25 April 2021, Volume 16 Issue 2

    

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Original Article
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  Construction and validation of human norovirus infectious clones

  CHEN Shuiye1, SONG Wuhui1，YI Zhigang1，2

  JOURNAL OF MICROBES AND INFECTIONS. 2021, 16(2): 71-78. https://doi.org/10.3969/j.isn.1673-6184.2021.02.001

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  The lack of a cultivation system for human noroviruses (HuNoV) is a major barrier to understand virus biology and the development of effective antiviral strategies. The study aims to verify the reported replication of the reverse genetics system of HuNoV G Ⅱ. 3 U201, and further to seek a better sequence from the HuNoV clinical strains to construct a highly efficient infectious clone. First the sequences of HuNoV GⅡ.3 U201 genome with T7 promoter and EF­1α promoter were synthesized, and inserted into the vector, named pU201 and pEF­1α­U201, respectively. Meanwhile, negative control plasmids with viral RNA polymerase activity inactivated were constructed. To verify whether these clones could replicate, T7 polymerase was co­transfected with pU201 and pEF­1α­U201 was transfected alone in COS 7 cells and Huh 7 cells, respectively, and HuNoV RNA level was determined by real­time quantitative polymerase chain reaction (RT­qPCR) in different time post transfection. The results showed that the HuNoV RNA levels of pU201 and pEF­1α­U201 were nearly two times and three times higher than those of the negative control group respectively. To facilitate the detection of HuNoV replication, full­length infectious clones with NanoLuc™ luciferase (Nluc) reporter gene were constructed by inserting Nluc sequence in front of viral genome, named pU201­Nluc and pEF­1α­U201­Nluc. COS 7 cells and Huh 7 cells were transfected and Nluc activity was determined in different time post transfection. The results showed that the Nluc activity of pEF­1α­U201­Nluc was nearly 2 times higher than those of the negative control group and the inhibitor­treated group, but there was no significant increase in pU201­Nluc. These results suggested that EF­1α promoter was superior to T7 promoter in initiating HuNoV GⅡ.3 U201 replication. Finally, to seek a better HuNoV sequence, a full­length infectious clone was constructed with a clinical isolate of HuNoV GⅡ.4 genome from a Taiwan patient and EF­1α promoter, named PCTc. COS 7 cells and Huh 7 cells were transfected and HuNoV RNA level was determined in supernatant and cells by RT­qPCR. And the viral RNA level between PCTc and control groups had no significant difference. The results suggested that full­length infectious clones and subgenomic clone of HuNoV GⅡ.3 U201 could only achieve limited and unsustainable replication in cells, and replication capacity may also be associated with different sequences of virus strains.
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  Establishment of a recombinant hepatitis B virus replicon expressing a split green fluorescent protein

  TIAN Qingyou, ZHU Yuanfei, CHANG Hao, YU Lin, DENG Qiang

  JOURNAL OF MICROBES AND INFECTIONS. 2021, 16(2): 79-87. https://doi.org/10.3969/j.issn.1673-6184.2021.02.002

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  In the present study, we developed a recombinant hepatitis B virus (rHBV) replicon in order to visualize virus-infected living cells via reporter gene expression. We first constructed an HBV replicon vector HBV1.1-ΔHBc113, with partial deletion of the sequence encoding HBV core (HBc). The vector demonstrated a competent viral DNA replication in transfected cells, only if HBc was provided in trans. However, the replication efficiency of ΔHBc113 vector was greatly impaired by insertion of a large foreign DNA fragment. Taking advantage of the property of intein-mediated protein splicing, we chose enhanced-green fluorescent protein (EGFP) and super folder GFP (sfGFP) as exteins, and developed EGFP^N1-8/EGFP^C9-11 and sfGFP^N1-10/sfGFP^C11 split system, respectively. We further constructed EGFP^C9-11 and sfGFP^C11 rHBV replicons based on the ΔHBc113 vector, the latter of which was proved to be competent for HBc-rescued, functional intracellular replication, and produced progeny virions in the culture medium. In cells co-expressing EGFP^N or sfGFP^N, rHBV replicon-expressed EGFP^C9-11 or sfGFP^C11 was capable of working with their counterparts, respectively, forming intact and functional GFP through intein-mediated protein splicing. We thus successfully developed a fluorescent rHBV replicon system, which can be not only used for HBV infection mechanism study, but also have an extensive application prospect for high-throughput antiviral screening.
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  Drug resistance of Helicobacter pylori among children in Shanghai from 2019 to 2020

  LI Chunling, HE Leiyan, FU Pan, CHEN Saige, WANG Aimin, ZHANG Lei, WANG Chuanqing

  JOURNAL OF MICROBES AND INFECTIONS. 2021, 16(2): 88-93. https://doi.org/10.3969/j.issn.1673-6184.2021.02.003

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  The present paper aims to analyze the resistance of Helicobacter pylori (H. pylori) infected by children in Shanghai from 2019 to 2020 to four commonly used antibacterial drugs. From January 2019 to October 2020, 1 605 children who came to Children’s Hospital of Fudan University in Shanghai and received gastroscopy and rapid urease test were selected. Gastric mucosal membrane samples were collected for H. pylori isolation, culture and identification. In vitro drug susceptibility test by E-test method was used to analyze H. pylori resistance in children in different age groups. The results showed that the overall positive rate of H. pylori of the enrolled children was 31.5% (506/1 605), and there was no difference in gender and age. The resistance rates of clinical H. pylori isolates to metronidazole, clarithromycin, levofloxacin, and amoxicillin were 42.3%, 25.1%, 8.1%, and 3.0%, respectively. Except that the resistance rate of H. pylori to clarithromycin in children aged 13-16 years (31.2%) was significantly higher than that of children aged 1-3 years (13.3%) (P＝0.040), there was no difference in age and gender. In addition, the double resistance rate of H. pylori to clarithromycin＋metronidazole was 18.0%, which was significantly higher than that to clarithromycin＋levofloxacin (5.1%), metronidazole＋levofloxacin (4.9%), metronidazole＋amoxicillin (1.4%) and amoxicillin＋levofloxacin (0.8%) (χ2＝172.706, P<0.01). Multi-drug resistance analysis showed that the resistance rate of H. pylori to levofloxacin＋clarithromycin＋metronidazole was 3.0%, which was significantly higher than that to levofloxacin＋amoxicillin＋clarithromycin (0.6%), amoxicillin＋metronidazole＋levofloxacin (0.6%) (χ2＝13.907, P＝0.01). It is suggested that H. pylori isolated from children in Shanghai from 2019 to 2020 has a higher resistance rate to metronidazole and clarithromycin, a lower resistance rate to amoxicillin and levofloxacin, and a higher dual resistance rate to metronidazole and clarithromycin.
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  Study on the expressive characteristic of interferon stimulated gene 15 mRNA from peripheral blood of patients with viral pneumonia

  WEI Dong1, CHEN Yongyan1, SHI Guochao2, ZHANG Xinxin1, WANG Ying3

  JOURNAL OF MICROBES AND INFECTIONS. 2021, 16(2): 94-102. https://doi.org/10.3969/j.issn.1673-6184.2021.02.004

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  This study analyzed the peripheral blood interferon stimulated gene 15(ISG15 mRNA) expression levels in viral pneumonia and bacterial pneumonia patients and discussed the possible relationship between ISG15 and the pathogenesis of viral pneumonia, providing further clinical evidences for host markers of specific respiratory virus infections. In this study, Peripheral blood samples were collected from 157 community acquired pneumonia (CAP) hospitalized patients and 40 healthy people (healthy controls) as well as their clinical data. The patients include 133 with viral respiratory tract infection and 24 patients with bacterial infection. The ISG15 mRNA expression levels for all samples were analyzed by real-time reverse transcription polymerase chain reaction(real-time RT-PCR). We validated the ISG15 mRNA expression by stimulating the peripheral blood mononuclear cell(PBMC) in vitro and analyzing the correlation between viral load and ISG15 mRNA levels. The expression level of ISG15 mRNA in the viral respiratory infection group was significantly higher than that in the healthy control group and the bacterial infection group. Receiver operating characteristic (ROC) curves analysis showed that the ISG15 mRNA in peripheral blood had a potential diagnostic value to distinguish viral respiratory infection and bacterial infection in adult CAP patients. In vitro studies also showed that there was a positive correlation between ISG15 mRNA expression in PBMC and viral replication. The peripheral blood ISG15 mRNA expression level was highly correlated with viral respiratory infection by analyzing the clinical data and in vitro experiments. This study demonstrated that ISG15 mRNA in peripheral blood may have an important clinical value in diagnosis of viral pneumonia.
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  Isolation and drug resistance of Mycobacterium from clinical specimens of acquired immunodeficiency syndrome patients in Guangxi

  MENG Zhihao1, WEI Yongzhong1, LIAO Guangfu1, LU Kangyan1, LU Xueping1, ZUO Yong1, LAN Ke1, TANG Yuelu1, SHEN Yinzhong2

  JOURNAL OF MICROBES AND INFECTIONS. 2021, 16(2): 103-109. https://doi.org/10.3969/j.issn.1673-6184.2021.02.005

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  To investigate the positive rate, species and drug resistance of Mycobacteria in clinical specimens of acquired immunodeficiency syndrome (AIDS) patients, the AIDS patients in a hospital in Guangxi during January 2010 and December 2019 were collected and their clinical specimens were cultured with acid-fast bacilli. The antimicrobial susceptibility testing was conducted with more than eight anti-tuberculosis drugs after isolation and identification. The overall positive rate was 15.68% (2 163/13 795). The positive rate was high in pus, secretions, various tissue specimens, pleural effusion, and ascites. The species identification results showed that Mycobacteria tuberculosis compound group accounted for 77.95% (1 442/1 850) and non-tuberculosis Mycobacterium (NTM) accounted for 22.05% (408/1 850). The latter accounted for 15.71%-26.07% over 10 years, with no statistically significant difference between years (χ2＝10.442, P>0.05). The overall drug resistance rate was 23.30% (336/1 442), with a significant difference over a 10-year period (χ2＝18.901, P＝0.026). The rates of resistance to isoniazid, rifampicin, streptomycin, ofloxacin, ethambutol, p-amino salicylic acid, kanamycin were 12.14% (175/1 442), 10.54% (152/336), 9.29% (134/1 442), 5.62% (81/1 442), 3.05% (44/1 442), 1.80% (26/1 442), 1.32% (19/1 442), respectively. The prevalence of multidrug-resistant tuberculosis (DR-TB) and extensively drug-resistant tuberculosis (XDR-TB) was 5.48% (79/1 442) and 0.28% (4/1 442), respectively. It is confirmed that the positive rate of Mycobacteria isolated from the clinical specimens of AIDS patients is high and NTM accounts for a high proportion. The drug resistance rate of Mycobacterium tuberculosis to commonly used anti-tuberculosis drugs is high. It is suggested that clinical Mycobacterium identification and antimicrobial susceptibility testing should be carried out to provide a reliable basis for clinical diagnosis and treatment.
Review
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  Research progress on Elizabethkingia meningoseptica and infection

  LU Yikai1, YUAN Shuying1, YANG Yuanbo1, WU Qile1, FANG Zixuan1, LIU Yan1, CHEN Li2, SUN Guiqin1

  JOURNAL OF MICROBES AND INFECTIONS. 2021, 16(2): 110-116. https://doi.org/10.3969/j.issn.1673-6184.2021.02.006

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  Elizabethkingia meningoseptica (EM), a non-fermented Gram-negative bacterium, is one of the most important pathogens of nosocomial infection. The detection rate has been on the rise in recent years. The susceptible people mainly include the patients with basic diseases, immunodeficiency or deficiency, long-term use of antibiotics, invasive procedures, and so on. EM could cause meningitis, bacteremia, lung infections, eye infections, etc. Its multi-drug resistance has burdened the clinical treatment. Animals could also be infected by EM, like frogs, fish, turtles, pigs, dogs and others. The biological characteristics, infection types, detection methods and risk factors are analyzed in the present paper, which would offer a new insight of EM. Hospitals should focus on the detection, drug resistance and pathogenesis of EM, which would provide the reference for prevention and control of nosocomial infection.
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  Application progress of metagenomics technology based next-generation sequencing platform in the diagnosis of infectious respiratory diseases pathogens

  WANG Huilin, LIU Xue

  JOURNAL OF MICROBES AND INFECTIONS. 2021, 16(2): 117-122. https://doi.org/10.3969/j.isn.1673-6184.2021.02.007

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  The incidence of respiratory infections is high, and early identification of infectious pathogens is the key to improving clinical results. Metagenomics next-generation sequencing (mNGS) has the advantages of being whole spectrum, time saving, no specific amplification of pathogens, and has high clinical application value in the identification of rare infections, mixed infections, immunosuppressive patient infection and the diagnosis of pathogens that are difficult to be detected by conventional methods. However, mNGS results of some cases have low specificity, lack recognized interpretation standards, have unclear relationships between sequencing results and treatment, and have difficulties testing drug-resistant genes. In clinical application present, mNGS and traditional microbial detection technology have a complementary effect and the combination of the two can improve the clinical diagnosis efficiency.
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  Research progress in mechanism of Fusobacterium nucleatum promoting colorectal cancer

  CHEN Zixian, DUN Yiting, LIU Chang, CHEN Qian

  JOURNAL OF MICROBES AND INFECTIONS. 2021, 16(2): 123-128. https://doi.org/10.3969/j.issn.1673-6184.2021.02.008

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  Fusobacterium nucleatum, a commensal bacterium in human oral cavity could cause opportunistic infections in certain conditions. Recently, the relationship between Fusobacterium nucleatum and colorectal cancer (CRC) has become a research focus. Clinical studies revealed that Fusobacterium nucleatum is implicated in CRC. Further studies on animals have proved that Fusobacterium nucleatum can promote the onset and progression of CRC and chemoresistance to treatments. This paper summarizes evidence for the association of Fusobacterium nucleatum with CRC and provides a comprehensive view of current mechanisms for the association.
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  No-antibiotic therapies of Pseudomonas aeruginosa infection

  TANG Xiaoli1, FENG Yi2, ZHUO Shaoyuan2, LE Ning2, SHANG Liguo2

  JOURNAL OF MICROBES AND INFECTIONS. 2021, 16(2): 129-136. https://doi.org/10.3969/j.issn.1673-6184.2021.02.009

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  Pseudomonas aeruginosa (P. aeruginosa) is a ubiquitous human pathogen causing different infections. Antibiotic therapy is the first clinical option for the infections caused by P. aeruginosa. Unfortunately, empiric antibiotic therapy may not work due to drug resistance. Non-antibitoc therapies for P. aeruginosa infections are getting extensive attention and promising research is being conducted. This review will focus on the progresses of non-antibitoc therapies including antigen-antibody immunotherapy, phage therapy and anti-virulence therapy. The problems of these new therapies in the clinical treatment and the possible strategies to overcome them are also discussed.
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  Research progress on vertical transmission of Chikungunya virus

  LIU Libo1, FAN Dongying1, AN Jing1, ZHOU Hongning2, WANG Peigang1

  JOURNAL OF MICROBES AND INFECTIONS. 2021, 16(2): 137-144. https://doi.org/10.3969/j.issn.1673-6184.2021.02.010

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  Chikungunya virus (CHIKV) is a mosquito-borne virus transmitted by Aedes aegypti and Aedes albopictus and belongs to the Alphavirus genus of the Togaviridae family. Since 2005, several high infection rate epidemics of CHIKV have been reported in Africa, Asia, and the Americas. CHIKV infection causes fever and arthritis which usually lasts for months or even years, resulting in a big burden to patients and society. Besides mosquito-borne transmission, vertical transmission from pregnant women infected with CHIKV during pregnancy to fetus or neonatal were occasionally reported, which may lead to severe neurological sequelae of the newborn. Here, we will summarize the vertical transmission cases of CHIKV reported so far, review the relevant clinical manifestations, propose underlying modes and mechanisms, analyze the advantages and disadvantages of the present animal model used in the field, and discuss the diagnosis, prevention and treatment of this disease.