Objective:To analyze the genus, drug resistance/virulence and phylogenetic characteristics of Brucella strains isolated from brucellosis surveillance sentinels in Henan Province from 2013 to 2022, and provide baseline data for the surveillance, early warning and outbreak tracing of brucellosis. Methods:Blood samples were collected from patients with Brucella infection for strain isolation, culture and species identification, drug susceptibility test, whole genome sequencing, splicing and assembly, functional/virulence/resistance gene prediction analysis and phylogenetic tree drawing based on single nucleotide polymorphism (SNP). Results:In 36 brucellosis patients, the majority were men (86.11%, 31/36), young adults aged 18-50 (88.89%, 32/36) and farmers/herdsmen (72.22%, 26/36). A total of 36 strains of Brucella melitensis were isolated, and average 1 305 functional proteins of 21 categories were predicted by strain genome; all the strains carried four main virulence factors (pmm, VirB group, BtpA/BtpB, BvrS/BvrR). The drug sensitivity rate was 100.00% to six types of antibiotics including levofloxacin, rifampicin, doxycycline, streptomycin, tetracycline and gentamicin, they showed different resistances to three antibiotics including compound trimethoprim-sulfamethoxazole, ciprofloxacin and ampicillin. The strains carried four types of resistance genes and two clusters of resistance genes, with four combinations of genotypes, the resistance mechanisms included antibiotic degradation/modification enzymes, resistant nodular cell differentiation (RND) efflux pumps, 16S/23S ribosomal rRNA binding site mutations, etc. The number of SNP differed in the genomes of 36 Brucellamelitensis strains ranged from 0 to 454 and phylogenetic tree was divided into three major branches, with relative branch distances between 0.000 0 and 0.498 6 for each strain. Conclusions:Human Brucellamelitensis strains isolated from surveillance sentinels in Henan from 2013 to 2022 carried multiple virulence and antibiotic resistance genes and had different drug resistance phenotypes. Single nucleotide polymorphism analysis and phylogenetic tree analysis showed significant differences in phylogenetic relationships among different strains.

Objective:To analyze the genetic evolution and molecular characteristics of H7N9 avian influenza virus (AIV) isolated in a live poultry market.Methods:Samples such as poultry feces, sewage, and hair removal machine and chopping board swabs were collected. Real-time fluorescent quantitative PCR was used to detect influenza A virus and H7N9 AIV in the samples. The whole genome of H7N9 AIV was amplified with influenza A virus universal primers and sequenced. BLAST and MEGA X were used for sequence alignment, phylogenetic analysis and molecular characterization.Results:Seven poultry-related environment samples were collected in the live poultry market in Xuchang city in February 2023, and four were positive for H7N9 AIV. The whole genome sequences of three H7N9 AIV isolates were successfully obtained, and the isolates shared high nucleotide identity in different genes (98.37%-100.00%). BLAST analysis showed they were highly identical to H7N9 strains isolated from domestic poultry in China from 2020 to 2021. Genetic evolution analysis showed that the three isolates clustered in the same branch and were closer to the recent environmental isolates than to the recent strains isolated from human or avian. Through comparison with the sequences of the representative strains in different periods, it was found that the isolated strains in this study showed high avian pathogenicity with four amino acids KRAA inserted at the cleavage site; the hemagglutinin receptor-binding site was QSG, which was an avian binding receptor; there was a G186I mutation in hemagglutinin. Mammalian-adaptive mutation E627K was not detected in polymerase basic protein 2. Mutations (R292K and I38T) associated with drug resistance to neuraminidase inhibitor (oseltamivir) and polymerase acidic protein inhibitor (baloshavir) were not detected, suggesting that these isolates remained susceptible to these drugs. A S31N mutation was found in M2 protein, indicating they were resistant to alkamines.Conclusions:The three H7N9 AIV strains isolated in the live poultry market have high avian pathogenicity, but there are no significant increase in mutations related to the binding ability to human receptors, mammalian pathogenicity, viral transmissibility, or drug resistance as compared with previous representative strains causing human or avian infection.

Objective:To analyze the epidemiological characteristics and spatiotemporal characteristics of human brucellosis in Henan Province.Methods:Data of human brucellosis in Henan Province from 2005 to 2021 were collected through the China Disease Prevention and Control Information System, and a descriptive epidemiological method was used to analyze the epidemic profile of brucellosis in Henan Province and the three distribution characteristics. Global and local spatial autocorrelation were used to analyze the spatial distribution and the hot spots of brucellosis in Henan Province, respectively, and spatiotemporal scanning was used to analyze the spatiotemporal clustering regions of brucellosis in Henan Province.Results:A total of 39 862 brucellosis cases were reported in Henan Province from 2005 to 2021, with an average annual incidence of 2.44/100 000, and the number of cases showed an overall increasing trend each year (χ 2trend = 11 127.85, P = 0.001). The onset months were mainly concentrated from March to July, accounting for 59.00% (23 517/39 862), with May as the peak (5 478 cases). Cases of brucellosis were reported in 157 counties (cities, districts) of the province. The ratio of male to female was 2.52∶1.00 (28 542/11 320). Farmers were the main occupation, with 32 985 cases (82.75%). The age of onset was mainly 45 to 65 years old, with 20 226 cases (50.74%). The global spatial autocorrelation analysis showed that the global Moran's I was > 0, Z > 1.96, and P < 0.05 in all years except 2006 - 2008, showing spatial clustering. Further local spatial autocorrelation analysis was performed, and high-high and low-low clustering areas existed in 2012 - 2021 ( P < 0.01). Spatiotemporal scanning analysis showed that there was one spatiotemporal cluster in the high incidence area and two spatiotemporal clusters in the low incidence area. The high incidence cluster was centered in Neixiang County, covering 48 counties (cities, districts) including Song County and Ruzhou City, and the aggregation time was from 2014 to 2021. The two low incidence clusters were centered in Yongcheng City and Boai County, covering 58 and 18 counties (cities, districts), respectively, and the aggregation time was 2016 - 2021 and 2005 - 2012, respectively. Conclusion:The overall incidence of brucellosis in Henan Province is on the rise from 2005 to 2021, with middle-aged and elderly male farmers as the main affected population, and there are spatiotemporal clusters of brucellosis in Henan Province.

Objective:To analyze the whole genome of Omicron variants causing the first local Omicron outbreak in Henan Province and to investigate the mutations in the SARS-CoV-2 genome for source tracing.Methods:Respiratory tract samples from COVID-19 cases in the Omicron outbreak in Henan Province from January 7 to 29, 2022 were subjected to whole-genome sequencing and sequence alignment analysis. Whole-genome identity, variations and evolution of the Omicron variants were analyzed.Results:Through high-throughput sequencing, the whole-genome sequences of SARS-CoV-2 were obtained from 120 cases, which accounted for 25.64% (120/468) of all COVID-19 cases in Anyang during the same period. Compared with the genome of Wuhan reference strain (NC_045512.2), there were 57-59 nucleotide mutation sites in the 120 whole genome sequences, and one or two nucleotide mutation sites were added to the shared 57 nucleotide sites. All of the 120 strains were VOC/Omicron (BA.1.1) variants and shared high homology. The whole-genome sequence obtained from the first case A contained 57 nucleotide mutation sites, while apart from the 57 identical nucleotide mutation sites, one specific mutation site (C1594T) was found in the whole-genome sequence obtained from the first case B, suggesting that the two cases were in the same transmission chain. After comparing with the database of domestic and imported cases by the Chinese Center for Disease Control and Prevention and the Henan Provincial Center for Disease Control and Prevention, it was found that the current outbreak was linked with the same transmission chain as the existing local epidemics in other provinces. Moreover, epidemiological investigation showed that on January 2, case A had come into contact with her cousin and his family who returned from an affected area outside the province.Conclusions:Based on the gene sequencing results and epidemiological investigation, the COVID-19 outbreak in Anyang city, Henan Province was a local epidemic and the source of it was a college student who returned to Anyang city from other province on December 28, 2021. These infections were linked to the same transmission chain as the existing local infection in other provinces.

Objective:To analyze the genome characteristics and variations in nucleotides and amino acids of SARS-CoV-2 causing an outbreak in Henan Province in November 2021 and perform the traceability analysis.Methods:In this study, throat swab specimens from cases in the acute phase were collected and tested for the nucleic acids of SARS-CoV-2 by real-time fluorescent RT-PCR. SARS-CoV-2 nucleic acid-positive samples were subjected to high-throughput genome sequencing and whole-genome alignment analysis.Results:The median Ct values of ORF1ab gene and N gene in 70 positive specimens was 26.41 (15.58 to 39.27) and 24.43 (12.04 to 39.74), respectively. Compared with the sequence of Wuhan-Hu(NC_045512) reference strain, 47 to 49 nucleotide mutations sharing 47 nucleotide mutation and 41 amino acid mutations were found in 63 strains of successfully sequenced SARS-CoV-2. Nine nucleotide mutations and 12 amino acid mutations were found in the spike protein. The index case shared 47 mutations with the Russian imported cases in Henan Province on October 14 and the local cases in Jiangxi Province in October. Moreover, their genomes were highly homologous and they all belonged to the Delta variant (AY.122 evolutionary branch).Conclusions:Continuous monitoring of imported COVID-19 cases and prolonging the period of quarantine were needed to reduce the risk of local outbreak and epidemic caused by imported COVID-19 cases. Analysis of the genomic characteristics of SARS-CoV-2 and the variations in nucleotides and amino acids was conducive to trace the origin of COVID-19 outbreak quickly and provide reference for precise control.

Objective:To investigate the performance of real-time RT-PCR for the detection of SARS-CoV-2 nucleic acid in clinical diagnosis of COVID-19.Methods:Laboratory test data and basic case information of Henan COVID-19 cases were collected from the China′s Infectious Disease Information System as of March 5, 2020. All information was entered by local hospitals and Center for Disease Control and Prevention (CDC). Local hospitals or country CDC were responsible for sampling and municipal CDC was responsible for nucleic acid testing.Results:A total of 6 714 specimens were detected and the positive rate of SARS-CoV-2 nucleic acid was 23.82%. The specimens were collected from 1 200 confirmed cases, 2 178 suspected cases and 77 asymptomatic cases. The nucleic acid diagnosis rate of COVID-19 was 36.96% (1 277/3 455). In all cases, the positive rates of SARS-CoV-2 nucleic acid in nasal swabs, sputum samples and throat swabs were 19.38%, 28.59% and 23.53%, respectively (χ 2=15.896, P<0.01). The positive rate of SARS-CoV-2 nucleic acid in confirmed COVID-19 cases was 63.10%. The positive rates in nasal swabs, sputum samples and throat swabs were 50.80%, 58.71% and 65.21 (χ 2=18.612, P<0.01). The positive rates of SARS-CoV-2 nucleic acid were 43.51%, 23.98%, 22.82%, 12.17%, 14.46% and 13.21% in samples collected on the day of symptom onset and one week, two weeks, three weeks, four weeks, five weeks and above five weeks after the onset, respectively. The positive rates in confirmed cases were respectively 89.03%, 86.57%, 52.30%, 17.53%, 17.69% and 24.14% at those time points. Conclusions:Real-time RT-PCR is the most effective method for early pathogenic diagnosis of COVID-19. The highest detection rate of nucleic acid is achieved within one week after the onset of COVID-19, and the latest time for nucleic acid detection is 38 d after the onset.

Objective:To investigate the etiological characteristics and drug resistance of non-typhoid Salmonella isolated from stool samples of children under 5 years old with diarrhea in Henan Province. Methods:Intestinal bacteria were isolated from fecal samples of 4 250 diarrhea children under five years old in five monitoring sites in Henan Province from 2015 to 2018. Serotypes and drug sensitivity of Salmonella strains were analyzed. The annual change in drug resistance was analyzed by Chi-square test and all data were analyzed retrospectively. Results:The detection rate of non-typhoid Salmonella in fecal samples was 8.73% (371/4 250). The highest detection rate was in the 0-1 age group (51.75%) and the peak season for Salmonella infection was from May to October. The most common serotype was Salmonella enteritidis (36.93%), followed by 4, 5, 12: i: - Salmonella (14.82%) and Salmonella typhimurium (14.02%). The non-typhoid Salmonella isolates were resistant to ampicillin and sulfamethoxazole with drug resistance rates of more than 80%, but more sensitive to ceftazidime, cefepime and cefoxitin. There were significant differences in drug resistance to cefepime, levofloxacin, ciprofloxacin, amikacin, doxycycline, chloramphenicol and compound neoforman among the strains isolated in different years ( P<0.05). Multidrug-resistant strains accounted for 86.52%. Conclusions:There was diversity in the serotypes of non-typhoid Salmonella in diarrheal children under five years old in Henan Province. The predominant serotype was Salmonella enteritidis. Drug resistance to common antibiotics was detected in the isolates, and most of them were multidrug-resistant.

Objective:To explore the serotype distribution and drug resistance of Shigella in stool samples of children under five years old with diarrhea from 2008 to 2017 in Sui County, Henan Province. Methods:A total of 4 721 stool samples of children under five years old with diarrhea were collected from Doufuyuan Clinic of Sui County during 2008 to 2017, and Shigella strains were isolated through bacterial culture. The slide agglutination test was used for serotyping of Shigella strains. Two hundred of seventy-one Shigella strains were selected in proportion, and multiple gradient polymerase chain reaction was used to detect virulence genes and Kirby-Bauer agar method was used for drug sensitivity. Trend chi square test was used to analyze the annual trend of drug resistance. Results:The detection rate of Shigella strains in 4 721 fecal samples was 20.69% (977/4 721). A total of 977 Shigella strains were divided into 13 serotypes in two groups, including 77.79%(760/977) Shigella flexneri strains and 22.21%(217/977) Shigella sonnei strains.The top three serotypes detected alternately every year.The dominant gene pattern of Shigella flexneri was Shigella enterotoxin ( shET)-1+ , shET-2+ , invasion plasmid antigen H ( ipaH)+ , invasion-associated locus ( ial)+ , accounted for 84.04%(179/213) and that of Shigella sonnei was shET-1-, shET-2+ , ipaH+ , ial+ , accounted for 46.55%(27/58). The drug resistance rates of 271 Shigella strains to ampicillin, nalidixic acid and tetracycline were more than 90% and the strains were more sensitive to cefepime and ceftazidime.The drug resistance rates to cefotaxime, cefepime, ciprofloxacin, chloramphenicol and sulfamethoxazole/trimethoprim increased year by year, and all had statistically significant differences ( χ2=24.027, 7.232, 6.039, 4.764 and 6.809, respectively, all P< 0.05). There were 98.52%(267/271) strains resistant to more than three kinds of drugs. The resistance rates of Shigella flexneri to ciprofloxacin, norfloxacin, and chloramphenicol were higher than those of Shigella sonnei, and the resistance rates to gentamicin and sulfamethoxazole/trimethoprim were lower than those of Shigella sonnei. The differences were statistically significant ( χ2=31.866, 14.868, 83.036, 68.534 and 14.738, respectively, all P<0.01). Conclusions:The major serotypes of Shigella in children under five years old in Sui County are constantly changing from 2008 to 2017. The dominant gene patterns of different serotypes are different. Most isolated strains have multiple drug resistances, and different serotypes have different resistance profiles.

To study the epidemiology and etiology characteristics of first imported Chikungunya fever case in Henan province, China, 2017. The patient was confirmed by Chikungunya virus (CHIKV) infected as CHIKV ribonucleotide was continuously detected in his serum specimens. BHK-21 cell line was used for virus isolation, the strain was named CHIKV/Henan001/2017. CHIKV/Henan001/2017 belonged to genotype ECSA. The highest ribonucleotide homology sequence of highly conserved region E1 with CHIKV/Henan001/2017 was hk02 strain (99.8%), who was an imported strain to Hong Kong, China, 2016. Epidemiological information and laboratory testing confirmed it was an imported Chikungunya fever case in Henan province, 2017. No secondary case has been reported.

Objective To investigate the distribution and etiological characteristics of Yersinia en-terocolitica in Henan province between 2011 and 2017 and to analyze the homology among pathogenic strains. Methods A total of 12728 samples, including stool specimens from patients with diarrhea and domestic animals, flies, and smear specimens from raw and cooked meat products, were collected. Cold enrichment method was used to isolate Yersinia enterocolitica. The isolated strains were analyzed by biochemical identifi-cation, biotyping, serotyping and virulence gene detection with PCR. Pulsed-field gel electrophoresis ( PFGE) was performed for molecular typing of pathogenic strains. Results There were 390 strains of Yersinia enterocolitica isolated from the 12728 specimens with a detection rate of 3. 06％, including 13 hu-man strains and 377 animal strains. Most of the strains were isolated from pig and chicken feces and both ac-counted for 25. 13％ (98/390). The predominant biotype was 1A and the serotypes of the strains were main-ly O : 5 and O : 8. Results of the virulence gene analysis showed that 21 strains of O : 3 serotype were path-ogenic, including one human strain and 20 pig strains. After NotⅠdigestion, these pathogenic strains were divided into three band types with a band similarity of 94％-100％. Conclusions Yersinia enterocolitica ex-isted in both human population and many kinds of animals in Henan province. Pig was the main host of path-ogenic strains and there was a high homology among these strains.