Objective To determine the viral carriage profiles of Rousettus leschenaultia in Yunnan, China, characterize a newly identified bocavirus (BoV), and elucidate its phylogenetic relationship, thereby providing new insights into viral source tracing and the prevention and control of associated diseases. Methods In September 2019, bats were captured from an orchard in Shuangjiang, Lincang City, Yunnan Province. After morphological identification, each bat was dissected to collect lung tissue, intestinal tissue, and intestinal contents. Homogenates were prepared from these tissues, and 200 μL of supernatant from each bat was pooled into two composite samples (pulmonary and intestinal). Samples were subjected to ultracentrifugation to concentrate viral particles, followed by nucleic acid extraction using a standardized viral RNA isolation kit. Viral nucleic acids were subjected to random PCR amplification, followed by high-throughput sequencing and de novo assembly of the amplicons. Based on the viral sequences obtained from contig assembly, primers were designed, followed by semi-nested PCR amplification and sequencing validation. Sequence alignments and phylogenetic reconstruction of the VP1 gene sequences were conducted using MEGA-X software with the neighbor-joining method. Results Five Rousettus leschenaultii were collected. A single 3 401-nucleotide (nt) contig was assembled from the intestinal composite sample, exhibiting 63.90% amino acid identity to the closest known bocaviruses by BLASTx analysis. No BoV-related sequences were detected in pulmonary tissues. Based on this sequence, five pairs of primers were designed to perform semi-nested PCR amplification on the intestinal tissues and contents of the five bats, respectively. Among these, positive amplification was observed in the intestinal tissues and contents of one Rousettus leschenaultii (ID: YNSJ03). Subsequent amplification yielded a 2 900-nt partial genome, designated YNSJ03-BtBoV. Phylogenetic analysis based on VP1 amino-acid sequences placed YNSJ03-BtBoV on an independent branch, closely related to Chinese bat bocaparvoviruses reported in 2017 and 2024. It shared 70.1% amino acid identity with BtMf-PV/HuB2013 strain and 71.0% nucleotide identity with YN2016 strain. Conclusion This study reports a novel bocavirus (YNSJ03-BtBoV) in the intestines of Rousettus leschenaultia in Yunnan, significantly expanding the known genetic diversity of bat-associated bocaviruses and providing critical insights into their evolutionary dynamics within zoonotic reservoir hosts.

Objective To investigate newly identify or overlooked arboviruses carried by Culicoides in Yunnan Province, providing a basis for human and animal health protection. Methods In July 2021, Culicoides were collected using UV light traps at a pigsty in Mengla, Yunnan and taken back to the laboratory in a low-temperature environment. Every 200 Culicoides was packed into one centrifuge tube, homogenized and the supernatants were inoculated into C6/36 cells to passage several generations for CPE. Total RNA of cells with CPE was extracted using TaKaRa RNAiso Plus, and electrophoresis was performed on 1% agarose gel. According to the electrophoresis band type, the isolates was identified by qRT-PCR using specific primers, and the type of positive isolates was identified. After determining the type of virus, primers were designed to amplify the viral genome. The obtained sequence was concatenated using Lasergene SeqMan, and the open reading frame was analyzed using NCBI ORF Finder. The gene sequence was downloaded from GenBank and a phylogenetic tree was constructed using MEGA 6.0 to analyze genetic evolutionary relationships. Results One isolate (ML38-1) was found to contain Oya virus (OYAV). The genome obtained sequence contains three fragments: the length is 986 nt for S fragment encoding 233 aa, 4 481 nt for M fragment encoding 1 433 aa, and 6 887 nt for L fragment encoding 2 261 aa. The strain of ML38-1 share the highest homology with OYAV (SZC50) isolated from Culicoides collected in Shizong, Yunnan in 2013, with similarities of 99.8% to 99.9% among the S, M, and L fragments. On the phylogenetic tree, the S fragment of ML38-1 belongs to the same evolutionary branch as OYAV isolated from Culicoides collected in Shizong,Yunnan, mosquitoes collected in China, and Culex spp. collected in Vietnam(GenBank accession numbers: OP672242.1, JX983192.1, MH484317.1), as well as Cat Que virus isolated from Culex collected in Vietnam(GenBank accession number: NC 024075.1); The M fragment belongs to the same evolutionary branch as OYAV isolated from Culicoides collected in Shizong, Yunnan and mosquitoes collected in China (GenBank accession numbers: OP672243.1, JX983192.1) ; The L fragment belongs to the same evolutionary branch as the Oya virus (GenBank accession numbers: P672244.1, JX983194.1, PQ463773.1, MK609491.1) and Cat Que virus (GenBank accession number: OP12970.1) isolated from China. Conclusion One strain of OYAV was isolated from Culicoides collected in Mengla, Yunnan in 2021, which is highly homologous to the OYAV isolated from Culicoides in Shizong, Yunnan. OYAV has spread among Culicoides in Yunnan Province, and monitoring should be continuously strengthened.

Objective To understand the pathogen carried by small mammals in the border port areas between China and Kazakhstan, as well as China and Mongolia, and the transmission mechanism and route of their natural foci, so as to provide a basis for controlling the spread of zoonoses. Methods From 2019 to 2023, small animals were captured using the rat trap method and the night trap method. Their species were identified by morphology, and the tissues inside the animals were collected. The pathogens and vectors carried by the animals were detected by serological and molecular biological methods, and the transmission modes were analyzed. Results A total of 1 619 small mammals, belonging to 5 orders, 13 families, 30 genera and 40 species, were collected. Among 354 long-tailed yellow rats collected from the Hongshanzui Port on the China-Mongolia border, 22 positive samples of 5 pathogens were detected, with a positive rate of 6.21% (22/354). Among 191 samples collected from the Takshken and Hongshanzui port areas on the China-Mongolia border, 39 positive samples of 4 myiasis pathogens were detected in 4 hosts, with a positive rate of 20.42% (39/191). Among 192 samples collected from three hosts in the border areas of Takshken and Jimunai, 60 positive samples of two intestinal parasitic disease pathogens were detected, with a positive rate of 31.25% (60/192). A total of 1 719 samples of 15 hosts were collected from 7 major border port areas. Among them, 201 positive samples of 17 pathogens were detected, with a total positive rate of 11.69% (201/1 719). Conclusion Among the dominant species of small mammals distributed in the border port areas of China, Kazakhstan and Mongolia in Xinjiang, 17 kinds of natural foci pathogens are carried. Diseases are transmitted through various infection vectors and various diffusion methods. It is necessary to strengthen the publicity and education of prevention and control knowledge for the local people and further carry out disease monitoring in this area. It provides important basis and clues for the prevention and control of major and emerging zoonotic diseases such as plague in border areas.

Objectives To investigate the species composition, distribution characteristics, and community structure of rodents and their ectoparasitic fleas at the border ports area of Tengchong City, Yunnan Province, and to provide a scientific basis for the monitoring and prevention of plague and other rodent-borne diseases at the border port regions. Methods Rodent surveys were conducted in three habitat types (residential areas, cultivated farmlands, and forest zones) located in and around the border port of Tengchong City. The night trapping method and cage night method were used to investigate and sample the rodents. The ectoparasitic fleas on the surface of the rodents were combed and collected, and both hosts and fleas were identified morphologically. The capture rate, flea infection rate, and flea index were calculated, and the community structure characteristics were analyzed by species diversity, dominance, and similarity coefficient. Results A total of 261 rodents were captured, representing 22 species, 13 genera, 5 families, and 3 orders, with an average capture rate of 10.44%(261/2 510). The four dominant species were Micromys minutus (15.71%), Niviventer fulvescens (13.41%), Anourosorex squamipes (11.88%), and Niviventer confucianus (11.11%). Dominant species differed among habitats: Rattus tanezumi in residential areas, Micromys minutus, Anourosorex squamipes, and Rattus nitidus in farmlands, and Niviventer fulvescens and Niviventer confucianus in forests. Capture rates were 1.68% (16/953), 19.27% (148/768), and 12.29% (97/789) in residential, farmland, and forest habitats, respectively (χ²=145.698, P<0.001). A total of 104 ectoparasitic fleas were detected, belonging to 8 species, 6 genera, 5 subfamilies, and 4 families. The average flea infection rate was 19.16% (50/261) with an overall flea index of 0.40. The dominant flea species were Palaeopsylla remote (45.19%, 47/104) and Neopsylla stevensi sichuanyunnana (31.73%, 33/104). Anourosorex squamipes and Rattus andamanensis showed relatively high flea infestation rates (50.00%, 16/32; and 46.15%, 6/13, respectively) and flea indices (1.47 and 0.77). Farmland and forest habitats exhibited higher species richness and diversity indices for both rodents and fleas, whereas residential areas showed higher ecological dominance. Similarity coefficients indicated moderate similarity (0.74) between farmland and forest rodent communities and very high similarity (0.77) between their flea communities. Conclusion There are significant habitat differences in the distribution of rodents and ectoparasitic fleas in the border areas of Tengchong City. Increased population density and ecological dominance of major hosts and flea vectors may elevate the risk of transmission of rodent-borne diseases such as plague. It is necessary to continuously strengthen the monitoring and prevention and control measures of rodent-borne diseases in border areas. The present findings offer reference data for the prevention and control of pestis along the Yunnan border.

Objective To investigate the infection status of Coxiella burnetii (Cb) in humans and small mammals in three counties/cities of Yunnan Province, including Lianghe County, Mangshi City, and Mile City, to provide a basis for the prevention and control of Q fever. Methods From July to August 2019, human serum samples were collected and small mammals were captured from Lianghe County, Mangshi City, and Mile City of Yunnan Province. Cb antibody in human serum was detected by indirect enzyme-linked immunosorbent assay (ELISA). In January 2023, DNA was extracted from the spleen tissues of the captured small mammals with a magnetic-bead method. A nested PCR targeting the Cb Com1 gene was performed; positive amplicons were sequenced, aligned for homology analysis and used to construct a phylogenetic tree. The chi-square test and Fisher's exact probability method were used in R software to analyze the factors influencing Cb infection in the humans and Cb carriage in small mammals. Results A total of 368 human serum samples were tested, with 32 positive cases, resulting in a positive rate of 8.70%. Among them, the positive rate was 10.00% in Lianghe County, 8.46% in Mangshi City, and 6.82% in Mile City. No statistically significant differences in Cb infection among the humans were observed across different regions, ages, genders, ethnic groups, occupations, or educational levels (P>0.05). Altogether 490 small mammals, belonging to 3 orders, 5 families, 11 genera, and 16 species, were trapped. Rattus tanezumi and Suncus murinus were the predominant species. Five positive samples were nested PCR-positive for the Cb Com1 gene, giving an overall carriage rate of 1.02% (1.29% in Lianghe, 1.05% in Mangshi, and 0% in Mile). Cb carriage showed no significant association with region, habitat, species, or sex (P>0.05). Phylogenetic analysis indicated that the gene sequences of the 5 samples clustered in the same branch, showing 88.7%-100.0% nucleotide identity with 11 reference strains in GenBank. Conclusion There is a history of Cb infection in the humans of Lianghe County, Mangshi City, and Mile City. The carriage rate of Cb in small mammals is low in Lianghe County and Mangshi City, while no Cb was detected in those from Mile City. Lianghe County and Mangshi City appear to be natural foci of Cb, whereas the route of Cb infection in the humans of Mile City warrants further investigation.

Objective Through whole genome sequencing and biological analysis of 2 Japanese encephalitis virus (JEV) strains from Tonghai County, Yunnan Province, to elucidate the genetic variation patterns, genotype characteristics, and phylogenetic relationships of local JEV, thereby providing scientific evidence for Japanese encephalitis (JE) control and prevention in Tonghai County. Methods In 2015, 2 JEV strains (HL-3 and HL-32) were obtained from Culex tritaeniorhynchus collected in Tonghai County, Yunnan Province. The viruses were recovered and inoculated into BHK-21 cell cultures. After characteristic cytopathic effects developed, supernatants were collected for RNA extraction and cDNA synthesis. Sixteen pairs of specific primers were designed for whole genome RT-PCR amplification and sequencing. Nucleotide and amino acid homology analysis, amino acid difference site comparison, and phylogenetic tree construction were performed. Results Complete genome sequences of 2 JEV strains were obtained, each with a length of 10 965 bp. The nucleotide and amino acid homology between 2 JEV was 99.9%. Nucleotide homology with GⅠ-b genotype ranged from 98.0% to 98.4%, while amino acid homology was 97.1% to 98.7%. Phylogenetic analysis showed that both JEV strains clustered in the GⅠ-b genotype evolutionary branch and demonstrated close genetic relationships with recent epidemic strains from Yunnan Province and Southeast Asian strains. Compared to the SA14-14-2 vaccine strain, the 2 JEV strains had 56 amino acid difference sites, including 17 in structural proteins and 39 in non-structural proteins. Conclusions Culex tritaeniorhynchus remains the primary vector for Japanese encephalitis virus (JEV) in Tonghai County. Both JEV strains belong to the GⅠ-b genotype, which aligns with the genotype conversion trends observed across mainland China. The 2 JEV strains demonstrate high genetic similarity and exhibit close evolutionary relationships with JEV strains from Yunnan Province and Southeast Asian countries. The overall genomic structural variations remain relatively conservative, and the SA14-14-2 vaccine continues to demonstrate protective efficacy. These research findings contribute valuable data to the local JEV knowledge base and provide essential molecular epidemiological evidence to support JEV prevention and control strategies.

Objective To investigate the species composition of rodents and their ectoparasitic fleas in Lianghe County, Yunnan Province, and to assess infection with Yersinia pestis and Rickettsia mooseri, thus providing a scientific basis for the prevention and control of local flea-borne diseases. Methods In April 2024, fleas were collected from the body surface of rodents captured by the cage-night method and clip-night method in Zhedao Town, Mangdong Town, Jiubao Town, Hexi Town, and Nangsong Town of Lianghe County, Yunnan Province, then the species of rodents and fleas were identified, and the flea infestation rate and flea index were calculated. The chi-square test and Fisher's exact probability method were employed to analyze flea infestation rates across different investigation sites. The total DNA was extracted from pooled flea samples, and ordinary PCR was applied to amplify the caf1 and pla genes of Yersinia pestis and the gltA gene of Rickettsia mooseri. Positive samples were sequenced and compared with GenBank entries, and a neighbor-joining phylogenetic tree was constructed with MEGA 6.06 software. Results A total of 163 rodents were captured in this study, belonging to 2 orders, 2 families, 8 genera, and 10 species. Rattus tanezumi (113, 69.33%) was the dominant rodent species in the area, followed by Crocidura attenuata (14), R. andamanensis (10), Mus caroli and Suncus murinus (8 each), R. nitidus (6), and four individuals of other species. A total of 127 ectoparasitic fleas were collected and classified into 1 order, 3 families, 3 genera, and 3 species. Leptopsylla segnis was the dominant local flea species, accounting for 74.02%, followed by Xenopsylla cheopis (22.05%), and Aviostivalius klossi bispiniformis (3.94%) was the least. The flea infestation rate in the residential area (38.57%) was significantly higher than in the Agricultural area (6.45%) (χ²=25.519, P<0.001). All 21 pooled flea samples were PCR-negative for Y. pestis, whereas three pools were positive for R. mooseri. The three positive gltA gene sequences (LHM10, LHM14, and LHM15) were identical, and Rickettsia mooseri gltA obtained in Lianghe County shared 99.14% identity with the Wilmington reference strain (U59714.1); phylogenetic analysis placed them in the same clade. Conclusions No Yersinia pestis infection was detected in ectoparasitic fleas in Lianghe County, Yunnan Province, but Rickettsia mooseri infection was detected, indicating an endemic focus of murine typhus in this region. Given the high density of rodents and fleas in certain areas, local residents are at risk and strengthened surveillance is warranted.

Objective To analyze the biological characteristics and genomic features of Yersinia enterocolitica phage vB_Yen_YN202-142, and to provide references for prevention and treatment of Yersinia enterocolitica. Methods Using Y. enterocolitica as the host strain, phages were isolated from the cecum of Apodemus chevrieri. Morphology was observed by transmission electron microscopy (TEM), and stability, optimal multiplicity of infection (MOI), one-step growth curve, and host range were determined. Open reading frames (ORFs), phylogenetic tree construction and comparative genomic analysis were predicted through whole-genome sequencing. Results A novel Y. enterocolitica phage, vB_Yen_YN202-142, was isolated. It formed transparent plaques (2.0-3.0 mm in diameter) surrounded by a 0.5 mm semi-transparent halo. TEM revealed a head and a contractile tail. The phage titer decreased significantly after 30 min of UV exposure and was inactivated by 75% ethanol within 1 h. It remained stable at 4-60 ℃ and pH 5.0-9.0, with an optimal MOI of 0.1, a latent period of 3 min, and a burst size of 83 PFU/cell. It showed lysis activity against multiple strains of Yersinia enterocolitica and Yersinia pestis of different serotypes. The genome was 51 163 bp, GC content: 46.93%, encoding 94 ORFs classified into five functional groups: structural proteins, DNA packaging, DNA metabolism/replication, host lysis, and hypothetical proteins. Conclusion A bacteriophage vB_Yen_YN202-142, isolated from cecum of an adult male A. chevrieri in a plague foci, represents a new member of Caudoviricetes. Its short latent period, robust stability, and broad lytic activity enrich the Y. enterocolitica phage repository and provide a scientific basis for phage therapy against yersiniosis and the control of Y. enterocolitica.

Objective To characterize the genetic evolution and molecular characteristics of 17 H5N6 avian influenza virus (AIV) strains isolated from live poultry markets (LPMs) in Changsha. Methods From 2016 to 2023, environmental samples were collected from LPMs, nucleic acid were tested for type A influenza virus and H5/N6 subtypes using RT-PCR. Subsequently, 17 H5N6 AIV-positive specimens were subjected to next-generation sequencing (NGS). The sequencing samples were subjected to sequence similarity comparison using GISAID database, followed by comprehensive characterization of key amino acid residues, phylogenetic relationships, and molecular characteristics using MEGA-X software. Results The nucleotide similarity of HA genes of 17 H5N6 subtype avian influenza viruses ranged from 99.88% to 96.63%. The HA protein contained a polybasic cleavage site of RERRRKR↓GLF, which is characteristic of highly pathogenic avian influenza (HPAI) viruses. While the receptor-binding sites Q226 and G228 remained conserved and retained their affinity for avian-type preference for α2,3-linked sialic acid receptors; mutations at S123P, I155T, and T160A were identified, which may potentially enhance binding affinity to human-type α2,6-linked receptors and increased the transmission ability. The nucleotide similarity of NA genes of 17 H5N6 subtype avian influenza viruses ranged from 99.93% to 98.66%, with stalk region deletion residues 59-69, that has been associated with increased virulence in murine models. No mutations E119V, H274Y, R292K, or N294S conferring resistance to neuraminidase inhibitors were detected, suggesting retained susceptibility to oseltamivir and zanamivir. Phylogenetic analysis classified the isolates into multiple clades: 2.3.4.4b (n=3), 2.3.4.4e (n=1), 2.3.4.4h (n=5), 2.3.4.4g (n=3), and unclassified 2.3.4.4 (n=5), demonstrating significant genetic diversity in the HA gene among H5N6 viruses in Changsha LPMs. Conclusion From 2016 to 2023, there were highly pathogenic H5N6 AIVs in the LPMs in Changsha City. Some receptor binding sites have changed, and the virus has differentiated into multiple sub-lineages, and continuous monitoring is therefore necessary.

Objective To analyze molecular tracing and evolutionary characteristics of H9N2 avian influenza virus (AIV) strains isolated from two human cases and related environmental samples collected from live poultry markets (LPMs) in Changsha City in April 2025, provide scientific basis for avian influenza prevention and control. Methods Gene sequencing was performed on H9N2 AIVs obtained from throat swabs of the two cases and environmental samples. Genetic evolution analysis on HA and NA genes, homology, receptor-binding specificity, protein activity, drug resistance, and N-glycosylation patterns were focused. Results A total of 10 H9N2 avian influenza virus (AIV) gene sequences were obtained, including 1 whole genome. Homology analysis revealed that the HA gene was most similar to strains isolated from Fujian Province, while the NA gene was most similar to isolates from Fujian and Chongqing. HA gene belonged to the Y280-like h9.4.2.5 lineage, featuring a cleavage site of PSRSSR↓GLF. The receptor-binding site harbored Q226L and Q227M substitutions, and multiple antigenic mutations were observed compared to Y280-like vaccine strains. N-glycosylation site prediction identified 7 potential sites in all strains. All NA gene exhibited a deletion at amino acid positions 55-57 in the stalk region. Key active-site mutations (K135E, I145T) were mutanted, along with one or more substitutions in head epitopes, but no antiviral resistance markers were found. N-glycosylation analysis revealed 4 potential sites in 9 strains and 3 in 1 strain, the latter possibly linked to an N78D substitution upon sequence alignment. Conclusion The H9N2 avian influenza virus exhibited diverse mutations in key sites and functional domains detected in Changsha in April 2025, with multiple transmission chains spreading locally. Multifaceted measures should be strengthened to enhance epidemic control and prevention.

Objective To investigate the prevalence of Borrelia burgdorferi, Leptospira interrogans, Rickettsia mooseri, and Orientia tsutsugamushi in rodents in the central and southern regions of Shanxi Province, thereby providing a reference for the prevention and control of local rodent-borne diseases. Methods In July 2024, rodents were captured using the night trapping method in 7 towns (townships) of Jinzhong, Yangquan, and Yuncheng Cities, Shanxi Province. The liver, spleen, and kidney tissues of rodents were collected under aseptic conditions for the detection of the above four pathogens by fluorescence quantitative PCR (qPCR). Chi-square or Fisher's exact tests were employed to compare pathogen-positive rates among different species, sexes, tissues, habitats, and sampling sites. Results In total, 301 rodents belonging to 8 species were captured, including Apodemus agrarius, Eothenomys inez, Apodemus draco, Mus musculus, Niviventer confucianus, Cricetulus longicaudatus, Rattus tanezumi, and Tscherskia triton. Real-time fluorescent quantitative PCR screening of liver, spleen, and kidney specimens of 301 rodents showed overall positivity rates of 16.94 % (51/301) for B. burgdorferi, 3.99 % (12/301) for L. interrogans, and 0 for both R. mooseri and O. tsutsugamushi. The detection rate of B. burgdorferi was higher than that of L. interrogans (χ²=117.430, P<0.01). B. burgdorferi was detected in four species of rodents, namely Apodemus draco, Apodemus agrarius, Mus musculus, and Niviventer confucianus. L. interrogans was detected in four species of rodents, including Apodemus agrarius, Eothenomys inez, Mus musculus, and Niviventer confucianus. Among tissues, kidneys showed the highest detection rate for B. burgdorferi (13.62 %), significantly higher than those of liver and spleen (χ²=35.999, P<0.01). There was no significant difference in the detection rates of L. interrogans among the three tissues (χ²=3.426, P=0.180). No pathogens were detected in the two rodents captured in the village habitat. Both pathogens were found in rodents from forest, farmland, and reforested cropland, with the highest B. burgdorferi positivity in reforested areas (21.28%) and the highest L. interrogans positivity in farmland (7.50 %). Conclusion Rodents in central and southern Shanxi Province are infected with B. burgdorferi and L. interrogans, indicating potential infection risks to the local human population. Relevant public-health authorities should pay close attention to these findings.

Objective To analyze clinical and epidemiological characteristics of the first human case of H5N6 avian influenza in Hefei City, and to provide a scientific basis for control and prevention of similar animal-origin influenza outbreaks in humans. Methods Field epidemiological investigation methods were used to collect and analyze basic information of the case, course of illness and medical consultation, history of poultry exposure, tracking and screening of close contacts, and testing results of relevant specimens. Results The case was an elderly female who presented with flu-like symptoms in the early stages of illness. The condition progressed rapidly, with time intervals from onset to first consultation, hospitalization, conversion to severe condition, use of anti-influenza drugs, laboratory confirmation, and death being 1 day, 2 days, 2 days, 4 days, 7 days, and 22 days, respectively. The patient had a clear history of exposure to live poultry (chickens), and the positive rate of H5N6 virus in the supply market environment was 20.00%. The results of gene sequencing showed that, the HA gene segment belongs to the same lineage as H5N6 strains isolated from chickens and crows in Japan in 2022 and 2023, and the H5N6 strain isolated from cats in South Korea in 2023; while the NA gene segment belongs to the same lineage as H5N6 strains isolated from mandarin ducks in South Korea in 2023 and from wild birds in Zhejiang, China in 2024. Expanded surveillance revealed that the positive rate of H5N6 in environmental samples from markets with live poultry sales was significantly higher than that in markets without live poultry sales (60.00% vs. 1.27%, χ²=88.364, P<0.001). No secondary cases were found among the 44 close contacts and co-exposed individuals. Conclusion This is the first human case of H5N6 avian influenza in Hefei City, with the source of infection being exposure to live poultry. The sale of live poultry is a risk factor for human infection with H5N6 avian influenza. It is recommended to prohibit the trade of live poultry in urban areas and even the entire city, promote centralized slaughter, cold chain transportation, and fresh poultry sales policies, strengthen public health education, encourage citizens to purchase and consume "qualified poultry", and reduce direct contact with live poultry to lower the risk of infection.

As a "mixer" of influenza viruses, pigs can be infected by both human-origin and avian-origin influenza viruses, allowing for the easy generation of novel viral strains through gene reassortment. Currently, the Eurasian avian-like H1N1 (EA H1N1) virus has spread widely among pigs in Europe and Asia, and has caused human infections in many countries. In recent years, human infections with the EA H1N1 virus have been reported in many provinces in China, raising public health concerns. In pigs, EA H1N1 virus undergoes genetic recombination with other swine influenza viruses, resulting in numerous genetic variants. Among them, the G4 EA H1N1 virus has possessed the essential characteristics for human infection, enhanced pathogenicity and transmissibility in mammalian models. Serological investigations also reveal that swine-exposed population have higher seroprevalence against G4 EA H1N1 virus than that of general population, suggesting a greater risk of infection. This article provides a comprehensive review of origin and evolution of EA H1N1 virus, the pathogenicity and transmission of different EA H1N1 genotypes on mammalian models, impact of key amino acid site mutations, confirmed human infection cases, and serological investigations of human infection against EA H1N1 virus. It aims to offer reliable basis for control and prevention of EA H1N1 virus and to guide the future research direction.

This article reports a fatal case of Japanese spotted fever (JSF) caused by Rickettsia japonica, providing reference experience for early identification and treatment of JSF in primary medical institutions. The patient presented fever of unknown cause for 6 days, with the highest temperature reaching 39.5 ℃, accompanied by chills, fatigue, and myalgia. The initial symptoms were similar to those of the common cold. Local treatment was ineffective, the patient was admitted to People's Hospital of Zigui County on April 27, 2021. On admission, laboratory tests revealed normal white blood cell count, thrombocytopenia, and varying degrees of abnormalities in coagulation function, liver function, myocardial enzyme spectrum, C-reactive protein, procalcitonin, and urine protein. Imaging showed a small amount of pleural effusion bilaterally. The patient's condition deteriorated rapidly the day after admission, and the patient died despite resuscitation efforts. The diagnosis of Rickettsia japonica infection was confirmed on the blood specimen by metagenomic next-generation sequencing and specific gene PCR techniques. This case highlights that in patients with non-specific clinical symptoms, laboratory tests, and imaging results, and fever of unknown and a history of outdoor activities, clinicians should be highly vigilant for possibility of rickettsial infection. Empirical anti-infective treatment should be initiated promptly before the pathogen detection report is available, with doxycycline being the preferred choice, which is crucial for improving patient prognosis. Meanwhile, it is essential to strengthen training for primary care physicians and public health education in potential epidemic areas.

Objective To analyze clinical and epidemiological characteristics of two cases of H5N6 avian influenza infection in Dazhou City in July 2021, to identify the transmission routes, and assess the risk of H5N6 avian influenza virus spread, to provide a reference for control and prevention of avian influenza. Methods Medical records of the H5N6-infected cases were collected in Dazhou City in July 2021, epidemiological investigations of their close contacts were conducted, and throat swabs from close contacts and dead poultry samples were collected for nucleic acid detection using real-time fluorescent RT-PCR. Results Epidemiological investigations revealed that both cases had a history of poultry exposure. One patient had long-term poultry breeding and routine cleaning of poultry pens, while the other had selected ducklings at a live poultry market 6 and 9 days before symptom onset. Clinically, both patients developed fever and cough after poultry exposure, with H5N6 nucleic acid positivity detected in bronchoalveolar lavage fluid samples on day 10 and 15, respectively. Among 149 environmental samples collected from live poultry markets in H County and K County, 120 tested positive for influenza A virus, with positive rate of 80.54%. Conclusion This study reports the first human H5N6 avian influenza cases in Dazhou City, both linked to poultry and poultry environment exposure. No human-to-human transmission was identified. Live poultry markets are high-risk areas for H5N6 transmission, highlighting the need for enhanced disinfection measures to prevent human infections.

Objective To establish a triple PCR method for the simultaneous, accurate, and rapid identification of three mosquito species, Anopheles crawfordi, Anopheles vagus, and Anopheles tessellatus. Methods Genomic DNA was extracted using a universal DNA extraction method from Anopheles peditaeniatus, Anopheles lesteri, Anopheles barbirostris, Anopheles splendidus, Anopheles maculatus, Anopheles aconitus, Anopheles crawfordi, Anopheles vagus, Anopheles tessellatus, and the concentration was determined. Primers were designed based on the mitochondrial COI gene of An. crawfordi, An. vagus, and An. tessellatus, with the other six mosquito species serving as controls. According to the primer design principles, primers capable of specifically amplifying the three species of mosquitoes were designed and synthesized by a professional institution. Single PCR was first performed using the extracted mosquito DNA as a template to verify primer specificity. Subsequently, the primer concentration, dNTPs concentration, and Taq enzyme amount in the triple PCR reaction system were optimized. The optimized PCR products were loaded following the capillary electrophoresis protocol, and the results were observed and recorded. The template DNA of An. crawfordi, An. vagus, and An. tessellatus was serially diluted 10-fold, ranging from 10 ng/μL to 10^-6 ng/μL, followed by sensitivity testing via triple PCR. Results The primer concentrations for An. crawfordi, An. vagus, and An. tessellatus were the same, showed excellent specificity at an annealing temperature of 58 ℃, amplifying specific bands of 118 bp, 187 bp, and 307 bp, respectively. No bands were observed for other mosquito species or the blank control. The detection limits for An. crawfordi, An. vagus, and An. tessellatus were all 10^-4 ng/μL. Conclusion This method enables the simultaneous, accurate, and rapid identification of An. crawfordi, An. vagus, and An. tessellatus samples, and is applicable to specimens with incomplete morphological features, such as damaged or non-adult stages. It provides a reliable technical tool for mosquito surveillance and disease prevention and control.

Objective To explored the functions of three kinds of CLIP domain serine proteases in the CLIP family of Aedes aegypti: Ae-5575351, Ae-CLIPB15 and Ae-CLIPB36 in mosquito immunity, and the interaction between these protein was preliminarily verified. Methods MEGA 11 and ESPript 3.0 were used for multiple sequence alignment of Ae-5575351, Ae-CLIPB15, Ae-CLIPB36 and CLIP in other insects. Yeast two-hybrid was used to verify the protein interaction between Ae-5575351 and Ae-CLIPB15, Ae-CLIPB36., The spatio-temporal expression profiles of Ae-5575351, Ae-CLIPB15 and Ae-CLIPB36 were constructed using real-time fluorescence quantitative polymerase chain reaction (qPCR). The functions of Ae-5575351, Ae-CLIPB15 and Ae-CLIPB36 in the process of Aedes aegypti's resistance to bacterial and fungal infection, and their effects on Toll pathway and IMD pathway were explored through RNA interference and pathogen infection experiments. Finally, the effects of Ae-5575351, Ae-CLIPB15 and Ae-CLIPB36 on the melanization were investigated by determining phenoloxidases (POs) activity. Results Ae-5575351, Ae-CLIPB15 and Ae-CLIPB36 were highly conserved compared with CLIP in other insects. All of them were highly expressed in the malpighian duct, salivary gland and hemolymph of Aedes aegypti, and they were mainly expressed in the middle and late stages in the development of Aedes aegypti. Ae-5575351 interacts with Ae-CLIPB15 and Ae-CLIPB36 at the protein level respectively. S. aureus and E. coli infections can induce increased expression of Ae-5575351, Ae-CLIPB15 and Ae-CLIPB36. Ae-5575351, Ae-CLIPB15 and Ae-CLIPB36 regulate the expression of transcription factors REL1 and REL2 in the process of Aedes aegypti larvae resisting B. bassiana and S. aureus infection. The knock-down of Ae-CLIPB15 and Ae-CLIPB36 significantly reduced enzymatic activity of POs in Aedes aegypti. Conclusion Ae-5575351, Ae-CLIPB15 and Ae-CLIPB36 may affect the ability of Aedes aegypti to resist bacterial and fungal infection by regulating Toll pathway, IMD pathway and melanization, and there may be some interaction between Ae-5575351 and Ae-CLIPB15 and Ae-CLIPB36.

Objective To investigate the influencing factors of negative results of interferon-γ release assay (IGRA) in patients with active tuberculosis (ATB), and to further enhance the correct interpretation of IGRA results in clinical practice, thereby providing a reference for the differential diagnosis of ATB. Methods Hospitalized ATB patients treated in Suzhou Fifth People's Hospital from January 1, 2018, to March 31, 2024, were retrospectively reviewed. Associations between IGRA results and patients' demographics, disease diagnosis information, testing period, blood routine indexes, and pathogenic indexes were analyzed. Results A total of 7 657 ATB patients were enrolled in the study, including 5 463 males and 2 194 females. The peripheral blood lymphocyte counts differed significantly among patients with different IGRA results (U=4 228 273; P<0.01), as did the neutrophil counts (U=4 494 928; P<0.01). Univariate analysis revealed that age (Wald χ²=188.123, P<0.01), hypertension (Wald χ²=20.679, P<0.01), immunodeficiency syndrome (Wald χ²=28.203, P<0.01), and testing period (Wald χ²=691.324, P<0.01) were factors influencing IGRA results in ATB patients. Multivariate analysis further demonstrated that advanced age (OR=1.023, 95% CI: 1.020-1.026, P<0.01), different testing periods (stage 2: OR=2.496, 95% CI: 2.179-2.860, P<0.01; stage 3: OR=1.673, 95% CI: 1.404-1.995, P<0.01), and elevated neutrophil-to-lymphocyte ratio (NLR) (OR=1.022, 95% CI: 1.013-1.031, P<0.01) were significant factors contributing to an increased IGRA-negative detection rate in ATB patients. Significant differences were observed in the distribution of IGRA results among ATB patients with varying tuberculosis culture results (Wald χ²=11.765, P<0.01) but not acid-fast staining smear results (Wald χ²=12.248, P=0.066). Conclusions In ATB patients, age, testing period, and NLR are critical influencing factors for IGRA test results. Therefore, when encountering IGRA-negative suspected cases, the possibility of ATB should not be excluded. A comprehensive analysis of the patient's immune status, incorporating laboratory indicators and imaging examinations, is recommended to avoid delayed treatment.

Objective To understand the effect of antiretroviral therapy among HIV/AIDS patients in Guizhou Province from 2016 to 2022, and to analyze the related influencing factors, to provide scientific advice for antiviral treatment measures against AIDS. Methods A retrospective study was conducted to collect the data of HIV/AIDS patients who received ART for the first time from the National HIV/AIDS Comprehensive Response Information System in Guizhou from 2016 to 2022. The socio-demographic characteristics and ART related information were analyzed to understand the effect of ART and related influencing factors. Results A total of 45 202 HIV/AIDS patients were included and followed up until the cut-off time. The overall mortality rate was 11.61%, and the overall loss to follow-up rate was 13.42%. The last viral load analysis result showed that the complete viral suppression rate was 84.20%. There were statistically significant differences in mortality rate, loss to follow-up rate and complete viral suppression rate among different follow-up years (P<0.001). Multivariate log-binomial regression analysis revealed that female (PR=1.05, 95%CI:1.04-1.05), primary school education (PR=1.01, 95%CI:1.01-1.02), junior high school education (PR =1.05, 95%CI: 1.05-1.07), high school or vocational school education (PR=1.07, 95%CI:1.06-1.07), college or above education (PR=1.12, 95%CI:1.11-1.12), homosexual transmission (PR=1.06, 95%CI:1.05-1.06), and no treatment plan change (PR=1.04, 95%CI:1.03-1.04) were protective factors for complete viral suppression; while divorced or widowed and unknown (PR =0.98, 95%CI: 0.97- 0.98), diagnosis to treatment time of 7 -30 days (PR=0.99, 95%CI:0.98-0.99), > 30- 365 days (PR=0.95, 95%CI: 0.94-0.95), > 365 days (PR=0.93, 95%CI:0.92-0.93), baseline WHO clinical stage of stage Ⅱ (PR=0.99, 95%CI: 0.98 -0.99), stage Ⅲ (PR=0.95, 95%CI: 0.94-0.96), stage Ⅳ (PR=0.98, 95%CI: 0.97-0.99), and initial treatment plan as a second-line regimen (PR=0.98, 95% CI: 0.98-0.99) were risk factors for complete viral suppression. Conclusion The complete viral suppression rate after antiviral treatment for AIDS is relatively high in Guizhou. However, it is necessary to reduce the risks of death and dropout. It is recommended that during the entire treatment, special attention should be paid to patients with late initiation of treatment and older age, to improve patients' medication compliance, and to further consolidate and enhance the treatment effect.

Objective To evaluate health status and carcinogenic risks of workers exposed to benzene in small and medium-sized enterprises in Hainan Province from 2022 to 2024, and to provide a scientific basis for control and prevention of occupational disease hazards of benzene in small and medium-sized enterprises in Hainan Province. Methods Occupational health examination data of workers exposed to benzene in small and medium-sized enterprises were collected in Hainan Province from 2022 to 2024. A total of 715 workers exposed to benzene were randomly selected for detection of concentration of trans, trans-muconic acid (tt-MA) as a biological monitoring indicator. The correlation between tt-MA and key indicators of blood routine examination was analyzed. The linear multi-stage carcinogenic model based on internal exposure concentration of benzene was used to evaluate carcinogenic risk of benzene exposure to workers. SPSS 26.0 software and R 4.2.0 language were used for statistical analysis of the data. Results From 2022 to 2024, a total of 11 662 workers exposed to benzene from 662 small and medium-sized enterprises in Hainan Province participated in the occupational health examination during their employment. The majority were male (9 772 people, accounting for 83.79%), and M（P₂₅，P₇₅） of age was 33.0 (27.0, 41.0) years old. Among the abnormal detection rates of routine examination indicators, blood routine examination had the highest rate (24.02%), followed by liver function (14.81%). The abnormal detection rate of blood routine special examination was 19.53%, the highest rate was in Haikou City (21.76%), in manufacturing industry (23.83%), in small-sized enterprises (20.47%), and the abnormal rate for females (29.74%) was higher than that for males (17.56%) （χ²=149.355，P<0.05）, and the age group of ≥ 55 years old had the highest abnormal rate (22.25%). The detection rates of low counts of three key blood routine indicators including white blood cells, neutrophils and platelets were 2.12%, 0.69% and 0.27%, respectively. The over-limit rate of tt-MA concentration among 715 biological monitoring subjects was 4.34%. The tt-MA concentration had a non-linear dose-response relationship with the neutrophil count (P_overall<0.001, P_non-linear<0.001). The carcinogenic risks of benzene exposure for all 715 workers exceeded the acceptable level of carcinogenic risk for occupational population（10^-4）. Among them, the top three positions were packaging position in the paper products manufacturing industry, painting position in the automotive parts and accessories manufacturing industry, and inspection position in the professional and technical service industry, with carcinogenic risks of 564.207(164.021-964.394)×10^-4, 309.869(2.652-3 288.607)×10^-4 and 208.668(2.656-3 868.348)×10^-4, respectively. Conclusion The occupational health risks of workers exposed to benzene in small and medium-sized enterprises in Hainan Province should not be underestimated. It is necessary to strengthen protection against benzene exposure in key industries and high-risk positions, and reduce the carcinogenic risks of benzene exposure to workers as much as possible.

Objective To analyze the epidemic characteristics and disease burden of brucellosis in Tangshan City from 2007 to 2022, explore the socioeconomic determinants of that burden, and provide a theoretical basis for further optimizing the prevention and control strategies of brucellosis and rationally allocating medical resources. Methods Based on monitoring data of brucellosis in Tangshan City from 2007 to 2022, the incidence rate, years lived with disability (YLD), standardized YLD rate, and indirect economic burden were calculated to assess the change of the disease burden. Joinpoint regression analysis was used to examine the trends in the health burden of brucellosis over time and by age. A generalized linear model was used to explore the socioeconomic factors associated with the burden. Results A total of 3 949 cases of brucellosis were reported from 2007 to 2022, yielding an average incidence rate of 3.23/10 million. Incidence was higher in males than in females and peaked in the 50-<60-year age group. The incidence rate was higher in the eastern areas such as Qian'an, Luannan, and Laoting. Over the 16 years, brucellosis caused 118.47 person-years of YLDs, corresponding to an overall YLD rate of 0.015 29‰. The 50-<60-year group had the greatest YLDs and YLD rate (36 person-years, 0.003 70‰). Males (88.71 person-years, 0.022 24‰) bore a heavier burden than females (29.76 person-years, 0.007 90‰), and the eastern region (75.48 person-years, 0.002 00‰) exceeded the central urban area (11.49 person-years, 0.000 43‰) and the western region (31.50 person-years, 0.000 62‰). Both the health burden and the indirect economic burden first rose and then declined, mirroring the incidence trend. Adults aged 30 years and older, and residents of the eastern region, carried the greatest indirect economic burden. The number of medical institutions and health manpower, rural per capita disposable income, mutton output, beef output, and milk output were identified as significant socioeconomic determinants of brucellosis burden. Conclusion Middle-aged and older men in the eastern regions of Tangshan experience the highest incidence and greatest health and economic burden of brucellosis. Local authorities should establish intersectoral information-sharing mechanisms, strengthen livestock quarantine and supply-chain supervision, enhance public education, and allocate medical resources and facilities rationally to further reduce the health and economic impact of brucellosis in Tangshan.

Objective This article traces the discovery history and epidemic situation of schistosomiasis in Wuhu area, explores the scientific control efforts and phased achievements from 1951 to 2024, and summarizes the prevention and control technologies and practical methods that were first explored and applied locally. It outlines the evolution of three main stages: a comprehensive prevention and control phase centered on Oncomelania hupensis eradication, a chemotherapy-based control phase, and a transmission-source control phase. Methods From the perspective of the history of science and technology, this study uses historical literature review and comparative analysis to examine three stages of schistosomiasis control: comprehensive control centered on Oncomelania hupensis eradication, key control based on chemotherapy, and precision control focusing on transmission-source management, and to evaluate their influencing factors and effectiveness. Results The study highlights the extensive experience Wuhu area has accumulated in Oncomelania hupensis management and population treatment, such as health education, feces management, and "one village, one policy", which has strengthened comprehensive management capabilities. Conclusion Through examining the historical evolution of schistosomiasis prevention and control and its influencing factors in Wuhu area, these practices offer valuable lessons for other lake-type, hilly-type, and water network-type epidemic areas in China, deepen the understanding of local public health governance experience, and provide practical support and scientific reference for current schistosomiasis prevention and control initiatives.

Objective To analyze the epidemiological characteristics and pathogen genetic features of a human metapneumovirus (hMPV) outbreak in a primary school in Ji'ning City, and to provide a scientific basis for the effective management of hMPV outbreaks in schools. Methods Descriptive analysis was used to study the epidemiological characteristics of the hMPV outbreak in Ji'ning City,2024. The attack rates between affected classes and genders were compared using the chi-square test. Whole-genome sequencing was performed on four hMPV-positive samples, and a phylogenetic tree was constructed based on the G protein gene sequences. Results The outbreak involved 31 students, with a student attack rate of 7.38% (31/420); no staff members were infected. Among the 31 cases, 17 were male and 14 were female (male-to-female ratio=1.21∶1), with no significant difference in attack rate between genders (P>0.05). All cases occurred in Grade 5, Class 2 (58.97%, 23/39) and Grade 6, Class 2 (22.22%, 8/36), and the difference in attack rates between the two classes was significant (χ²=10.43, P<0.01). The main common symptoms of the cases were cough (67.74%, 21/31), sore throat (58.06%, 18/31), and fever (35.48%, 11/31). Multiplex nucleic acid testing showed that five samples were positive for hMPV, with four being solely positive for hMPV and one being co-positive for hMPV and Haemophilus influenzae. Whole-genome analysis revealed that all four hMPV strains belonged to the B2 subtype, with significant variations in the G gene. Conclusion Subtype B2 hMPV with significant G-gene mutations was the primary pathogen responsible for this outbreak. Although the symptoms in students were relatively mild, there is a need to strengthen respiratory infectious disease prevention and health monitoring in schools.