Capacity assessment of the health laboratory system in two resource-limited provinces in China.

BACKGROUND: Strong laboratory capacity is essential for detecting and responding to emerging and re-emerging global health threats. We conducted a quantitative laboratory assessment during 2014-2015 in two resource-limited provinces in southern China, Guangxi and Guizhou in order to guide strategies for strengthening core capacities as required by the International Health Regulations (IHR 2005). METHODS: We selected 28 public health and clinical laboratories from the provincial, prefecture and county levels through a quasi-random sampling approach. The 11-module World Health Organization (WHO) laboratory assessment tool was adapted to the local context in China. At each laboratory, modules were scored 0-100% through a combination of paper surveys, in-person interviews, and visual inspections. We defined module scores as strong (> = 85%), good (70-84%), weak (50-69%), and very weak (< 50%). We estimated overall capacity and compared module scores across the provincial, prefecture, and county levels. RESULTS: Overall, laboratories in both provinces received strong or good scores for 10 of the 11 modules. These findings were primarily driven by strong and good scores from the two provincial level laboratories; prefecture and county laboratories were strong or good for only 8 and 6 modules, respectively. County laboratories received weak scores in 4 modules. The module, 'Public Health Functions' (e.g., surveillance and reporting practices) lagged far behind all other modules (mean score = 46%) across all three administrative levels. Findings across the two provinces were similar. CONCLUSIONS: Laboratories in Guangxi and Guizhou are generally performing well in laboratory capacity as required by IHR. However, we recommend targeted interventions particularly for county-level laboratories, where we identified a number of gaps. Given the importance of surveillance and reporting, addressing gaps in public health functions is likely to have the greatest positive impact for IHR requirements. The quantitative WHO laboratory assessment tool was useful in identifying both comparative strengths and weaknesses. However, prior to future assessments, the tool may need to be aligned with the new WHO IHR monitoring and evaluation framework.

Correction to: Genetic characterization of Bacillus anthracis in Guizhou Province, Southwest of China.

ERRATUM: Upon publication of the original article (1) it was highlighted by the authors that a grant awarded to support the research work of the study was missed in the acknowledgements. It should also be acknowledged that the grant titled "Genotyping and Molecular Epidemiological Characteristic of Bacillus anthracis in Guizhou Province" awarded by the Program of Natural Science Foundation of Guizhou Province (Grant No. Qian Ke He J Word [2015] 2084)also contributed to the resources for this research. This has since been formally noted in this correction article.

Genetic characterization of Bacillus anthracis in Guizhou Province, Southwest of China.

BACKGROUND: Bacillus (B.) anthracis is the pathogen that causes fatal anthrax. Guizhou Province is an old foci of anthrax in the southwest of China. Human anthrax has also been frequently reported in Guizhou in recent year. However, there is limited information on the genetic background of local B. anthracis isolates in Guizhou Province. Strain-specific detection of this bacterium using molecular approaches has enhanced our knowledge of microbial genetics. In the present study, we employed Multiple Locus Variable Number Tandem Repeats (VNTR) Analysis (MLVA) assay to analyze the genetic characteristics of B. anthracis strains isolated in Guizhou Province and their relationships to worldwide distributed isolates. RESULTS: A total of 32 isolates of B. anthracis from soil, human, cattle, dog and water of different anthrax epidemics in Guizhou Province from 2006 to 2011 were confirmed with phage lysis test, penicillin inhibition test and PCR. MLVA-8 discriminated them into 28 unique MLVA types (MT G1 - G28), which were novel MTs compared with the previous reports. Cluster tree based on 32 isolates from Guizhou Province and 76 worldwide distributed isolates (30 MTs) showed they were divided into three clusters, designated A, B and C. All the 32 isolates were distributed in cluster A, which were further grouped into A1, A2, A3 and A4 sub-clusters. 32 isolates from Guizhou Province were closely grouped in each of the sub-clusters, respectively. Minimum Spanning Tree (MST) based on the MLVA data showed that the 28 MLVA profiles of isolates from Guizhou Province and 30 MLVA profiles of worldwide distributed isolates formed three clonal complexes (CCs) and ten singletons. CONCLUSIONS: 28 novel MTs of B. anthracis from Guizhou were revealed and their relationships to worldwide isolates were showed. The results will provide important information for prevention of anthrax and also enhances our understanding of genetic characteristics of B. anthracis in China.

[Molecular typing of 12 Brucella strains isolated in Guizhou province in 2010-2013].

OBJECTIVE: To identify and characterize the Brucella strains from Guizhou province in 2010-2013. METHODS: A total of 12 strains of Brucella suspicious bacteria were isolated in Guizhou province from 2010 to 2013. Four strains (GZLL3, GZLL4, GZLL11 and SH2) were isolated from goat blood samples and eight strains (SH4, GZZY, GZSQ, GZZA, BR13001, BR13004, BR13005 and BR13006) were isolated from blood samples of patient 12 Brucella suspicious strains were identified and characterized using conventional methods. Brucella genus specific gene BCSP31-based PCR (BCSP31-PCR) was used to identify the genus of Brucella and IS711 insert sequence-based PCR (AMOS-PCR) was applied to identify the species of Brucella strains. Goats and patients originated Brucella strains were comparatively analysed using Pulse-field Gel Electrophoresis (PFGE). RESULTS: Both of conventional methods and PCR identified the 12 Brucella suspicious strains as B. melitensis biotype 3. BCSP31-PCR identification results showed that a specific DNA bands (223 bp) were detected in all the 12 strains and positive control samples with no DNA band in negative samples. AMOS-PCR amplified a 731 bp-DNA bands in all the 12 strains, with 731 bp, 498 bp and 275 bp in M5, S2 and A19 strains, respectively, and no DNA band was detected in the negative control samples. PFGE analysis showed that 12 Brucella isolates from patients and goats showed consistent PFGE patterns with the digestion of restriction enzyme Xba I. CONCLUSION: The epidemic species/type of Brucella in both human and animal in Guizhou province was B. melitensis biotype 3 and goat was the main animal source of infection of brucellosis in Guizhou province.

Effects of Combined Nitrogen-Phosphorus on Biomass Accumulation, Allocation, and Allometric Growth Relationships in Pinus yunnanensis Seedlings after Top Pruning.

Pinus yunnanensis (Franch), a species endemic to southwestern China, provides significant ecological and economic benefits. The quality of afforestation can be enhanced by promoting high-quality sprout growth. This study analyzed the effects of six fertilization treatments following top pruning: T1 (N: 0 g/plant-1; P: 0 g/plant-1), T2 (N: 0 g/plant-1; P: 2 g/plant-1), T3 (N: 0 g/plant-1; P: 4 g/plant-1), T4 (N: 0.6 g/plant-1; P: 0 g/plant-1), T5 (N: 0.6 g/plant-1; P: 2 g/plant-1), and T6 (N: 0.6 g/plant-1; P: 4 g/plant-1). The accumulation and allocation of aboveground biomass in roots, stems, and leaves of P. yunnanensis were measured, as well as changes in biomass per plant at 90 days (early stage), 180 days (middle stage), and 270 days (late stage) post-fertilization. At 90 days, root biomass accumulation in T3 was significantly higher, by 13.31%, compared to that in T1 (p < 0.05). The growth rates of stem and plant biomass followed the order T6 > T1 > T3 > T5 > T4 > T2. The biomass of sprouts and individual plants exhibited allometric growth under T1, T5, and T6 treatments. At 180 days, needle biomass allocation in T1 and T4 increased by 7.47% and 8.6%, respectively, compared to 90 days. Combined nitrogen-phosphorus fertilization significantly influenced aboveground biomass allocation, promoting growth more effectively than other treatments. By 270 days, the stem and individual biomass in T2 and T3 treatments showed significant differences (p < 0.01) compared to other treatments. Overall, root, stem, and sprouts were primarily influenced by phosphorus fertilization, while nitrogen fertilization notably promoted needle and leaf growth in later stages. The aboveground components were more affected by phosphorus fertilization. The combination of nitrogen and phosphorus fertilizers enhanced early-stage stem and sprouts, as well as late-stage root development.

Metagenomic analysis of individual mosquito viromes reveals the geographical patterns and drivers of viral diversity.

Mosquito transmitted viruses are responsible for an increasing burden of human disease. Despite this, little is known about the diversity and ecology of viruses within individual mosquito hosts. Here, using a meta-transcriptomic approach, we determined the viromes of 2,438 individual mosquitoes (81 species), spanning ~4,000 km along latitudes and longitudes in China. From these data we identified 393 viral species associated with mosquitoes, including 7 (putative) species of arthropod-borne viruses (that is, arboviruses). We identified potential mosquito species and geographic hotspots of viral diversity and arbovirus occurrence, and demonstrated that the composition of individual mosquito viromes was strongly associated with host phylogeny. Our data revealed a large number of viruses shared among mosquito species or genera, enhancing our understanding of the host specificity of insect-associated viruses. We also detected multiple virus species that were widespread throughout the country, perhaps reflecting long-distance mosquito dispersal. Together, these results greatly expand the known mosquito virome, linked viral diversity at the scale of individual insects to that at a country-wide scale, and offered unique insights into the biogeography and diversity of viruses in insect vectors.

Source tracking of human leptospirosis: serotyping and genotyping of Leptospira isolated from rodents in the epidemic area of Guizhou province, China.

BACKGROUND: Sustained human leptospirosis as well as death cases has been reported in Qiandongnan Prefecture, Southeast of Guizhou, China, recently, but these human patients were only clinically diagnosed, and leptospires have never been isolated from patients in these epidemic regions, In order to track the source of infection and understand the etiologic characteristic of leptospirosis, we performed rodent carrier surveillance for leptospirosis in the epidemic area in 2011. The population distribution of rodents in the epidemic regions was revealed. RESULTS: Four strains of leptospire were isolated from Apodemus agrarius. Microscopic agglutination test (MAT) confirmed the four isolates belonged to leptospiral serogroup Icterohaemorrhagiae. Multilocus sequence typing (MLST) indicated that all the four strains were defined as sequence type 1(ST1), which is identical to the three strains isolated from Rattus tanezumi in Rongjiang County in 2007. Clustering analysis of the MLST data indicated that the local isolates exactly matched with reference strain of leptospiral serovar Lai strain 56601, which is consistent with anti-Leptospira antibody detection of patients using MAT. CONCLUSIONS: Apodemus agrarius may be the potentially important carrier of leptospirosis and the potential source of leptospiral infection in human, and serovar Lai maybe the epidemic serovar of Leptospira in the localities.

Metagenomic analysis of individual mosquitos reveals the ecology of insect viruses.

Mosquito transmitted viruses are responsible for an increasing burden of human disease. Despite this, little is known about the diversity and ecology of viruses within individual mosquito hosts. Using a meta-transcriptomic approach, we analysed the virome of 2,438 individual mosquitos (79 species), spanning ~4000 km along latitudes and longitudes in China. From these data we identified 393 core viral species associated with mosquitos, including seven (putative) arbovirus species. We identified potential species and geographic hotspots of viral richness and arbovirus occurrence, and demonstrated that host phylogeny had a strong impact on the composition of individual mosquito viromes. Our data revealed a large number of viruses shared among mosquito species or genera, expanding our knowledge of host specificity of insect-associated viruses. We also detected multiple virus species that were widespread throughout the country, possibly facilitated by long-distance mosquito migrations. Together, our results greatly expand the known mosquito virome, linked the viral diversity at the scale of individual insects to that at a country-wide scale, and offered unique insights into the ecology of viruses of insect vectors.

[Analysis of the genetic differences in the nucleoprotein between rabies virus and its vaccine strains in Guizhou province from year 2005 to 2010].

OBJECTIVE: This study was to explore the differences in the nucleoprotein gene between rabies virus (RABV) and its vaccine strains in Guizhou province from year 2005 to 2010. METHODS: Samples from 4 rabies patients and cerebral tissue samples of 28 rabies infected dogs were collected from different districts in Guizhou province between year 2005 and 2010. Direct Immunofluorescence Assay (DFA) and RT-nested PCR assay were applied to detect the overall length of N gene sequence. Meanwhile, based on the comparison between the homology and phylogenetic tree, the differences in N gene sequence between the prevalent RABV and the RABV vaccine strains collected from NCBI database in these years. RESULTS: According to DFA and RT-nested PCR assay, the antigen and nucleic acid of the 21 dogs and 4 human samples were both confirmed positive; whose full length of N gene sequences were both 1353 bp. The homological analysis showed that the 25 strains of RABV virus and the RABV type I virus stored by GenBank database shared a high homology in N gene nucleotide and amino acid sequences, which were 89%-100% and 98%-100%, respectively. Besides, the homology between the 25 strains of RABV virus and its vaccines in nucleotide and amino acid sequences were separately 86%-95% and 96%-100%. The N gene of vaccines for livestock shared the highest homology with HEP-Flury strain in the nucleotide and amino acid, which were 88%-89% and 98%-99%, respectively. The vaccines for human use showed its greatest homology with the CTN strain in nucleotide (86%-100%) and amino acid (96%-100%). The phylogenetic tree analysis indicated that the 25 strains of RABV virus, RABV type I virus and the CTN vaccine strains constituted one individual cluster, which was least different from the CTN vaccine for human use. CONCLUSION: The prevalent RABV virus, the vaccine HEP-Flury for livestock and the vaccine CTN for human use were found to be highly similar in N gene expression in Guizhou province from 2005 to 2010.

Ehrlichia, Coxiella and Bartonella infections in rodents from Guizhou Province, Southwest China.

Rodents are generally recognized to be the reservoir hosts of a great many zoonotic pathogens. In some areas of China, rodent-borne pathogens, as well as the role of rodents in the natural cycle of these pathogens, are still poorly investigated. To increase our knowledge on the distribution and epidemiology of rodent-borne bacterial pathogens, 81 rodent liver samples were collected in three locations of Guizhou province located in Southwest China, and screened for the presence of Ehrlichia, Coxiella, and Bartonella in them. A putative novel Ehrlichia species was identified in 5 Berylmys bowersi samples (100%, 5/5). Its 16S rRNA, gltA, and groEL genes have highest 99.84%, 89.11%, and 98.02% identities to those from known Ehrlichia species, and form distinct clades in the phylogenetic trees. Herein we name it "Candidatus Ehrlichia zunyiensis". Bartonella was tested positive in 8 A. agrarius (striped field mouse), 2 A. chevrieri (Chevrier's field mouse), 1 R. norvegicus (Norway rat), 1 N. confucianus, and 1 N. lotipes, with a total positive rate of 16.05% (13/81). Sequence analysis indicated high genetic diversity in these Bartonella strains. Unexpectedly, two Coxiella strains were identified from the rodents (1 Niviventer confucianus and 1 Mus pahari). Genetic and phylogenetic analysis indicated that both of them are closely related to the Coxiella endosymbiont of ticks. This result supported previous conjectures that vertebrate hosts such as rodents may play a role in the preservation and transmission of Coxiella endosymbiont of ticks.

Diversity of Rickettsiales in Rhipicephalus microplus Ticks Collected in Domestic Ruminants in Guizhou Province, China.

Rhipicephalus microplus ticks are vectors for multiple pathogens infecting animals and humans. Although the medical importance of R. microplus has been well-recognized and studied in most areas of China, the occurrence of tick-borne Rickettsiales has seldom been investigated in Guizhou Province, Southwest China. In this study, we collected 276 R. microplus ticks from cattle (209 ticks) and goats (67 ticks) in three locations of Guizhou Province. The Rickettsia, Anaplasma, and Ehrlichia were detected by targeting the 16S rRNA gene and were further characterized by amplifying the key genes. One Rickettsia (Ca. Rickettsia jingxinensis), three Ehrlichia (E. canis, E. minasensis, Ehrlichia sp.), and four Anaplasma (A. capra, A. ovis, A. marginale, Ca. Anaplasma boleense) species were detected, and their gltA and groEL genes were recovered. Candidatus Rickettsia jingxinensis, a spotted fever group of Rickettsia, was detected in a high proportion of the tested ticks (88.89%, 100%, and 100% in ticks from the three locations, respectively), suggesting the possibility that animals may be exposed to this type of Rickettsia. All the 16S, gltA, groEL, and ompA sequences of these strains are 100% identical to strains reported in Ngawa, Sichuan Province. E. minasensis, A. marginale, and Candidatus Anaplasma boleense are known to infect livestock such as cattle. The potential effects on local husbandry should be considered. Notably, E. canis, A. ovis, and A. capra have been reported to infect humans. The relatively high positive rates in Qianxinan (20.99%, 9.88%, and 4.94%, respectively) may indicate the potential risk to local populations. Furthermore, the genetic analysis indicated that the E. minasensis strains in this study may represent a variant or recombinant. Our results indicated the extensive diversity of Rickettsiales in R. microplus ticks from Guizhou Province. The possible occurrence of rickettsiosis, ehrlichiosis, and anaplasmosis in humans and domestic animals in this area should be further considered and investigated.

Prevalence of Spotted Fever Group Rickettsia and Candidatus Lariskella in Multiple Tick Species from Guizhou Province, China.

Rickettsiales (Rickettsia spp., Ehrlichia spp., and Anaplasma spp., etc.) are generally recognized as potentially emerging tick-borne pathogens. However, some bacteria and areas in China remain uninvestigated. In this study, we collected 113 ticks from mammals in Guizhou Province, Southwest China, and screened for the Rickettsiales bacteria. Subsequently, two spotted fever group Rickettsia species and one Candidatus Lariskella sp. were detected and characterized. "Candidatus Rickettsia jingxinensis" was detected in Rhipicephalus microplus (1/1), Haemaphysalis flava (1/3, 33.33%), Haemaphysalis kitaokai (1/3), and Ixodes sinensis (4/101, 3.96%), whereas Rickettsia monacensis was positive in H. flava (1/3), H. kitaokai (2/3), and I. sinensis ticks (74/101, 73.27%). At least two variants/sub-genotypes were identified in the R. monacensis isolates, and the strikingly high prevalence of R. monacensis may suggest a risk of human infection. Unexpectedly, a Candidatus Lariskella sp. belonging to the family Candidatus Midichloriaceae was detected from Ixodes ovatus (1/4) and I. sinensis (10/101, 9.90%). The gltA and groEL gene sequences were successfully obtained, and they show the highest (74.63-74.89% and 73.31%) similarities to "Candidatus Midichloria mitochondrii", respectively. Herein, we name the species "Candidatus Lariskella guizhouensis". These may be the first recovered gltA and groEL sequences of the genus Candidatus Lariskella.

Epidemiological Investigation and Etiological Analysis of a Cutaneous Anthrax Epidemic Caused by Butchering Sick Cattle in Guizhou, China.

A suspected human cutaneous anthrax epidemic caused by butchering sick cattle occurred in Zhijin County of Guizhou Province, Southwest of China, in 2016. Epidemiological investigation and etiological analysis were performed to provide a scientific basis for the source tracking of the epidemic. The epidemic was epidemiologically investigated, and skin blister samples collected from patients and soil samples collected from the butchering spots were used for Bacillus anthracis isolation. The suspicious B. anthracis isolates were identified using conventional methods and PCR, followed by genotyping using multiple-locus variable-number tandem repeats (VNTRs) analysis (MLVA-15) and canonical single-nucleotide polymorphism (canSNP). The genetic relationship of epidemic strains and isolates collected from other regions was analyzed. Epidemiological investigation results showed that the patients may be infected by B. anthracis during butchering sick cattle. Two suspected B. anthracis strains were isolated from blood samples and blister fluids, respectively. Conventional methods identified the two suspected isolates as B. anthracis, while PCR results showed that anti-protective antigen (PA) and capsule (CAP) gene were positive in the two isolates. MLVA-15 showed that the MLVA profiles of the two isolates were 9-20-12-53-16-2-8-8-8-4-4-4-4-10-4, which is different from the MLVA profiles of representative strains from other regions. CanSNP analysis showed that the two strains belonged to cluster A.Br.001/002. Clustering analysis and minimum spanning tree (MST) demonstrated that the two isolates were clustered with strains previously isolated from Guizhou Province. The results indicated that B. anthracis was the pathogen for this epidemic, and the patients were infected during butchering the sick. The genetic characteristics and the relationship of the B. anthracis isolates to strains from other regions indicated that the epidemic was a local occurrence.

Genetic analysis of group A streptococcus isolates recovered during acute glomerulonephritis outbreaks in Guizhou Province of China.

In this study, 68 group A streptococcus (GAS) isolates associated with two outbreaks of acute glomerulonephritis (AGN) in China were analyzed by emm typing. A total of 11 different emm types were identified. Analysis of emm type distribution suggested that AGN outbreaks in two counties were caused by emm60.1- and emm63.0-type GAS. These two types were further characterized by pulsed-field gel electrophoresis, multilocus sequence typing, sof sequence typing, and PCR-based identification of streptococcal pyrogenic exotoxin A, B, and C (speA, speB, and speC) genes. In antimicrobial susceptibility tests, all outbreak strains were resistant to erythromycin and tetracycline, and the rates of resistance of nonoutbreak strains to the two antibiotics were 63.6% and 90.9%. This study is also the first to report a nephritogenic M63 GAS strain.

Novel genospecies of Borrelia burgdorferi sensu lato from rodents and ticks in southwestern China.

By using multilocus sequence analysis, five Borrelia valaisiana-related strains isolated from rodents and ticks in southwestern China were eventually classified as a new genospecies of B. burgdorferi sensu lato rather than B. valaisiana. The finding explained the differences in transmission cycle and phenotype between B. valaisiana strains from Europe and B. valaisiana-related strains from eastern Asia.

Leptospira interrogans infection leads to IL-1ß and IL-18 secretion from a human macrophage cell line through reactive oxygen species and cathepsin B mediated-NLRP3 inflammasome activation.

Leptospirosis is a worldwide zoonosis caused by spirochetes from the genus Leptospira. Although there is a large diversity of clinical signs and symptoms, a severe inflammatory response is common to all leptospirosis patients. The mechanism of IL-1ß secretion during Leptospira infection has been previously studied in mouse macrophages. However, the outcome of Leptospira infection is very different in human and murine macrophages, and the mechanisms responsible for IL-1ß secretion in human macrophages had not been investigated. This study therefore examines the effects of Leptospira interrogans infection on inflammasome activation and proinflammatory cytokine expression in human macrophages. Increased mRNA and protein expression of NLRP3 was observed by real time RT-PCR and flow cytometry at 1 h after co-cultivation. Enzyme-linked immunosorbent assay (ELISA) determination showed that IL-1ß and IL-18 are released in the culture supernatants at 1 h after cultivation. The inhibition assay showed that glybenclamide (a K+ efflux inhibitor that blocks NLRP3 inflammasome activation) and N-benzyloxycarbony-Val-Ala-Asp (O-methyl)-fluoromethylketone (Z-VAD-FMK; a caspase-1 inhibitor) and NLRP3 depletion with siRNAs reduced the levels of IL-1ß and IL-18 release. Moreover, the levels of IL-1ß and IL-18 production decreased in CA-074 (a cathepsin B inhibitor) and NAC (an anti-oxidant) pretreated human macrophages, compared to untreated controls. This study suggests that L. interrogans infection leads to reactive oxygen species (ROS)- and cathepsin B-dependent NLRP3 inflammasome activation, which subsequently mediates caspase-1 activation and IL-1ß and IL-18 release.

Feasibility study of enforcing immunization certificate check before primary school or kindergarten enrollment in Guizhou Province, China.

OBJECTIVE: To study the feasibility of enforcing immunization certificate check before children enroll in primary schools or kindergartens in Guizhou Province. METHODS: Quantitative and qualitative studies were conducted. The multi-stage and cluster sampling approach was adopted for the quantitative part of the study. A questionnaire was designed and 996 children and their keepers were interviewed. Principals, doctors or teachers of the primary schools, directors and child care nurses of kindergarten, and staff of immunization agencies were invited to take part in 12 focus group discussions; meanwhile, face-to-face individual in-depth interviews with 16 officials of the Health, Education and Governmental Departments at various levels were conducted. RESULTS: The total number of subjects was 996. 16.7% of the children in the study completed all the procedures of the National Immunization Programme. 34.3% of them had immunization certificates while the remainder 44.7% registered in immunization agencies. Factors, including the migrant children, doubt about vaccine efficiency, mother's occupation and educational background, knowledge of the National Immunization Programme on targeted vaccines, played an important role in obtaining or not immunization certificates. 95% of the keepers interviewed thought the immunization certificates were useful; 94.8% of them considered the check was critical while only 3.6% of them thought it unnecessary. The first reason from those who found it unnecessary was that they feared that repeated immunization might affect their children's health. The second reason was the cost of immunization, which some of them could not afford to pay. However, the Health Department expressed a favorable attitude to the checking scheme. Though the Education Department agreed that the scheme was essential, they worried that it would affect the enrollment rate. CONCLUSION: In spite of the difficulty in administering immunization certificate check, the effort would be rewarding for raising the immunization coverage rate among the children in Guizhou Province.

The role of reactive oxygen intermediates in the intracellular fate of Leptospira interrogans in the macrophages of different hosts.

BACKGROUND: Pathogenic species of Leptospira cause leptospirosis, a global zoonotic disease. Our previous work showed that leptospires survive and replicate in human macrophages but are killed in murine macrophages. However, the mechanism responsible for the different intracellular fates of leptospires within the macrophages of different hosts remains unclear. RESULTS: The present study demonstrates that infection with Leptospira interrogans caused significant up-regulation of reactive oxygen species (ROS) and superoxide in J774A.1 cells but did so to a lesser extent in THP-1 cells. The up-regulation of ROS and superoxide was significantly inhibited by the NADPH oxidase inhibitor apocynin. The damaged leptospires and remnants of leptospires within membrane-bound vacuoles were significantly inhibited by apocynin in J774A.1 cells but were less inhibited in THP-1 cells. In addition, apocynin significantly prevented damage to leptospires and the co-localization of L. interrogans with lysosomes in J774A.1 cells but did so to a lesser extent in THP-1 cells. Furthermore, the relative fluorescence intensity levels of intracellular leptospires and the viability of the intracellular leptospires increased in apocynin pretreated J774A.1 and THP-1 cells after 2 h of infection. CONCLUSIONS: The present study, based on our previous findings, further demonstrated that ROS contributed substantially to the bactericidal ability of mouse macrophages to kill intracellular leptospires. However, ROS did not contribute as much in human macrophages, which partially explains the different intracellular fates of L. interrogans in human and mouse macrophages.

Simultaneous virus identification and characterization of severe unexplained pneumonia cases using a metagenomics sequencing technique.

Many viruses can cause respiratory diseases in humans. Although great advances have been achieved in methods of diagnosis, it remains challenging to identify pathogens in unexplained pneumonia (UP) cases. In this study, we applied next-generation sequencing (NGS) technology and a metagenomic approach to detect and characterize respiratory viruses in UP cases from Guizhou Province, China. A total of 33 oropharyngeal swabs were obtained from hospitalized UP patients and subjected to NGS. An unbiased metagenomic analysis pipeline identified 13 virus species in 16 samples. Human rhinovirus C was the virus most frequently detected and was identified in seven samples. Human measles virus, adenovirus B 55 and coxsackievirus A10 were also identified. Metagenomic sequencing also provided virus genomic sequences, which enabled genotype characterization and phylogenetic analysis. For cases of multiple infection, metagenomic sequencing afforded information regarding the quantity of each virus in the sample, which could be used to evaluate each viruses' role in the disease. Our study highlights the potential of metagenomic sequencing for pathogen identification in UP cases.

Extensive diversity of coronaviruses in bats from China.

To help reveal the diversity and evolution of bat coronaviruses we collected 1067 bats from 21 species in China. A total of 73 coronaviruses (32 alphacoronaviruses and 41 betacoronaviruses) were identified in these bats, with an overall prevalence of 6.84%. All newly-identified betacoronaviruses were SARS-related Rhinolophus bat coronaviruses (SARSr-Rh-BatCoV). Importantly, with the exception of the S gene, the genome sequences of the SARSr-Rh-BatCoVs sampled in Guizhou province were closely related to SARS-related human coronavirus. Additionally, the newly-identified alphacoronaviruses exhibited high genetic diversity and some may represent novel species. Our phylogenetic analyses also provided insights into the transmission of these viruses among bat species, revealing a general clustering by geographic location rather than by bat species. Inter-species transmission among bats from the same genus was also commonplace in both the alphacoronaviruses and betacoronaviruses. Overall, these data suggest that high contact rates among specific bat species enable the acquisition and spread of coronaviruses.