Lessons Learned from SARS-CoV and MERS-CoV: Preparation for SARS-CoV-2 induced COVID-19

Coronaviruses (CoVs) are the largest positive-sense RNA viruses with a wide range of natural hosts. To date, seven types of coronaviruses (HCoV-NL63; Human coronavirus NL63, HCoV-229E; Human coronavirus 229E, HCoV-OC43; Human coronavirus OC43, HCoV-HKU1; Human coronavirus HKU1, SARS-CoV; Severe acute respiratory syndrome-related coronavirus, MERS-Co; Middle East respiratory syndrome coronavirus, and SARS-CoV-2; Severe acute respiratory syndrome-related coronavirus) are known to cause disease in humans, and three of the CoVs (SARS-CoV, MERS-CoV, and SARS-CoV-2) cause severe, occasionally fatal, respiratory infections in humans. In November 2002, the case of severe acute respiratory syndrome (SARS), a new respiratory illness caused by SARS-CoV, was first reported in Guangdong Province, China. For the next several months, the SARS outbreak resulted in more than 8,000 cases of infection and 800 deaths. In June 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) was first identified in Saudi Arabia with 2,373 reported viral infections and 823 associated deaths until February 2019. The outbreak of the MERS-CoV pandemic also occurred in South Korea in May 2015. In late December 2019, another novel coronavirus called SARS-CoV-2, genetically linked to SARS-CoV, emerged in Wuhan, Hubei Province of China that has spread worldwide. Outbreaks of coronavirus-infections are occurring frequently in the 21st century; therefore, it seems very likely that another pandemic of coronavirus can emerge anytime in the future. In this review, we outlined the biological characteristics of coronaviruses and summarized the status of vaccine development against SARS-CoV-2, SARS-CoV, and MERS-CoV in preparation for the unpredictable emergence of coronavirus pandemic.

Evaluation of Multiplex Polymerase Chain Reaction Assay for the Simultaneous Detection of Sexually Transmitted Infections Using Swab Specimen

Sexually transmitted infections (STIs) are caused by the spread of pathogens via sexual activity and can cause serious complications if left untreated, regardless of their symptoms. Therefore, early diagnosis of STI is important, and molecular diagnostic methods for rapid detection and monitoring are needed. In this study, we evaluated a multiplex polymerase chain reaction (PCR) kit for simultaneously detecting 13 different bacterial, fungal, and viral microorganisms that cause STIs. The kit performance was evaluated for its sensitivity, lot-to-lot variation, and interference in detecting different pathogens. Additionally, its clinical usefulness was evaluated by estimating its sensitivity and specificity for clinical samples. The limit of detection (LOD) was 0.021–50.104 copies for each pathogen. In the tests of lot-to-lot, 100% of positive samples were detected at low concentrations and negative samples all showed negative results. This result confirms that there is no the variation of lot-to-lot. In the test for interference between pathogens, the efficiency of amplification for each pathogen was not significantly reduced and no nonspecific amplification product was formed. We tested 322 vaginal swab samples using the multiplex PCR kit and confirmed that its clinical sensitivity and specificity were 100% for all pathogens. This multiplex PCR kit can be used widely for rapid diagnosis and monitoring of STIs.

Diagnosis of Viral Infection Using Real-time Polymerase Chain Reaction

The laboratory-based diagnosis of viral infection has been evolving over the years, to increase objectivity, accuracy, and sensitivity via the continuous development of various technologies. Cell culture, which is one of the methods used for the diagnosis of viral infection, is a “gold-standard” approach; however, it is time consuming and is associated with a high risk of contamination. To overcome these shortcomings, molecular biology methods, such as conventional polymerase chain reaction (cPCR), real-time PCR, and sequencing, have been used recently for virus diagnosis. Realtime PCR has higher accuracy and sensitivity compared with cPCR. Moreover, realtime PCR can quantify viral nucleic acids by confirming the amplification using the threshold cycle, which is the initial amplification point. Real-time PCR applications for the detection of various types of viruses in clinical settings should be based on the use of appropriate samples, nucleic acid extraction according to virus characteristics, and selection of diagnostic methods using sensitivity and specificity targets. In addition, the implementation of real-time PCR requires to evaluate the performance of the test protocol by measuring sensitivity, specificity, accuracy, and reproducibility. The verified real-time PCR method is an easy, fast, and accurate method for monitoring the diagnosis and treatment outcomes in a clinical setting. In this review, we summarize the characteristics of the typical diagnostic methods for viral infection, especially of the advanced real-time PCR method, to detect human pathogenic viruses.

Evaluation of EZplex MTBC/NTM Real-Time PCR kit: diagnostic accuracy and efficacy in vaccination

PURPOSE: Tuberculosis (TB) is mainly caused by Mycobacterium tuberculosis, which is a pathogenic mycobacterial species grouped under Mycobacterium tuberculosis complex (MTBC) with four other pathogenic mycobacterial species. The mycobacteria not included in MTBC are known as nontuberculous mycobacteria (NTM), and cause several pulmonary diseases including pneumonia. Currently, NTM occurrences in TB-suspected respiratory specimens have increased, due to which, precise detection of MTBC and NTM is considered critical for the diagnosis and vaccination of TB. Among the various methods available, real-time PCR is frequently adopted for MTBC/NTM detection due to its rapidness, accuracy, and ease of handling. In this study, we evaluated a new real-time PCR kit for analytical and clinical performance on sputum, bronchial washing, and culture specimens. MATERIALS AND METHODS: For assessing its analytical performance, limit of detection (LOD), reactivity, and repeatability test were performed using DNA samples. To evaluate clinical performance, 612 samples were collected and clinically tested at a tertiary hospital. RESULTS: LOD was confirmed as 0.584 copies/µL for MTBC and 47.836 copies/µL for NTM by probit analysis (95% positive). For the reactivity test, all intended strains were detected and, in the repeatability test, stable and steady results were confirmed with coefficient of variation ranging from 0.36 to 1.59. For the clinical test, sensitivity and specificity were 98.6%–100% and 98.8%–100% for MTBC and NTM, respectively. CONCLUSION: The results proved the usefulness of the kit in TB diagnosis. Furthermore, it could be adopted for the assessment of vaccine efficacy.

Human Rhinoviruses: the Forgotten but Still Important Viruses

Human rhinoviruses (HRVs) are responsible for many of the characteristic symptoms of the common cold, such as a sore throat, runny nose, nasal congestion, sneezing, and coughing. However, despite the high detection rate in children, most HRV infections are asymptomatic. As a result, these viruses are generally ignored, even though a close association between HRV infections in early life and the subsequent induction of asthma has been reported. Therefore, it is necessary to conduct further research into HRV diagnostics, treatments, epidemiology, and vaccines. This review describes recent studies of HRVs, including their genomic diversity, surveillance systems, taxonomy, and immune responses, as well as vaccines.

Human Rhinoviruses: the Forgotten but Still Important Viruses

Human rhinoviruses (HRVs) are responsible for many of the characteristic symptoms of the common cold, such as a sore throat, runny nose, nasal congestion, sneezing, and coughing. However, despite the high detection rate in children, most HRV infections are asymptomatic. As a result, these viruses are generally ignored, even though a close association between HRV infections in early life and the subsequent induction of asthma has been reported. Therefore, it is necessary to conduct further research into HRV diagnostics, treatments, epidemiology, and vaccines. This review describes recent studies of HRVs, including their genomic diversity, surveillance systems, taxonomy, and immune responses, as well as vaccines.

The Characteristics of RNA Vaccine; its Strengths and Weaknesses

Since 1990 when Wolff and co-authors proved that both RNA and DNA expression vectors containing interest gene were directly injected into mouse muscle and expressed the protein in vivo, the concept of gene vaccine has been broadly tested in the vaccine field. However, due to the limitations of technology and the misconception about RNA, most previous studies have focused on the DNA vaccine. Recently, the RNA vaccine has emerged as a new game-changing disruptive innovation technology in the vaccine field. This review has covered the characteristics of the RNA vaccine, including its strengths and weaknesses. Finally, we have suggested future directions for the RNA vaccine.

Cardiovascular Screening in Asymptomatic Adolescents with Metabolic Syndrome

BACKGROUND: In recent days, the prevalence of childhood metabolic syndrome (MS) has increased substantially due to the increasing rate of childhood obesity on a global scale. The aims of this study were to detect the important parameters and provide the screening system to prevent cardiovascular disease in adolescents with MS. METHODS: Ninety one male adolescents were divided into two groups based on the presence or absence of MS. Anthropometric measurement and laboratory study were studied. Intimal medial thickness and pulse wave velocity were estimated. Left ventricular mass index (LVMI), ejection fraction, myocardial velocity, strain and strain rate were measured by tissue Doppler imaging and strain rate imaging. RESULTS: The prevalence of MS was 7.7%. Weight, body mass index (BMI), waist circumference (WC), glucose, insulin, homeostasis model assessment of insulin resistance, triglyceride and LVMI were significantly increased in the MS group. High density lipoprotein-cholesterol (HDL-C), peak early diastolic myocardial velocity (e'), systolic myocardial velocity (s') and global longitudinal strain were significantly lower in the MS group. In univariant analysis, LVMI was significantly correlated with BMI, WC, fat %, fat mass, systolic blood pressure, alanine aminotransferase, total cholesterol (TC) and low density lipoprotein-cholesterol. e' was significantly correlated with BMI, fat %, fat mass, and HDL-C. Global circumferential strain had significant correlation with glucose and TC. Basal anterolateral strain rate was significantly correlated with weight, BMI, WC, fat %, and fat mass. CONCLUSION: LVMI, strain and strain rate are practical and accurate parameters for assessment of left ventricular function in adolescents with MS.

Influence of the Host Factors on Human Papillomavirus Infection and Vaccine Efficacy

Human papillomavirus (HPV) is associated with cervical cell changes, genital warts, recurrent respiratory papillomatosis, laryngeal papillomatosis, head and neck cancer, and cervical cancer. Two commercial HPV vaccines have successfully been made available in the clinical field. This review covers the progress of cervical disease by understanding the nature of HPV infection, as well as the relationship between the host factors and HPV vaccine effectiveness. Among these host factors, microbiota has been revealed to influence the development and function of both the innate and adaptive immune systems. Therefore, the composition of the microbiome may ultimately affect vaccine efficacy. Understating the relationship between host factors and HPV infection/vaccine efficacy may prove to be useful in earlier diagnosis, as well as disease prophylaxis.

Associations of matrix metalloproteinase (MMP)-8, MMP-9, and their inhibitor, tissue inhibitor of metalloproteinase-1, with obesity-related biomarkers in apparently healthy adolescent boys / 소아과

PURPOSE: Matrix metalloproteinases (MMPs) have been implicated in atherosclerosis, and therefore, are considered risk factors for metabolic dysfunction in adults. However, there is little data on circulating levels of MMPs and tissue inhibitors of MMPs (TIMPs) with regard to obesity-related biomarkers in the general adolescent population. In the present study, we determined the associations of MMP-8, MMP-9, and TIMP-1 levels and MMP-8/TIMP-1 and MMP-9/TIMP-1 ratios with obesity-related biomarkers in apparently healthy adolescent boys. METHODS: We measured MMP and TIMP concentrations in plasma samples using the enzyme-linked immunosorbent assay and analyzed their associations with obesity-related biomarkers, such as liver enzymes and lipid profiles, in a sample of 91 Korean boys aged 13-14 years who participated in a general health check-up. RESULTS: The mean age of the boys was 13.8+/-0.3 years; 72 boys were normal weight and 19 were overweight/obese. The Pearson correlation coefficients revealed a significant correlation between MMP-8 and aspartate aminotransferase (r=0.217, P=0.039) and alanine aminotransferase (r=0.250, P=0.017) and between TIMP-1 and aspartate aminotransferase (r=0.267, P=0.011). In a multivariate linear regression analysis, serum alanine aminotransferase was positively associated with the MMP-8 level. There were no significant differences in the MMP-8, MMP-9, and TIMP-1 levels or MMP-8/TIMP-1 and MMP-9/TIMP-1 ratios between control and overweight/obese subjects. CONCLUSION: We found a significant association between the MMP-8 level and alanine aminotransferase in the apparently healthy adolescent boys. These findings indicate that there may be a pathophysiological mechanism underlying the relationship between MMP-8 and liver enzymes in young adolescents.

Is Obesity One of Physiological Factors which Exert Influenza Virus-induced Pathology and Vaccine Efficacy?

Obesity has been considered a risk factor for infectious diseases including the influenza virus. Most epidemiological investigations indicated that obesity is connected to the severity of influenza, although there are some exceptions. Many studies using obese humans and animal models showed that immune response was impaired in the obese group, increasing susceptibility and severity of influenza virus. However, the exact mechanism by which obesity inhibits anti-viral immune response remains unknown. This review discusses current studies about the properties of immune cells in obesity. In obesity, the balance of adipokines is disrupted and the level of proinflammatory cytokine is increased compared with non-obese control. Moreover, macrophages induced systemic inflammation by secreting cytokines such as TNF-alpha and IL-6, antigen presenting capacity of dendritic cells was diminished which affect T cell responses, and influenza-specific antibody production seems reduced and decreased even faster after vaccination in obese mouse. The number of circulating T cells and proliferation of mitogen-stimulated T cells dropped and T cell memory was significantly low in influenza infected obese mouse. Therefore, obesity may be one of factors for disease progression in influenza virus infection and vaccine efficacy.

Prophylactic and therapeutic vaccines for obesity

Chronic diseases such as obesity and diabetes are major causes of death and disability throughout the world. Many causes are known to trigger these chronic diseases, and infectious agents such as viruses are also pathological factors. In particular, it is considered that adenovirus 36 infections may be associated with obesity. If this is the case, a vaccine against adenovirus 36 may be a form of prophylaxis to combat obesity. Other types of therapeutic vaccines to combat obesity are also being developed. Recently, hormones such as glucagon-like peptide-1, ghrelin, and peptide YY have been studied as treatments to prevent obesity. This review describes the ongoing development of therapeutic vaccines to treat obesity, and the possibility of using inactivated adenovirus 36 as a vaccine and an anti-obesity agent.

Novel Role of Invariant Natural Killer T-cell in Glycemic Control: Regulation by human Adenovirus 36

Obesity is associated with a state of chronic low-grade inflammation. This abnormal inflammation state may cause metabolic dysfunction. Many studies have supported the claim that immune cells such as adipose tissue macrophage and invariant natural killer T-cells (iNKT) are related to the development of metabolic diseases like diabetes. It has recently been reported that while human adenovirus 36 (Ad36) infection is associated with human obesity, it also helps to improve the serum level of lipid factors (glycemic control). However, the detailed cellular mechanism remains unclear. This study (The Journal of Clinical Investigation, 2012;122:3343-54) showed that iNKT cell-deficient mice on a low-fat diet used as a control for high-fat diet boasted insulin resistance phenotype without adipose tissue inflammation. The results of this study offer insight into the possibility of a novel role for iNKT related to the improvement of metabolic diseases, especially insulin resistance, and hint that Ad36-induced inflammation may be associated with iNKT in adipose tissue, while also playing a role in the improvement of glycemic control.

Regulation of Obesity and Non-alcoholic Fatty Liver Diseases by Modulation of the Gut Microbiota Through Inflammasome; its Mechanism and Potential for Clinical Use

The revelation that gut microbes are associated with the pathogenesis of human diseases such as obesity, colon cancer, inflammatory bowel disease and liver-related diseases has resulted in the role of gut microbes becoming a novel research topic in basic and clinical science. Recently, emphasis has been placed on the role of gut microbes in non-alcoholic fatty liver disease (NAFLD) and its progressive form, non-alcoholic steatohepatitis (NASH). Researchers have suggested that inflammasome deficiency-changed dysbiosis is associated with exacerbating NAFLD/NASH progression. This particular study also showed a direct 'gut-liver axis' regulated by modulation of gut microbiota. This paper (Nature 2012;482: 179-185) was summarized herein and the potential clinical applications were discussed.

Infectobesity: a New Area for Microbiological and Virological Research

Obesity is connected with numerous diseases, such as type 2 diabetes, atherosclerosis, cancer, and nervous system dysfunctions. Obesity is affected by genetic, environmental, and cultural factors. However, numerous studies indicate that several pathogens might cause obesity. This review discusses recent data and the characteristics of pathogens that are implicated in obesity. In particular, human adenovirus 36 (Ad36) is the most clearly implicated virus in human obesity. It was recently shown that obese groups from the USA, Korea, and Italy have a higher prevalence of serum antibodies against Ad36. The mechanisms of Ad36-induced obesity remain unclear. However, glucose uptake and inflammation are possible mechanisms of Ad36-induced obesity. Overall, this new understanding of causes of obesity has developed into the concept of 'infectobesity' and the possibility of developing a 'vaccine' or 'therapeutic agents' for obesity.

Development of a Gene Therapy Method for Cervical Cancer Using Attenuated Coxsackievirus B3 as a Vector System

We previously reported the development of an attenuated coxsackievirus B3, known as YYFF, which functioned as a viral vector system for foreign gene expression. In this study, we demonstrated the potential use of YYFF as a gene therapy vector. Recombinant YYFF was constructed to express the human papillomavirus 16 (HPV16) E7 gene, referred to as YYFF-HPV16-E7. Growth of YYFF-HPV16-E7 resembled the wild type, YYFF, and it expressed HPV16-E7 in cell culture. When YYFF-HPV16-E7 was directly injected into TC-1-transplanted C57/BL6 mice, there was no reduction in tumor size, because of the non-growth of YYFF in C57/BL6 mice. However, when YYFF-HPV16-E7-induced immune cells/serum that originated from BALB/c mice was passively delivered into BALB/c background TC-1-transplanted nude mice, it reduced the size of cervical tumors in the nude mice. This study indicates the potential use of YYFF-HPV16-E7 as a gene therapy agent for treating HPV-induced cervical cancer.

Regulation of Innate Immunity via MHC Class II-mediated Signaling; Non-classical Role of MHC Class II in Innate Immunity

MHC class II has long been known to play a classical role in antigen presentation and to act as a signal transducer capable of inducing the adaptive immunity needed to produce pathogen specific antibodies. However, it has recently been revealed that MHC class II can also promote the activation of Toll-like receptor mediated signaling by functioning as an adapter. This means that in addition to its classical function of adaptive immunity, MHC class II also plays an intriguing role in the mechanisms that regulate innate immunity. That being the case, queries inevitably arise regarding the fact that many pathogens have tried to control the induction of MHC class II so as to escape the host immune response. Liu et al (Nat Immunol 2011;12:416-424) demonstrated that intracellular MHC class II interacted with Btk, and that this activated Btk promoted TLR signaling via Myd88 and TRIF. The results of this study provide insight regarding the possibility of a novel role for MHC class II, which was heretofore regarded solely as a classical molecule involved in adaptive immune responses, as a regulator of innate immune responses.

Development of Neutralization Assay using Murine Leukemia Virus (MuLV) Pseudotyped with Japanese encephalitis Virus (JEV) env Gene

The envelope (E) glycoprotein of JEV is the major antigen to elicit neutralizing antibody (NAb) against JEV infection. In order to develop a rapid and safe neutralization assay system for evaluation of the JEV vaccine strains, we constructed JEV-pseudotyped viruses with JEV env genes (Nakayama-NIH, Beijing-1). The titers of JEV-pseudotyped viruses with NK and BJ strains were 4.0x10(4) IFU/ml and 1.3x10(5) IFU/ml in Vero cell cultures, respectively. We have analyzed the neutralization activity of immunized mouse sera with JEV-NK and JEV-BJ pseudotyped viruses. The neutralizing antibody titers of NK and BJ (50% reduction of virus) were about 1:10,000 at each immunized sera. Compared with conventional plaque reduction neutralization test (PRNT), the method using JEV-pseudotyped virus has desirable advantages such as more rapid, easier, and non-biohazardous. This neutralization assay system might be useful to evaluate NAb activity against JEV vaccine strains or vaccine candidates.

Neutralizing Antibody Induction and Cytotoxic T Lymphocyte Response to Nakayama-NIH and Beijing-1 as Japanese Encephalitis Virus Vaccine Strains

The Japanese encephalitis virus (JEV), a member of the Flaviviridae family and Flavivirus genus, is transmitted by mosquitoes. JEV, of which some 35,000 cases are recorded every year, is a positive RNA virus. Two types of JEV vaccines have been developed to prevent the onset of encephalitis in humans, namely formalin-inactivated and liveattenuated vaccines. JEV inactivated vaccines are usually made using the Nakayama-NIH or Beijing-1 strains of the JEV virus. In this study, the immunological response to the Nakayama-NIH and Beijing-1 strains was analyzed as part of the effort to compile basic data which could lead to the selection of a suitable vaccine strain. To this end, the virus titer of Beijing-1 was found to be two-fold higher than that of Nakayama-NIH by plaque assay. Moreover, Beijing-1-induced neutralizing antibodies showed a higher level of titers when confronted by Korean JEV isolates than Nakayama-NIH-induced neutralizing antibodies (1:320 vs. 1:160, respectively). However, as a minimum ratio of 1:10 neutralizing antibody titers are required to protect against JEV infection, both strains in effect exhibited a sufficient level of neutralizing antibody titers. What's more, Beijing-1 was found to induce a somewhat higher cytotoxic T lymphocyte (CTL) response than Nakayama-NIH. Taken together, this can be taken to mean that Beijing-1 may in fact be a more effective vaccine candidate strain when it comes to inducing a high level of protective immunity against JEV infection.

Host Gene Profiling of Coxsackievirus B3 H3- and 10A1-infected Mouse Heart

Coxsackievirus B3 (CVB3) is a non-enveloped virus that has a single-stranded RNA genome. CVB3 induces myocarditis, and ultimately, dilated cardiomyopathy. A myocarditis variant of CVB3 (CVB3 H3) and its antibody-escape mutant (CVB3 10A1) were studied previously; H3 was found to induce myocarditis and 10A1 was found to be attenuated in infected mice. Although amino acid residue 165, located in a puff region of VP2, was found to be altered (i.e., the H3 asparagine was altered to aspartate in 10A1), the detailed mechanism of attenuation was not clearly elucidated. Here, DNA microarray technology was used to monitor changes in mRNA levels of infected mouse hearts after CVB3 H3 and 10A1 infection. This tool was used to elucidate the pathogenic mechanisms of viral infection by understanding virus-host interactions. We identified several genes, including protein tyrosine kinases, Ddr2 and Ptk2, as well as Clqb and Crry, involved in complement reactions, which may be involved in these viral processes. Thus, gene profiling can provide an opportunity to understand host immune responses to viral infection for gene therapy and may contribute to the identification of the target gene that is modified during treatment of viral myocarditis.