Importation of a novel Indian Ocean lineage carrying E1-K211E and E2-V264A of Chikungunya Virus in Zhejiang Province, China, in 2019.

The chikungunya virus (CHIKV) is widespread. In Zhejiang province, China, CHIKV infection is often associated with travelers from tropical and subtropical countries. In the present study, three CHIKV isolates from serum samples of travelers in Zhejiang province in 2019 were sequenced, and phylogenetically analyzed to study their molecular characteristics. Sequence analysis showed that the non-structural protein and the structural protein had 37 and 28 amino acid mutations, respectively; no mutation site was found at the E1-A226 residue, which could increase the adaptability of CHIKV to Aedes albopictus. All three samples carried two mutations, namely, E1-K211E and E2-V264A, which were introduced to Bangladesh around late 2015 and Thailand in early 2017. Phylogenetic analysis revealed that these three CHIKVs were Indian Ocean lineage of the East Africa/Central/South Africa genotype (ECSA) and that the MF773566 strain from Bangladesh (Australia/Bangladesh 2017) had the closest evolutionary relationship. The three CHICKs imported into Zhejiang province in 2019 belonged to the ECSA genotype and had multiple amino acid variation sites. The variation in the three samples provides a certain reference for the subsequent research on CHIKV evolution.

Case Report: A case of Chlamydia psittaci infection in an HIV patient.

Chlamydia psittaci is the pathogen of psittacosis and infects a wide range of birds and even humans. Human infection occurs most commonly in those with a history of contact with birds or poultry. We describe a case of psittacosis in a human immunodeficiency virus infected patient in Zhejiang Province for the first time. C. psittaci infection was confirmed by nested polymerase chain reaction (PCR) and Real-Time PCR. Phylogenetic analysis revealed that the sequences from the patient's samples clustered with genotype A in the same branch. Our study highlights the possibility of diagnosing psittacosis in patients with a chronic disease such as HIV-infected patients, and should increase awareness and surveillance of psittacosis in China.

Corrigendum: Antimicrobial resistance and genomic characterization of Escherichia coli from pigs and chickens in Zhejiang, China.

[This corrects the article DOI: 10.3389/fmicb.2022.1018682.].

Antimicrobial resistance and genomic characterization of Escherichia coli from pigs and chickens in Zhejiang, China.

Escherichia coli is considered an opportunistic pathogen and an indicator for antimicrobial resistance (AMR) monitoring. Despite many reports on its AMR monitoring, studies based on genome-based analysis of AMR genes are still insufficient. Here, 181 E. coli strains were isolated from anal swab samples collected from pigs and chickens of animal farms located in Eastern China and sequenced through the Illumina platform. The results showed that 87.85% (159/181) of the E. coli isolates were multidrug-resistant (MDR). Ampicillin (AMP)- spectinomycin (SPT)- tetracycline (TET)- florfenicol (FFC)- sulfisoxazole (SF)- trimethoprim/sulfamethoxazole (SXT) was the predominant AMR pattern. By whole-genome sequencing, we found that ST10 (10.49%, 19/181) and ST48 (7.18%, 13/181) were major sequence types. IncFIB and IncX1 were the most prevalent plasmid replicons. The AMR genes bla NDM-5 (1.10%, 2/181), mcr-1 (1.10%, 2/181), tet(X4) (1.10%, 2/181), and cfr (6.08%, 2/181) were also found in these isolates. In addition, among the 169 virulence genes detected, we identified astA (37.02%, 67/181), hlyA (1.66%, 3/181), hlyB (1.66%, 3/181) and hlyD (1.66%, 3/181), which were closely related to heat-stable enterotoxin 1 and α-hemolysin. In addition, there were 33 virulence genes associated with the iron uptake system, and 46 were adhesion-related genes. Our study highlighted the need for routine surveillance of AMR with advanced genomic approaches, providing up-to-date data on the prevalence of AMR for the development and execution of antimicrobial stewardship policy.

Severe community-acquired pneumonia caused by Chlamydia psittaci genotype E/B strain circulating among geese in Lishui city, Zhejiang province, China.

Between November 2021 and January 2022, four patients of community-acquired pneumonia were admitted to the hospitals in Lishui city, Zhejiang province, China. Their main clinical manifestations were fever and dry cough as well as radiographic infiltrate, but the empiric antimicrobial therapy or traditional Chinese medicine was not effective for their illness. Clinical specimens from the patients as well as environmental and poultry specimens were collected for the determination of the causative pathogen. The ompA gene and seven housekeeping genes of Chlamydia psittaci were successfully amplified from all the patients, and the sequences of each gene were identical to one another, suggesting that they were infected by the same strain of C. psittaci. A novel strain of C. psittaci (LS strain) was isolated from the bronchoalveolar lavage fluid of patient 2 and its whole genome was obtained. Phylogenetic analyses based on the whole-genome sequences showed that the isolate is most closely related to the strain (WS/RT/E30) identified as genotype E/B. In addition, The ompA gene and four housekeeping genes of C. psittaci were also amplified from two of four faeces samples of geese at the home of patient 2, and the sequences from geese were 100% identical to those from the patients. Accordingly, these cases could be attributed to a circulating C. psittaci strain of genotype E/B in the local geese. Therefore, there is an urgent need to strengthen the regional surveillance on C. psittaci among poultry and humans for prevention and control of the outbreak of psittacosis in the city.

Case Report: Metagenomic Next-Generation Sequencing Clinches the Diagnosis of Acute Q Fever and Verified by Indirect Immunofluorescence Assay.

Q fever is a zoonotic infectious disease caused by Coxiella burnetii. The clinical symptoms of acute Q fever are usually atypical, and routine serological tests of C. burnetii are not readily available, making the diagnosis of Q fever a challenge. In this case, we report a male patient who had repeated fevers and was administered empirical anti-infective treatment, but the effect was poor. After conducting relevant laboratory and imagological examinations, the etiology has not yet been confirmed. Subsequently, metagenomic next-generation sequencing (mNGS) identified the sequence reads of C. burnetii from the patient's peripheral blood within 48 h, and then the diagnosis of acute Q fever was established. Moreover, the serological test of indirect immunofluorescence assay (IFA) of the C. burnetii antibody was further performed in the Centers for Disease Control, certifying the result of mNGS. The patient was ultimately treated with doxycycline and recovered well. mNGS is an unbiased and comprehensive method in infrequent or culture-negative pathogen identification. To our knowledge, this is the first case of acute Q fever identified by mNGS and confirmed by IFA in Taizhou, China. A further large-scale prospective clinical cohort study is worth carrying out to compare the diagnostic efficiency of mNGS with traditional serological methods and PCR in acute Q fever.

Detection and typing of dengue virus by one-step RT-PCR-based high-resolution melting assay.

Dengue is a rapidly spreading arboviral disease that can be transmitted through any of the four types of dengue virus (DENV) serotypes. Previous studies have observed that individuals who have a pre-existing secondary infection due to a different dengue serotype, experience severe forms of this disease. During a DENV outbreak, a time-sensitive preliminary diagnosis of the origin of DENV might be useful in controlling the epidemic. Here, we developed a rapid and accurate one-step TB Green RT-PCR-based high-resolution melting (HRM) assay to identify and serotype DENV using serotyping primers based on the alignment with the E gene. This assay had a detection limit of 7.7 × 102 (DENV 1), 3.8 × 102 (DENV 2), 6.2 × 102 (DENV 3), and 1.2 × 103 (DENV 4) RNA copies/mL. No cross-reactivity with the Chikungunya, Zika, and Japanese encephalitis viruses was observed. The feasibility of using this assay for clinical diagnosis was evaluated in DENV-positive patient sera. The HRM assay and the RT-qPCR had complete matched results derived from DENV detection, including 51 serum positive and 20 serum negative. Additionally, eight DENV 2 strains in the same serotype were successfully differentiated by an HRM assay. Thus, this assay facilitated accurate detection and serotyping of DENV, along with the time-sensitive identification of the infectious focus of different DENVs.

Abdominal-B contributes to abdominal identity in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae).

The homeotic complex gene Abdominal-B (Abd-B) is involved in regulating the development of posterior abdomens and has been extensively studied in holometabolous insects. However, the function of Abd-B in hemimetabolous insects is not fully understood. Here, we functionally characterize an Abd-B homologue in the brown planthopper (BPH), Nilaparvata lugens. The full-length cDNA of the N. lugens Abd-B homologue (NlAbd-B) is 2334 nt, with an open reading frame of 1113 bp. NlAbd-B has the highest expression level at the egg stage relative to the nymphal and adult stages and is mainly expressed in the fourth to the ninth abdominal segment of embryos. RNA interference (RNAi)-mediated knockdown of NlAbd-B in nymphs disrupted the development of genitalia both in females and males and caused a genitalia-to-leg transformation. Parental RNAi of NlAbd-B in both female and male adults caused an extra abdominal segment in offspring nymphs, while parental RNAi of the N. lugens abdominal-A homologue in both female and males adults led to embryos with leg-like appendages on the second to the eighth abdominal segment. These findings suggest that NlAbd-B plays a pivotal role in genital development and posterior abdominal patterning and thus highlight the conservational role of Abd-B in holometabolous and hemimetabolous insects.

A cluster of Psittacosis cases in Lishui, Zhejiang Province, China, in 2021.

Introduction: Psittacosis, caused by Chlamydia psittaci, is widespread throughout the world. In humans, C. psittaci infection may lead to severe conditions and complications, including sepsis and multiple organ failure. We report a cluster of cases caused by C. psittaci in Zhejiang Province, 2021, which led to one death and three cases of hospitalization. Methods: The cases were confirmed by nest-PCR, RT-PCR, and mNGS. Results: The four cases were related and the sequences obtained from the samples were closely correlated with those from Taiwan. Discussion: This study is the first to report on the case of death from psittacosis in Zhejiang Province, and our results help to assess the disease and recommend effective measures to prevent further spread of C. psittaci.

Molecular Characterization of Dengue Virus Type 1 in Zhejiang in 2019.

Dengue fever (DF) is a mosquito-borne viral disease caused by the dengue virus (DENV), which is considered one of the most important arboviruses in the world. This study aimed to determine the molecular, epidemiological, and phylogenetic characterization of 174 DENV-1 (132 indigenous cases and 42 imported cases) isolated from nine municipalities of Zhejiang province in 2019. The analyses of phylogenetics, haplotypes, and amino acid substitutions were conducted based on the full envelope (E) gene sequences. Sixty-four haplotypes were clustered into two main clades, with isolates from Wenzhou and Taizhou mainly clustered into clade I and Hangzhou and Ningbo cases clustered into clade II. Six sites of amino acid substitutions including A88T, F96L, M297V, T339S, I378L, and V436I were only observed in strains isolated from Ningbo and Hangzhou, while two sites of amino acid substitutions including V312L and V380I were observed in strains from Taizhou and Wenzhou. In our study, strains were in high homology with the strains from Southeast Asian countries, thus cases in Zhejiang were probably imported from Southeast Asian countries. The strains from different regions in Zhejiang were clustered in the same branch which may be caused by the continuous import of cases in the same country at different time periods. After the continuous outbreak in Zhejiang province, some sites of the dengue gene have mutated, and the effects need further study.

Simultaneous Detection of Ebola Virus and Pathogens Associated With Hemorrhagic Fever by an Oligonucleotide Microarray.

Ebola virus infection causes severe hemorrhagic fever, and its mortality rates varied from 25 to 90% in the previous outbreaks. The highly infectious and lethal nature of this virus highlights the need for reliable and sensitive diagnostic methods to distinguish it from other diseases present with similar clinical symptoms. Based on multiplex polymerase chain reaction (PCR) and oligonucleotide microarray technology, a cost-effective, multipathogen and high-throughput method was developed for simultaneous detection of Ebola virus and other pathogens associated with hemorrhagic fever, including Marburg virus, Lassa fever virus, Junin virus, Machupo virus, Rift Valley fever virus, Crimean-Congo hemorrhagic fever virus, malaria parasite, hantavirus, severe fever with thrombocytopenia syndrome virus, dengue virus, yellow fever virus, Chikungunya virus, influenza A virus, and influenza B virus. This assay had an excellent specificity for target pathogens, without overlap signal between the probes. The limit of detection was approximately 103 pathogen copies/µl. A total of 60 positive nucleic acid samples for different pathogens were detected, a concordance of 100% was observed between microarray assay and real-time PCR analysis. Consequently, the described oligonucleotide microarray may be specific and sensitive assay for diagnosis and surveillance of infections caused by Ebola virus and other species of hemorrhagic fever pathogens.

A SARS-CoV-2 variant with the 12-bp deletion at E gene.

The coronavirus disease 2019 (COVID-19) pandemic is still ongoing and has become an important public health threat. This disease is caused by a new coronavirus named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection, and so far, little is known about this virus. In this study, by using plaque purification, we purified two SARS-CoV-2 virus strains from the same specimen, one named F8 containing a 12-bp deletion in the E gene and the other named 8X containing the wild-type E gene. There was no significant difference in the viral titer and infectivity of these two strains. The S protein content of the F8 viral culture was 0.39 µg/ml, much higher than that of 8X. An inactivated vaccine made from the F8 strain could trigger high levels of the IgG titer and neutralizing antibody titer, which could last for at least 6 weeks and were significantly higher than those from the 8X strain at 1 and 3 weeks post vaccination, respectively. In conclusion, we reported that both the E gene mutant and wild-type SARS-CoV-2 strains were isolated from the same clinical sample by plaque purification. A 12-bp deletion in the E gene was important for SARS-CoV-2 replication and immunogenicity.

Contamination-free visual detection of SARS-CoV-2 with CRISPR/Cas12a: A promising method in the point-of-care detection.

The outbreaks of the infectious disease COVID-19 caused by SARS-CoV-2 seriously threatened the life of humans. A rapid, reliable and specific detection method was urgently needed. Herein, we reported a contamination-free visual detection method for SARS-CoV-2 with LAMP and CRISPR/Cas12a technology. CRISPR/Cas12a reagents were pre-added on the inner wall of the tube lid. After LAMP reaction, CRISPR/Cas12a reagents were flowed into the tube and mixed with amplicon solution by hand shaking, which can effectively avoid possible amplicon formed aerosol contamination caused by re-opening the lid after amplification. CRISPR/Cas12a can highly specific recognize target sequence and discriminately cleave single strand DNA probes (5'-6FAM 3'-BHQ1). With smart phone and portable 3D printing instrument, the produced fluorescence can be seen by naked eyes without any dedicated instruments, which is promising in the point-of-care detection. The whole amplification and detection process could be completed within 40 min with high sensitivity of 20 copies RNA of SARS-CoV-2. This reaction had high specificity and could avoid cross-reactivity with other common viruses such as influenza virus. For 7 positive and 3 negative respiratory swab samples provided by Zhejiang Provincial Center for Disease Control and Prevention, our detection results had 100% positive agreement and 100% negative agreement, which demonstrated the accuracy and application prospect of this method.

Muscle destruction caused by coxsackievirus A10 in gerbils: Construction of a novel animal model for antiviral evaluation.

The morbidity and mortality of coxsackievirus A10 (CVA10)-associated hand, foot, and mouth disease (HFMD) have been increasing in recent years, while few studies on the vaccine and animal model of CVA10 have been reported. Here, we first established a CVA10-infected gerbil model and employed it to evaluate the immunoprotective effect of an inactivated CVA10 vaccine. The results showed that gerbils up to the age of 14 days were fully susceptible to CVA10, and all died within five days post-infection by intraperitoneal inoculation. Lethargy, wasting, hind-limb paralysis, and even death could be observed in the CVA10-infected gerbils. Pathological examination suggested that CVA10 has a strong tropism toward muscle tissue, and muscle bundle fracture and muscular fibers necrosis were observed in the limb muscles. Additionally, active immunization results showed that gerbils immunized with the inactivated CVA10 vaccine were 100 % protected from lethal CVA10 challenge. The antisera from vaccinated gerbils also showed high neutralizing titers against CVA10. Based on these results, the CVA10-infected gerbil model was a suitable tool for analyzing the pathogenesis of CVA10 and assessing the protective efficacy of CVA10 candidate vaccines.

Epidemic Trend and Molecular Evolution of HV Family in the Main Hantavirus Epidemic Areas From 2004 to 2016, in P.R. China.

Hemorrhagic fever with renal syndrome (HFRS) is caused by hantavirus (HV) infection, and is prevalent across Europe and Asia (mainly China). The genetic variation and wide host range of the HV family may lead to vaccine failure. In this study, we analyzed the gene sequences of HV isolated from different regions of China in order to trace the molecular evolution of HV and the epidemiological trends of HFRS. A total of 16,6975 HFRS cases and 1,689 HFRS-related deaths were reported from 2004 to 2016, with the average annual incidence rate of 0.9674 per 100,000, 0.0098 per 100,000 mortality rate, and case fatality rate 0.99%. The highest number of cases were detected in 2004 (25,041), and after decreasing to the lowest numbers (8,745) in 2009, showed an incline from 2010. The incidence of HFRS is the highest in spring and winter, and three times as many men are affected as women. In addition, farmers account for the largest proportion of all cases. The main hosts of HV are Rattus norvegicus and Apodemus agrarius, and the SEOV strain is mainly found in R. norvegicus and Niviventer confucianus. Phylogenetic analysis showed that at least 10 HTNV subtypes and 6 SEOV subtypes are endemic to China. We found that the clustering pattern of M genome segments was different from that of the S segments, indicating the possibility of gene recombination across HV strains. The recent increase in the incidence of HFRS may be related to climatic factors, such as temperature, relative humidity and hours of sunshine, as well as biological factors like rodent density, virus load in rodents and genetic variation. The scope of vaccine application should be continuously expanded, and surveillance measures and prevention and control strategies should be improved to reduce HFRS infection in China.

An adult gerbil model for evaluating potential coxsackievirus A16 vaccine candidates.

A suitable animal model of CVA16 infection is crucial in order to understand its pathogenesis and to help develop antiviral vaccines or screen therapeutic drugs. The neonatal mouse model has a short sensitivity period to CA16 infection, which is a major limitation. In this study, we demonstrate that adult (60-day-old) gerbils are susceptible to CVA16 infection at high doses (108.0 TCID50). A clinical isolate strain of CVA16 was inoculated intraperitoneally into adult gerbils, which subsequently developed significant clinical symptoms, including hind limb weakness, paralysis of one or both hind limbs, tremors, and eventual death from neurological disorders. Real-time RT-PCR revealed that viral loads in the spinal cord and brainstem were higher than those in other organs/tissues. Histopathological changes, such as neuronal degeneration, neuronal loss, and neuronophagia, were observed in the spinal cord, brainstem, and heart muscle, along with necrotizing myositis. Gerbils receiving both prime and boost immunizations of alum adjuvant inactivated vaccine exhibited no clinical signs of disease or mortality following challenge by CVA16, whereas 80% of control animals showed obvious clinical signs, including slowness, paralysis of one or both hind limbs, and eventual death, suggesting that the CVA16 vaccine can fully protect gerbils against CVA16 challenge. These results demonstrate that an adult gerbil model provides us with a useful tool for studying the pathogenesis and evaluating antiviral reagents of CVA16 infection. The development of this animal model would also be conducive to screening promising CVA16 vaccine candidates as well as further vaccination evaluation.

Global Gene Expression Analysis of the Brainstem in EV71- and CVA16-Infected Gerbils.

Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the two most important pathogens of hand, foot, and mouth disease (HFMD). However, the neuropathogenesis of EV71 and CVA16 has not been elucidated. In our previous study, we established gerbils as a useful model for both EV71 and CVA16 infection. In this work, we used RNA-seq technology to analyze the global gene expression of the brainstem of EV71- and CVA16-infected gerbils. We found that 3434 genes were upregulated while 916 genes were downregulated in EV71-infected gerbils. In CVA16-infected gerbils, 1039 genes were upregulated, and 299 genes were downregulated. We also found significant dysregulation of cytokines, such as IP-10 and CXCL9, in the brainstem of gerbils. The expression levels of 10 of the most upregulated genes were confirmed by real-time RT-PCR, and the upregulated tendency of most genes was in accordance with the differential gene expression (DGE) results. Our work provided global gene expression analysis of virus-infected gerbils and laid a solid foundation for elucidating the neuropathogenesis mechanisms of EV71 and CVA16.

Epidemiological Characterization of the 2017 Dengue Outbreak in Zhejiang, China and Molecular Characterization of the Viruses.

Dengue, a mosquito-borne disease caused by the dengue virus (DV), has been recognized as a global public health threat. In 2017, an unexpected dengue outbreak occurred in Zhejiang, China. To clarify and characterize the causative agent of this outbreak, data on dengue fever cases were collected from the China Information System for Disease Control and Prevention in Zhejiang province for subsequent epidemiological analysis. A total of 1,229 cases were reported, including 1,149 indigenous and 80 imported cases. Most indigenous cases (1,128 cases) were in Hangzhou. The epidemic peak occurred in late August and early September, and the incidence rate of elderly people (4.34 per 100,000) was relatively high. Imported cases were reported all year round, and most were from South-East Asia and Western Pacific regions. Young people and men accounted for a large fraction of the cases. Acute phase serums of patients were collected for virus isolation. And 35 isolates (including 25 DV-2, 8 DV-1, 1 DV-3, and 1 DV-4) were obtained after inoculation and culture in mosquito C6/36 cells. The E genes of the 35 new DV isolates and the complete genome of a DV-2 isolate (Zhejiang/HZ33/2017), and the E gene of a DV-2 isolate from Ae. albopictus (Zhejiang/Aedes-1/2017) were determined. Phylogenetic analyses were performed using the neighbor-joining method with the Tajima-Nei model. Phylogenetically, DVs of all four serotypes with multiple genotypes (mainly including 21 Cosmopolitan genotype DV-2, 4 Asian I genotype DV-2, 6 genotype I DV-1, and 2 genotype V DV-1) were present in the indigenous and imported cases in Zhejiang during the same period. Most of the isolates probably originated from South-East Asia and Western Pacific countries. The imported cases, high density of mosquito vector, and missed diagnosis might contribute to the 2017 outbreak in Zhejiang.

Risk Factors and Preventions of Breast Cancer.

Breast cancer is the second leading cause of cancer deaths among women. The development of breast cancer is a multi-step process involving multiple cell types, and its prevention remains challenging in the world. Early diagnosis of breast cancer is one of the best approaches to prevent this disease. In some developed countries, the 5-year relative survival rate of breast cancer patients is above 80% due to early prevention. In the recent decade, great progress has been made in the understanding of breast cancer as well as in the development of preventative methods. The pathogenesis and tumor drug-resistant mechanisms are revealed by discovering breast cancer stem cells, and many genes are found related to breast cancer. Currently, people have more drug options for the chemoprevention of breast cancer, while biological prevention has been recently developed to improve patients' quality of life. In this review, we will summarize key studies of pathogenesis, related genes, risk factors and preventative methods on breast cancer over the past years. These findings represent a small step in the long fight against breast cancer.