clinical trials.FundingHealth and Medical Research Fund (RRG-09) of the Hong Kong Special Administrative Region, China.

Objective To provide scientific evidences for Zika virus detection by clarifying the means by which Zika virus was discharged and the duration of corresponding processes. Methods Various samples of Zika cases were collected at different times and detected by using real-time RT-PCR. The positive samples were inoculated into cells and suckling mice through intracranial injection. The whole genome se-quences of those isolated Zika virus strain were sequenced and the results were further analyzed by comparing with the sequences of Zika virus from GenBank. Results The positive rates of Zika virus in urine, saliva and serum samples were 82. 4% (14/17), 82. 4% (14/17) and 52. 9% (9/17) respectively. The longest period of detected presence of Zika virus was found in urine samples amongst the three types of samples, fol-lowed by saliva and serum samples. Six Zika virus strains were isolated from 9 positive serum samples. Phy-logenetic analysis showed that the six genomes of Zika virus all belonged to Asia lineage, but located in two branches by Samoa and Venezuela strains. Conclusion This study indicated that urine, saliva and serum all could be used as the samples for routine detection of Zika virus. Urine and saliva samples showed higher detection rates of Zika virus RNA in comparison to serum samples, while Zika virus could be easily isolated from positive serum samples. Suckling mice were better for Zika virus isolation than cell lines.

Objectives The third largest historical outbreak of dengue occurred during July to December 2014, in 20 of 21 cities of Guangdong, China. The epidemiological and molecular characteristics of the introduction, expansion and phylogeny of the DENV isolates involved in this outbreak were investigated. Methods A combination analyses of epidemiological characteristics and genetic diversity of dengue virus was performed in this study. Results In total, 45,236 cases and 6 fatalities were reported. Unemployed individuals, retirees and retailers were the most affected populations. A total of 6024 cases were verified to have DENV infections by nucleic acid detection, of which 5947, 74 and 3 were confirmed to have DENV-1, -2, and -3 infections, respectively. Phylogenetic analyses of DENV-1 isolates were assigned into three genotypes (I, IV, and V). Genotype V was the predominant genotype that likely originated from Singapore. The DENV-2 isolates were assigned to the Cosmopolitan and Asian I genotypes. A unique DENV-3 isolate (genotype III) shared high similarity with isolates obtained from Guangdong in 2013. Conclusions A combination analyses demonstrated the multiple geographical origins of this outbreak, and highlight the importance of early detection, the case management and vector surveillance for preventing further dengue epidemics in Guangdong.

目的 调查广东省揭阳市一个建筑工地登革热暴发疫情的流行病学特征及评价疫情处置效果.方法 采用现场流行病学调查方法对病例进行调查分析,于2014年10月9日至1 1月1 1日采集患者血液进行抗体检测和核酸检测,应用布雷图指数开展蚊媒密度调查.结果 首例发病为2014年9月26日,末例发病10月12日,流行期16d,共发病22例,罹患率为14.7％(22/150),采取防控措施后,蚊媒密度明显下降,疫情得到有效控制.登革热病毒进化分析显示工地病例分离株序列与广州市流行株有极高的遗传相似度.结论 该起疫情由登革热1型病毒引起,传染源可能来自广州市,早期病例的误诊是导致疫情在工地内传播的主要原因,政府部门及时应对和防蚊措施有效落实是成功防控登革热的关键.

Objective To establish a method for the isolation of Zika virus and to gather experi-ences for viral isolation. Methods Suckling mice at age 1-3 days were inoculated with serum samples posi-tive for Zika virus through intracranial injection. All mice were sacrificed 6 days after the injection. Viral nu-cleic acids were extracted from brain, heart, liver, spleen, lung, kidney, muscle, skin and intestine tissue samples and analyzed by real-time RT-PCR. The supernatants of brain tissues positive for Zika virus were used for subculturing. Nested PCR was performed to amplify the NS5 gene of the isolated virus. The se-quences of NS5 gene were analyzed by using MEGA6. 0 software. Results All of the tissue samples were positive for Zika virus. Higher viral loads were detected in heart and brain tissue samples with cycle thresh-old (Ct) values of 24. 4 and 25. 3, respectively. The second generation of Zika virus was identified in suck-ling mice brain tissues 2 days after infection by using real-time RT-PCR. The amplified product of nested PCR was 972 bp in length. Sequencing analysis showed that the isolated Zika virus ( GDZ16002 strain) be-longed to the Asian lineage. Conclusion A strain of Zika virus was successfully isolated in China by using intracranial injection via a suckling mouse model. The isolated Zika virus belonged to the Asian lineage.

目的 建立一种含内参的,针对登革病毒和基孔肯雅病毒核酸检测的3重荧光定量PCR方法. 方法 针对登革病毒3'端非结构蛋白编码区和基孔肯雅病毒5'端非结构蛋白编码区设计引物与探针,建立一套含有内参的并且能同时检测登革病毒和基孔肯雅病毒的3重实时荧光定量RT-PCR方法.对其最低检测限和特异度进行验证,通过检测血清标本对该方法进行临床应用评估. 结果 建立的3重荧光定量PCR登革病毒与基孔肯雅病毒绝对定量标准曲线显示,病毒拷贝数的对数值与CT值之间具有良好的线性关系,梯度稀释后的标准品核酸检测均呈现典型的S型曲线.检测日本脑炎病毒、西尼罗病毒、黄热病毒、辛德毕斯病毒各1株,结果均为阴性,特异度为100％.用该方法检测294份登革热患者血清,灵敏度为100％,特异度为89.3％,阳性预测值为92.1％,阴性预测值为100％;测定33份基孔肯雅热病患者血清的灵敏度、特异度及阴、阳性预测值均为100％. 结论 建立的含有内参的能同时检测登革病毒与基孔肯雅病毒的3重荧光定量PCR方法具有较高的灵敏度和特异度,可用于登革病毒与基孔肯雅病毒感染检测,为早期筛查和鉴别登革病毒和基孔肯雅病毒感染提供依据.