Abstract: Objective To conduct a molecular epidemiological tracing and analysis of Yersinia pestis strains isolated from two human plague outbreaks with unknown sources in Gansu Province, China. The results of this analysis would provide a basis for isolating and controlling the sources of Yersinia pestis. Methods The strains of Yersinia pestis isolated from two human plague outbreaks occurring on December 12, 2017, and September 27, 2019 were genotyped by the different region (DFR) and the clustered regularly interspaced short palindromic repeats (CRISPR). The repeat numbers of the variable number tandem repeat (VNTR) loci in the tested strains of Yersinia pestis were calculated by the multiple variable number tandem repeats analysis (MLVA), and the location of the phylogenetic tree of the tested strains was determined with the method of minimum spanning tree (MST) by the software BioNumerics 6.6. Results The strain of 20171212 lacked DFR01, DFR02, DFR03, DFR04, DFR13, DFR23, and the DFR type was identified as type 8. The space sequence of YPa was a1'-a2-a3-a4-a5-a6-a7-a35, the space sequence of YPb was b1-b2-b3-b4, the space sequence of YPc was c1-c2-c3, the gene cluster of CRISPR was Ca35', the genotype of CRISPR was 26'. MLVA clustering analysis showed that the strain clustered within in the cluster of Yuerhong pasture in Subei County and formed an independent branch. On the other hand, the strain of 20190927 lacked DFR01, DFR13 and DFR23, with the DFR type identified as type 1b. The space sequence of YPa was a1-a2-a3-a4-a5-a6-a7, the space sequence of YPb was b1-b2-b3-b4, the space sequence of YPc was c1-c2-c3, the gene cluster of CRSIPR was Ca7, the genotype of CRSIPR was 22 MLVA clustering analysis showed that the strain was located close to the cluster of Dangjinshan in Akesai County, and relatively distant from the cluster of Yuerhong pasture in Subei County. Conclusions The genotypes of strain 20171212 by DFR and CRISPR were consistent with the main genotypes of Y. pestis from Himalayana Marmota foci in Subei County, which confirmed that the human plague cases were naturally occurring locally. However, the strain gathered the cluster of Yuerhong pasture in Subei County, which indicated that the source of infection was not in Yanchiwan Town, but in the surrounding area of the Yuerhong pasture. The genotypes of strain 20190927 by DFR and CRISPR were in accordance with the main genotype of Y. pestis from Himalayana Marmota foci in Akesai County and were closer to the cluster of Dangjinshan in Aksai County than to

Objective:To study the genotyping and regional distribution characteristics of Yersinia pestis by single nucleotide polymorphism (SNP) in Gansu Province. Methods:A total of 52 strains of Yersinia pestis isolated from Himalaya Marmot plague foci and Spermophilus alaschanicus plague foci in Gansu Province from 1962 to 2017 were selected for culture and extraction of DNA. The genomic DNA of Yersinia pestis was sequenced by the second generation of Illumina PE150 to identify the SNP sites. The species characteristics of Yersinia pestis in Gansu Province was determined by the Kimura-2-parameter model of neighbor joining of Mega 10.0 software based on the SNP sites. The molecular evolutionary tree of the groups was determined by Hasegawa-Kishino-Yano model of maximum likelihood method according to the SNP sites. Results:A total of 103 SNP sites were identified in 52 strains of Yersinia pestis in Gansu Province, including 28 intergenic loci, 43 non-synonymous mutations, 31 synonymous mutations and 1 nonsense mutation. The 52 strains of Yersinia pestis were divided into 2 biotypes and 3 groups, which were ancient type (1.IN2, 3.ANT) and medieval type (2.MED). Among them, 35 strains belonged to 1.IN2 group, 13 strains belonged to 3.ANT group, and 4 strains belonged to 2.MED group. The 1.IN2 group was further divided into 5 subgroups: the groups of Yuerhong Town and Dangchengwan Town in Subei County, the groups of Mati Town and Dahe Town in Sunan County, and the group of Xiahe County. The 3.ANT group was further divided into 2 subgroups: the groups of Hongliuwan Town in Aksay County and Machang in Dangchengwan Town of Subei County. Conclusion:The SNP method can be used to genotype Yersinia pestis from different plague foci in Gansu Province, which has certain regional characteristics.

Objective:To summarize the clinical characteristics, diagnosis and treatment of caspase recruitment domain-containing protein 9 (CARD9) gene deficiency associated invasive candidiasis, and report a novel mutation in CARD9 gene.Methods:The clinical characteristics, laboratory tests, treatment and the outcome of follow-up in a boy with invasive candidiasis were described. The boy′s main clinical manifestations were central nervous system infection and retroperitoneal mass. Whole-exome sequencing was performed and Sanger sequencing was verified to identify the CARD9 gene mutations in the patient and his parents. A literature search for “CARD9”and “invasive candidiasis”was conducted in PubMed, Wanfang and CNKI databases from their establishment to May 2020.Results:A 10-year-old boy suffered onset symptom of chronic diarrhea, which lasted for two months. The symptom was followed by progressive neurological symptoms such as headache, vomiting, seizures and disorder of consciousness. His unusual medical history was absent. Candida albicans were cultured several times in cerebrospinal fluid and blood, and yeast-like fungi were found in the stool high power field of vision. Cerebral magnetic resonance imaging indicated obstructive hydrocephalus and abdominal CT scan showed retroperitoneal mass and thickening of the intestinal wall. The whole-exome sequencing analyses of blood samples from the boy and his parents were performed. The results showed that there was a homozygous mutation of c.952-12_956delinsAG in the CARD9 gene, which was an unreported pathogenic mutation. This was confirmed by Sanger sequencing. There was no significant relief from intravenous combined antifungal medications. After lateral ventricular drainage surgery and injection of amphotericin B into the lateral ventricle, improvement of clinical symptoms and cerebral spinal fluid abnormalities was observed after nine weeks, and the retroperitoneal mass shrank. At follow-up after four-month oral combined antifungal medications, the child had no complaint except fatigue. However, cerebral spinal fluid analysis showed increased protein level and decreased glucose. Persistent hydrocephalus and periventricular white matter abnormal signals were revealed on the brain magnetic resonance imaging and the smaller retroperitoneal mass than before on the abdominal CT scan. In addition to this case, totally 21 cases with CARD9 gene deficiency associated invasive candidiasis have been reported worldwide, most of which featured central nervous system infections.Conclusions:CARD9 gene deficiency is an autosomal recessive primary immunodeficiency that confers human susceptibility to fungal disease. The associated invasive candidiasis often affects the central nervous system and makes the patient severely ill. Adequate systemic antifungal therapies should be given, and patients with hydrocephalus need surgical treatment. A novel mutation is reported that expands the variant diversity of CARD9 gene. For patients with unexplained invasive candidiasis, including those without a history of previous recurrent infection, genetic testing is recommended for primary immunodeficiency including CARD9 gene deficiency.

Objective:To understand the plague epidemic characteristics in the natural foci of the Qilian Mountains-A-erh-chin Mountains Himalayan marmot plague in Gansu Province, and to provide scientific basis for innovative prevention and control of the plague in combination with local conditions. Methods:A retrospective study was used to collect the monitoring data of the natural foci of plague in Gansu Province from 2011 to 2018 (from the epidemic surveillance files of the Center for Disease Control and Prevention of Gansu Province and direct network reporting information). Descriptive epidemiological method was used to analyze the plague epidemic characteristics of natural foci of the Qilian Mountains-A-erh-chin Mountains Himalayan marmot plague in Gansu Province from 2011 to 2018, including the distribution of host animals, pathogenic and serological testing of the plague bacteria, and the epidemic characteristics of human plague. Results:From 2011 to 2018, the total average marmot density in the natural foci of the Qilian Mountains-A-erh-chin Mountains Himalayan marmot plague in Gansu Province was 0.21/hm 2, of which Tianzhu County had the highest average marmot density of 0.58/hm 2, and Jiayuguan City had the lowest average marmot density of 0.01/hm 2. A total of 381 strains of Yersinia pestis were isolated in the foci, of which 4 were isolated from human corpses, 298 were host animals, and 79 were infectious vectors. Among them, the top 3 counties (cities) of isolated strains were Aksai County (38.85%, 148 strains), Subei County (31.50%, 120 strains) and Yumen City (16.27%, 62 strains). A total of 6 860 marmot serum, 1 769 dog serum were tested, the F1 antibody positive rates were 2.70% (185/6 860), 8.42% (149/1 769); and the F1 antigen positive rate of 814 animal materials was 4.30% (35/814), respectively. There were 4 times of human plague, 4 cases occurred and 4 cases died; 3 times occurred in Subei County and 1 time in Yumen City. The onset months were July, September, November and December. Active contact with infected animals such as shepherd dogs was the main route of infection, and migrant herders were the key occupation population. Conclusions:The animal epidemic situation in the natural foci of the Qilian Mountains-A-erh-chin Mountains Himalayan marmot plague in Gansu Province is active, and the plague presents different epidemic states in different regions. The prevention and control measures should be taken according to local conditions and guided by classification to strictly prevent the occurrence and transmission of the plague.

Objective:To investigate the effect of intercellular adhesion molecule-1 (ICAM1) targeted miR-133a-5p on lipopolysaccharide (LPS)-induced alveolar epithelial A549 cell injury.Methods:Dual lucife-rase reporter assay was used to verify the ICAM1 targeted effect of miR-133a-5p.A549 cells were induced by LPS in vitro and divided into the control group, LPS group, LPS+ negative control(miR-NC) group, LPS+ miR-133a-5p group, LPS+ small interfering RNA(si)-NC group, and LPS+ si-ICAM1 group.The expression levels of miR-133a-5p and ICAM1 mRNA were detected by real-time fluorescent quantitative PCR.Apoptosis was detected by flow cytometry.The expression levels of ICAM1, Bcl-2, Bax and cleaved caspase-3 protein that could activate cysteine were detected by Western blot.The expression levels of interleukin-6 (IL-6) and tumor necrosis factor α(TNF-α) were detected by enzyme linked immunosorbent assay kit. Results:Compared with the control group, the LPS group had a decreased expression level of miR-133a-5p (0.39±0.04 vs.1.00±0.09) in A549 cells, increased expression of ICAM1 (0.86±0.08 vs.0.39±0.03), an increased apoptotic rate [(27.65±2.47)% vs.(8.13±0.89)%], and increased secretion of IL-6 [(624.59 ± 51.42) ng/L vs.(194.25±18.43) ng/L] and TNF-α [(548.35±51.42) ng/L vs.(174.26±19.43) ng/L]. The differences were significant (all P<0.05). Compared with the LPS+ miR-NC group, the apoptosis rate of A549 cells [(13.46±1.38)% vs.(28.71±2.54)%] in LPS+ miR-133a-5p group were significantly decreased, and the secretion of IL-6 [(296.43±23.51) ng/L vs.(635.86±55.41) ng/L] and TNF-α [(321.14±30.56) ng/L vs.(563.24±49.52) ng/L] was significantly decreased (all P<0.05). Compared with LPS+ si-NC group, the apoptosis rate of A549 cells [(13.65±1.64)% vs.(23.51±2.33)%] in LPS+ si-ICAM1 group was significantly decreased, and the secretion of IL-6 [(324.15±29.41) ng/L vs.(625.39±52.59) ng/L] and TNF-α [(334.65±20.46) ng/L vs.(534.97±51.42) ng/L] were significantly decreased (all P<0.05). Conclusions:miR-133a-5p can alleviate LPS-induced alveolar epithelial cell injury, and the mechanism may be related to down-regulation of ICAM1 expression.

Objective:To analyse the effects of meteorological factors on plague epidemic in Himalayana marmot plague natural foci in Subei and Sunan counties of Gansu Province. Methods:The surveillance data of plague from 1973 to 2016 in Himalayan marmot foci of Subei and Sunan counties (from Gansu Provincial Center for Disease Control and Prevention) and the meteorological factors (rainfall, temperature, relative humidity) of current, preceding first, second and third years (the first, second and third years before the reference year, from China Meteorological Data Sharing Service System) were collected. In order to build generalized additive model (GAM), the positive rate of plague was used as dependent variable while meteorological factors of the current, preceding first, second and third years were used as independent variables. The influence of meteorological factors on plague was quantitatively analyzed by using R2.3.2 software. Results:In 2006 - 2016, compared with 1974 - 1983, the average temperature in Subei rose by 0.99 ℃, the average relative humidity rose by 3.55%RH, and the average rainfall rose by 12.16 mm; in Sunan County, the average temperature rose by 1.00 ℃, the average relative humidity rose by 2.01%RH, and the average rainfall rose by 14.60 mm. From 1973 to 2016, 11 cases of human plague with 7 cases of dead (12 cases of attack) occurred in the investigated district. The average rainfall of the preceding third year of Subei rose every 1.00 mm within a certain range, the animal plague occurred rose by 0.40%. The average relative humidity of preceding second year of Subei rose every 1.00%RH within a certain range, animal plague occurred rose by 11.66%. The average rainfall of the preceding first year of Sunan rose every 1.00 mm within a certain range, the animal plague occurred rose by 1.32%. The average relative humidity of preceding third year of Sunan rose every 1.00%RH within a certain range, animal plague occurred rose by 11.96%. The average temperature of the preceding second year of Subei, Sunan rose every 1.00 ℃ within a certain range, animal plague rose 73.17%, 70.18%, respectively.Conclusions:The occurrence and prevalence of plague in Himalayana marmot are closely related to meteorological factors, and the temperature within a certain range (but not continuously) not only promotes the epidemic of plague, but also has a lag effect. In addition, the effects of different types of climate in the same foci on the prevalence of animal plague vary.

Objective:To study the clustered regularly interspaced short palindromic repeats (CRISPR) loci polymorphism of Yersinia pestis and its area distribution in Gansu province. Methods:A total of 203 strains of Yersinia pestis isolated from 1962 to 2014 were selected for the culture and extraction of DNA. Three pairs of CRISPR primers were used to amplify the strain DNA by PCR, and the PCR products were sequenced. The groups and genotypes of strains were determined according to the spacer and spacer arrangement of CRISPR loci in the strain. Cluster analysis was done by using the software BioNumerics 5.10. Results:A total of 16 spacers, including 9 species of YPa loci, 4 species of YPb loci and 3 species of YPc loci, were found in the 203 strains of Yersinia pestis. A new spacer of a1′ was found. The 203 strains were divided into 5 CRISPR genotypes and classified into 5 CRISPR clusters (Cb2, Ca7, Ca7′, CaΔ5′ and Ca35′). Each cluster showed significant area-specific characteristics, Cb2 was mainly distributed in Huining country and Pingchuan district, Ca7 was mainly found in Aksai Kazak autonomous country, Ca7′ was mainly found in Xiahe country, Ca35′ was mainly found in Subei Mongolia autonomous county and Yumen city and CaΔ5′ was mainly distributed in Sunan Yugur autonomous county. Conclusions:The strains from different plague foci in Gansu were distinguished by CRISPR, all kinds of clusters showed the obvious area specific characteristics. It is important to study the evolution of Yersinia pestis in Gansu and trace the molecular biology origin of human plague.

Objective To analyze the relationship between meteorological variables and the prevalence of marmot plague in the Himalayan marmot foci of Subei County,Gansu Province.Methods The surveillance data (from Gansu Provincial Center for Disease Control and Prevention) on human plague,the meteorological factors (average rainfall,temperature,relative humidity) data (from China Meteorological Data Sharing Service Network) of the current and preceding (relative to current year as a reference) first,second and third years and meteorological factors data of the current and preceding first,second and third months in Subei of Himalayan marmot foci from 1973 to 2014 were collected.The association of positive rate of plague bacteriological detection and meteorological factors was evaluated by using SAS 9.3 software.The influence of meteorological factors on the epidemic of marmot plague was analyzed by the logistic regression analysis.Results From 1973 to 2014,6 human plague cases with 5 dead cases occurred in Subei County.The positive rate of plague bacteriological detection was positively correlated with the average relative humidity of current year and the average temperature of preceding first year (r =0.366,0.385,P ＜ 0.05),and it was negatively correlated with the average relative humidity of preceding third year (r =-0.304,P ＜ 0.05);the positive rate of plague bacteriological detection was positively correlated with the average rainfall of preceding first month (r =0.212,P ＜ 0.05),and it was negatively correlated with the average relative humidity of preceding second and third months (r =-0.196,-0.201,P ＜ 0.05).The risk factor of animal plague epidemic in marmot foci in Subei County was average temperature of current month [odds ratio (OR) =1.304,95％ confidence intervals (CI):1.195-1.422],and the protective factor was average relative humidity of preceding third month (OR =0.960,95％CI:0.932-0.988).Conclusions The prevalence of marmot plague is closely associated with meteorological factors in the natural plague foci of Subei County of Gansu Province.The temperature not only plays a role in promoting the prevalence of plague,but also has a lagging effect.Therefore,the meteorological factors can be considered as a reference of the plague prevalence in Himalayan marmot plague foci.

Objective To explore the effects of meteorological factors on population densities of the host and the prevalence of animal plague in the natural plague foci of Spermophilus alaschanicus.Methods The surveillance data of seroprevalence rate in natural plague foci of Spermophilus alaschanicus of Gansu Province in current year,one previous year (relative to current year as a reference),as well as two and three previous years’ meteorological data such as average annual rainfall,temperature,relative humidity were collected from 1962 to 2014.SAS 9.3 software was used for correlation and regression analysis of the influencing meteorological factors on the density of Spermophilus alaschanicus and prevalence of animal plague;generalized additive model (GAM) was used for quantitative study of meteorological factors on density of Spermophilus alaschanicus;logistic regression analysis was used to study the effect of meteorological factors on prevalence of Spermophilus alaschanicus plague.Results Serum positive rate of Spermophilus alaschanicus was positively correlated with average annual rainfall of current and previous one year,and with relative humidity of current year (r =0.279,0.341,0.654,P ＜ 0.05);serum positive rate was negatively correlated with average annual temperature of current and preceding first,second and third years (r =-0.360,-0.339,-0.273,-0.323,P ＜ 0.05).The average annual rainfall of preceding second and third years was significantly associated with the densities of Spermophilus alaschanicus by GAM parameter estimation (t =2.88,2.42,P ＜ 0.05).The average annual rainfall of preceding second and third years was significantly associated with the densities of Spermophilus alaschanicus by GAM nonparameter estimation (x2=31.580,13.428,P ＜ 0.05),but the effect was nonlinear.Multivariate logistic regression analysis showed that average annual rainfall of preceding first year [odds ratio (OR) =2.475,95％ confidence interval (C/):1.175-5.213] was risk factor for the prevalence of the animal plague in the natural plague foci of Spermophilus alaschanicus.Conclusions Rainfall is risk factor for the prevalence of the animal plague in the natural plague foci of Spermophilus alaschanicus.There is a lag effect of rainfall on the numbers of hosts and the prevalence of Spermophilus alaschanicue plague.When the rainfall in the region (not continuously) reaches a certain amount,it will provide favorable conditions for the spread of Yersinia pestis.

Objective To investigate the relationship between fleas density of Spermophilus alaschanicue and host density as well as meteorological factors.Methods The densities of fleas of Spermophilus alaschanicue and meteorological temperature,precipitation,relative humidity data in the same period from 1977 to 2014 in plague natural foci of Spermophilus alaschanicue of Gansu Province were collected.In order to further establish the multiple linear regression and the stepwise regression analysis model,SAS 9.3 software was used to explore the linear correlation analysis between fleas index number,density of Spermophilus alaschanicue and meteorological factors.Results The average body flea index positively correlated with the density of Spermophilus alaschanicue (r =0.34,P ＜ 0.05),in addition,hole fleas index was in negative correlation with the average temperature of the current and preceding first,second and third years (r =-0.44,-0.39,-0.45,-0.54,P ＜ 0.01).However,hole fleas index positively correlated with the average rainfall of preceding first year (r =0.38,P ＜ 0.05).More specifically,the monthly body flea index was negatively correlated with the average rainfall and the average temperature of current,preceding first,second,and third months,and the average relative humidity of current month (r =-0.23,-0.34,-0.43,-0.29,-0.25,-0.33,-0.36,-0.38,-0.26,P ＜ 0.01).After multiple linear regression analysis,the following equations were obtained:body fleas index =3.983 1 + 0.695 1 × Sperrnophilus density-0.004 9 × average annual rainfall;hole fleas index =10.780 4 + 0.009 1 × average annual rainfall-0.216 2 × annual average relative humidity.Conclusions The relationship between fleas density of Spermophilus alaschanicue and the environment is complex,they have mutual connection as well as mutual restriction.Except Spermophilus density affects the number of fleas,the two most important meteorological factors which affect the flea body index are temperature and rainfall.In addition,the relationship between meteorological factors and flea body index shows somewhat lagging behind for two to three months.