Establishment and application of a rapid assay for GII.4/GII.17 NoV detection based on the combination of CRISPR/Cas13a and isothermal amplification.

Introduction: Norovirus (NoV) is one of the most important agents responsible for viral acute gastroenteritis, among which GII.4 NoV is the predominant strain worldwide, and GII.17 NoV surpassed GII.4 in some epidemic seasons. Rapid and accurate gene recognition is essential for a timely response to NoV outbreaks. Methods: In the present study, the highly conserved regions of GII.4 and GII.17 NoVs were identified in the junction of open reading frame (ORF) 1 and ORF2 and then amplified by isothermal recombinase-aided amplification (RAA), followed by the cleavage of CRISPR-Cas13a with screened CRISPR RNAs (crRNAs) and RAA primers. The entire detection procedure could be completed within 40 min using a thermostat, and the results could be read out by the naked eye under a portable blue light transilluminator. Discussion: The assay showed a high sensitivity of 97.96% and a high specificity of 100.0%. It offered a low limit of detection (LOD) of 2.5×100 copies/reaction and a coincidence rate of 96.75% in 71 clinical fecal samples. Overall, rapid and inexpensive detection of GII.4/GII.17 NoVs was established, which makes it possible to be used in areas with limited resources, particularly in low-income countries. Furthermore, it will contribute to assessing transmission risks and implementing control measures for GII.4/GII.17 NoVs, making healthcare more accessible worldwide.

Development and evaluation of a new luciferase immunosorbent assay to detect GII.6 norovirus-specific IgG in different domestic and wild animals.

Noroviruses (NoVs) are the leading viral pathogens globally causing acute gastroenteritis (AGE) in humans, posing a significant global health threat and economic burden. Recent investigations revealed that human NoVs had been detected in different animals, which raises concerns about whether NoVs are potential zoonotic diseases. This study developed a novel luciferase immunosorbent assay (LISA) to detect GII.6 NoV IgG based on P protein of VP1. The LISA showed high specificity (99.20%) and sensitivity (92.00%) with 4-16 times more sensitivity compared with an ELISA. NoV-LISA was reproducible with human serum regarding the inter- and intra-assay coefficient of variance values. Potential cross-reactivity was also evaluated using mice serum immunized by other antigens, which showed that NoV-LISA could differentiate GII.6 NoV from rotavirus and various genotypes of NoV. Specific GII.6 NoV IgG was widely detected in different domestic and wild animals, including dogs, pigs, bats, rats, and home shrews, with various IgG-positive rates ranging from 2.5 to 74.4%. In conclusion, our newly developed NoV-LISA assay is suitable for NoV-specific IgG detection in humans and animals. The wide distribution of IgG antibodies against human NoV indicates potential zoonotic transmission between humans and animals.

A retrospective analysis of Q fever osteomyelitis in children, with recommendations.

Osteomyelitis in children due to Q fever is a rare occurrence. In this study, we review 25 pediatric cases of this disease entity. The mean age of these 25 patients was 3.7years (range, 2-7 years). The risk factor for infection was history of contact with animals, such as cattle and sheep (23/25, 92.0%). The most common sites of infection were foot and ankle (12/25, 48.0%). The diagnosis of Q fever was confirmed by serologic testing in 25 children. On PCR, Coxiella burnetii was detected in 17 cases and it was not detected in the remaining five cases. A total of 22 children were treated with antibiotics, of which 15 received surgical treatment. The composition and duration of treatment varied greatly. Only 18 patients achieved significant recovery of joint mobility after drug and/or surgical treatment. Our current literature review show that the clinical manifestations of Q fever osteomyelitis in children are atypical. In the absence of serological test results, mNGS identification may be a good option. Compared with the guidelines that recommend doxycycline combined with hydroxychloroquine, we recommend that ciprofloxacin-based treatment combined with rifampicin or sulfamethoxazole/trimethoprim should be preferred in children under the age of 8 years, and the need for performing combined surgical debridement and determining the treatment duration should be evaluated according to their actual clinical situation.

Mycobacterium xenopi related spine infections: A case report and systematic literature review.

Background and purpose: Nontuberculous mycobacteria (NTM) disease is an important infection disease throughout the world. Mycobacterium xenopi (M. xenopi) is a common NTM. Extrapulmonary infections due to M. xenopi, particularly spine infections, are a rare occurrence, but lack of research is cited as a constraint for implementing NTM control in such patients. The purposes of this paper are to describe a case of spondylodiscitis, to review the published literature on cases of M. xenopi spine infections, and to summarize the predisposing factors, diagnosis, and treatment of infection. Methods: A case of spondylodiscitis was caused by M. xenopi in a patient with systemic lupus erythematosus (SLE). Research was conducted using the PubMed, ScienceDirect, Embase, Wiley Online Library, and Scopus databases using the following search terms: "Mycobacterium xenopi", "vertebral", "spinal", "spondylodiscitis", "infection", and "osteomyelitis". Results: We retrieved 14 cases published before August 2022. The risk factors for infection were iatrogenic infections (3/14, 21.43%), SLE (4/14, 28.57%), AIDS (4/14, 28.57%), and immunocompetence without any comorbidities (3/14, 21.43%). The most common sites of infection were thoracic vertebrae (10/14, 71.43%) and lumbar vertebrae (4/14, 28.57%). A total of 14 cases were isolated and identified as M. xenopi from a toad by mycobacterial culture. The identification time was 55.00 ± 7.55 days (the present report identification time of metagenomic next generation sequencing (mNGS) was only 2 days). All patients were treated with antibiotic therapy, and the duration of treatment was 13.18 ± 2.13 months. Clarithromycin-based therapy showed a higher improvement rate (5/6, 83.33%). Surgical intervention was performed in 5 patients. Only 1 patient did not show any improvement after surgical treatment. Conclusion: M. xenopi spine infection in humans presents with atypical clinical symptoms. mNGS identification may be a good choice. M. xenopi may be considered in immunocompromised patients with spinal infection. We recommend a clarithromycin-containing regimen and prolonging the duration of treatment to ensure effectiveness.

Serological surveillance of GI norovirus reveals persistence of blockade antibody in a Jidong community-based prospective cohort, 2014-2018.

Introduction: Herd immunity against norovirus (NoV) is poorly understood in terms of its serological properties and vaccine designs. The precise neutralizing serological features of genotype I (GI) NoV have not been studied. Methods: To expand insights on vaccine design and herd immunity of NoVs, seroprevalence and seroincidence of NoV genotypes GI.2, GI.3, and GI.9 were determined using blockade antibodies based on a 5-year longitudinal serosurveillance among 449 residents in Jidong community. Results: Correlation between human histo-blood group antigens (HBGAs) and GI NoV, and dynamic and persistency of antibodies were also analyzed. Seroprevalence of GI.2, GI.3, and GI.9 NoV were 15.1%-18.0%, 35.0%-38.8%, and 17.6%-22.0%; seroincidences were 10.0, 21.0, and 11.0 per 100.0 person-year from 2014 to 2018, respectively. Blockade antibodies positive to GI.2 and GI.3 NoV were significantly associated with HBGA phenotypes, including blood types A, B (excluding GI.3), and O+; Lewis phenotypes Leb+/Ley+ and Lea+b+/Lex+y+; and secretors. The overall decay rate of anti-GI.2 antibody was -5.9%/year (95% CI: -7.1% to -4.8%/year), which was significantly faster than that of GI.3 [-3.6%/year (95% CI: -4.6% to -2.6%/year)] and GI.9 strains [-4.0%/year (95% CI: -4.7% to -3.3%/year)]. The duration of anti-GI.2, GI.3, and GI.9 NoV antibodies estimated by generalized linear model (GLM) was approximately 2.3, 4.2, and 4.8 years, respectively. Discussion: In conclusion, enhanced community surveillance of GI NoV is needed, and even one-shot vaccine may provide coast-efficient health benefits against GI NoV infection.

Under-five mortality in Sierra Leone and possible associated factors: evidence from the 2019 Demographic and Health Survey.

High under-five mortality rate remains one of the public health challenges, especially in Sub-Saharan Africa, accounting for more than half of all global cases. Sierra Leone was and is still one of the countries with the highest under-five mortality rate. Using the latest 2019 Sierra Leone Demographic and Health Survey data, we investigated factors associated with under-five mortality in Sierra Leone. A total of 9771 mothers aged 15-49 years in the country were interviewed and included in the analysis. The dependent variable is child status (dead = 1; alive = 0). A total of 871 (9%) children died before their fifth birthday. The maternal age of 20-24 years [adjusted odds ratios (AOR) = 0.46; 95% confidence interval (CI) = 0.33-0.64; P < 0.001] up to 40-44 years (AOR = 0.43; CI = 0.27-0.7; P = 0.001), currently breastfeeding (AOR = 0.20; CI = 0.17-0.24; P < 0.001), maternal media exposure and usage of reading newspapers/magazines less than once a week (AOR = 0.48; CI = 0.28-0.85; P = 0.011) were more likely to enhance child survivability through their fifth birthday. Also, the child sex being female (AOR = 0.68; CI = 0.59-0.79) was more likely to survive under-five mortality compared to their male counterpart. On the other hand, mothers who listened to radio at least once a week (AOR = 1.31; CI = 1.08-1.59; P = 0.007) watched television less than once a week (AOR = 1.48; CI = 1.16-1.90), had two (AOR = 3.4, CI = 2.78-4.16; P < 0.001) or three and above birth (AOR = 8.11; CI = 6.07-10.83; P < 0.001) in five years, had multiple birth children (AOR = 1.41; CI = 1.08-1.86) and very small-sized child at birth (AOR= 1.95; CI = 1.41-2.70) were more likely to lose their children below the age of 5 years. The factors contributing to under-five mortality in Sierra Leone are critical to ensuring child survival and improving maternal health. Breastfeeding, maternal age, media exposure, child's sex, multiple birth type, very small-sized child and the total number of births in 5 years were significant drivers of under-five mortality. The result affirms the need for attention to be focused on enhancing the survival rate of under-five children in Sierra Leone.

Seroepidemiology of Chlamydia trachomatis Infection in the General Population of Northern China: The Jidong Community Cohort Study.

A longitudinal serological study to investigate the seropositive frequency, incidence, and antibody dynamics of Chlamydia trachomatis infection in the general population of China is urgently needed in order to optimize the strategies for surveillance and precise prevention of C. trachomatis infection. This longitudinal study enrolled 744 subjects aged 18-65 years from Jidong Community of Northern China from 2014 to 2018. Seropositive frequency, incidence, and reinfection of C. trachomatis were determined by detecting antibody against C. trachomatis Pgp3 using "in-house" luciferase immunosorbent assay (LISA). The dynamic of anti-Pgp3 antibody was analyzed using the Generalized Estimating Equation (GEE) model. The overall Pgp3 seropositive frequency among the 18-65-year-old population was 28.1% (95% CI 24.9-31.5), and significantly increased from 12.0% in those aged 18-29 years to 48.6% in the 60-65 years old. The seropositive frequency was slightly higher in women than in men (31.3% vs. 25.4%) without statistical significance. The C. trachomatis incidence and reinfection rate were 11 and 14 per 1,000 person-years, respectively, and showed no significant difference with respect to age, gender, ethnicity, marital status, and education levels. Furthermore, anti-Pgp3 antibody remained detectable in 93.3% (195/209) of the seropositive subjects during the 5 years of follow-up. The overall decay rate for anti-Pgp3 antibody for CT-infected persons was -0.123 Log2 RLU/year, which was dramatically slower than in CT new infection (-3.34 Log2 RLU/year) or reinfection (-1.1 Log2 RLU/year). In conclusion, at least one quarter of the people aged 18-65 years have been infected with C. trachomatis over their lifetime while all age groups are susceptible to C. trachomatis infection in the community of Northern China. Therefore, comprehensive prevention strategies are urgently needed.

Serological surveillance of noroviruses in a community-based prospective cohort: a study protocol.

INTRODUCTION: Noroviruses are the leading cause of viral acute gastroenteritis affecting all age groups. Since 2014, the previous rarely reported GII.P17-GII.17 and recombinant GII.P16-GII.2 norovirus emerged, replacing GII.4 predominant genotype, causing increased outbreaks in China and other countries. Meanwhile, GII.4/2012 Sydney strain has re-emerged as the dominant variant in many places in 2015-2018. The role of herd immunity as the driving force during these new emerging or re-emerging noroviruses is poorly defined. Serological surveillance studies on community-based prospective cohort on norovirus are highly needed. METHODS AND ANALYSES: This study will include 1000 out of 9798 participants aged 18 years and above from Caofeidian district, Tangshan city, northern China. Baseline data on sociodemographic characteristics and blood samples were collected in 2013-2014. Blood collection will be replicated annually throughout the cohort until 2023. Saliva samples were also collected in 2016. The seroprevalence and seroincidence of blockade antibodies against norovirus genotypes of GII.P17-GII.17, GII.P16-GII.2, the re-emerged GII.4/2012 and potential novel pandemic variants will be evaluated by ELISA. Associations between genotype blockade antibodies and sociodemographic factors and human histo-blood group antigens will be evaluated using univariate and multivariate analysis. The dynamics of herd immunity duration will be estimated in this longitudinal surveillance. ETHICS AND DISSEMINATION: The study has been approved by the Ethical Committees of the Staff Hospital of Jidong oil-field of China National Petroleum Corporation. This study will provide insight into the seroprevalence and seroincidence of noroviruses, and their relationships with sociodemographic characteristics and genetic susceptibility. It will also explain herd immunity of the emerged and re-emerged genotypes or variants. The study will further enable an understanding of the mechanism driving the replacement of norovirus genotypes. Research findings will be disseminated in peer-reviewed journals and at scientific meetings.

Characterization of Antigenic Relatedness Among GI Norovirus Genotypes Using Serum Samples From Norovirus-Infected Patients and Mouse Sera.

Characterizing diversity and the antigenic relatedness of norovirus remains a primary focus in understanding its biological properties and vaccine designs. The precise antigenic and serological features of GI genotypes have not been studied. The study represented an investigation on a gastroenteritis outbreak related to GI.3 norovirus and the three most detected GI genotypes, GI.2 (belonging to immunotype B), GI.3 and GI.9 (belonging to immunotype C), were selected to characterize their phylogenetic relationship, HBGA binding profiles and antigenic relatedness within (intra-immunotype), and between (inter-immunotypes) genotypes using mouse sera and patient's serum samples from the GI.3 related outbreak. Wide HBGA binding profiles and evolution of binding affinity were observed in the three GI genotypes studied. A low specific blockade antibody to GI.3 in the population generated the pool of susceptible individuals and supported virus spread in the outbreak. We found strong blockade immune response in homologous strains, moderate intra-immunotype blockade but weak inter-immunotypes blockade in humans following GI.3 norovirus infections. These findings further support the immunotypes grouping and will be valuable for optimizing the design of norovirus vaccine.

[Preparation and characterization of monoclonal antibodies against GII.4 norovirus P domain].

Objective To prepare monoclonal antibodies (mAbs) against GII.4 norovirus P domain by multiple antigens in an immunization program. Methods BALB/c mice were immunized with the multiple GII.4 NoV P domain, namely 1996cluster (VA387), 2004cluster, 2006b cluster and 2010 cluster. The spleen cells from the immunized mice were fused with SP2/0 cells and the hybridoma cells were screened by ELISA. The supernatant of the mAbs was collected and purified by the limiting dilution assay. Its subtype was identified, and the specificity and neutralization were analyzed by indirect ELISA and HBGA blocking, respectively. Results We obtained thirteen hybridoma cell lines that stably secreted mAbs against GII.4 NoV P domain. Their titers reached above 10-4 after purification. The subtypes of the mAbs were identified as IgG1. Indirect ELISA showed that all the mAbs specifically bound to all GII.4 norovirus variants. Five mAbs specifically bound to GII.17, GII.3 and GII.6 variants. Three mAbs specifically bound to GII.2 variants and strongly blocked NoV P particle from binding to the histo-blood group antigen (HBGA) receptors. Conclusion The mAbs against GII.4 norovirus P domain have been obtained by combined antigens immunization program. Multi-antigen immunization can enhance immune response significantly and cross-react with other GII.4 norovirus variants. The findings provide a basis for further development of novel GII.4 norovirus vaccines and for the optimization of the immunization programs of combined multi-antigen vaccine candidates.

Prevalence of sexually transmitted infections among foreigners living in Guangzhou, China: a cross-sectional study (2010-2017).

BACKGROUND: The prevalence of HIV/HCV/HBV/ Treponema pallidum is an essential health issue in China. However, there are few studies focused on foreigners living in China. This study aimed to assess the prevalence and socio-demographic distribution of HIV, HBV, HCV, and T. pallidum among foreigners in Guangzhou in the period of 2010-2017. METHODS: A cross-sectional study was conducted to screen serological samples of 40,935 foreigners from 2010 to 2017 at the Guangdong International Travel Health Care Center in Guangzhou. Samples were tested for hepatitis B surface antigen (HBsAg), anti-HCV, syphilis antibody (anti-TPPA) and anti-HIV 1 and 2. We collected secondary data from laboratory records and used multiple logistic regression analyses to verify the association between different factors and the seroprevalence of HIV/HBV/HCV/ T. pallidum. RESULTS: The prevalence of HBV/HCV/HIV/ T. pallidum was 2.30, 0.42, 0.02, and 0.60%, respectively, and fluctuated slightly for 7 years. The results of multiple logistic regression showed that males were less susceptible to HBV than females (odds ratio [OR] = 0.77, 95% CI: 0.67-0.89). Participants under the age of 20 had a lower risk of HBV (OR = 0.25, 95% CI: 0.18-0.35), HCV (OR = 0.06, 95% CI: 0.02-0.18), and T. pallidum (OR = 0. 10, 95% CI: 0.05-0.20) than participants over the age of 50. Participants with an education level below high school were more likely to have HBV (OR = 2.98, 95% CI: 1.89-4.70) than others, and businessmen (OR = 3.02, 95% CI: 2.03-4.49), and designers (OR = 3.83, 95% CI: 2.49-5.90) had a higher risk of T. pallidum than others. Co-infection involved 58 (4.20%) total cases, and the highest co-infection rate was observed for HBV and T. pallidum (2.60%). CONCLUSION: The prevalence of HBV/HCV/HIV/ T. pallidum was low among foreigners in Guangzhou. Region, gender, age, educational level, and occupation were risk factors for positive infection.

Epidemiology and HBGA-susceptibility investigation of a G9P[8] rotavirus outbreak in a school in Lechang, China.

Rotaviruses cause severe gastroenteritis in infants, in which the viruses interact with human histo-blood group antigens (HBGAs) as attachment and host susceptibility factors. While gastroenteritis outbreaks caused by rotaviruses are uncommon in adolescents, we reported here one that occurred in a middle school in China. Rectal swabs and saliva samples were collected from symptomatic and asymptomatic students, and samples were also collected from the environment. Using PCR, followed by DNA sequencing, a single G9P[8] rotavirus strain was identified as the causative agent. The attack rate of the outbreak was 13.5% for boarders, which was significantly higher than that of day students (1.8%). Person-to-person transmission was the most plausible transmission mode. The HBGA phenotypes of the individuals in the study were determined by enzyme immunoassay, using saliva samples, while recombinant VP8* protein of the causative rotavirus strain was produced for HBGA binding assays to evaluate the host susceptibility. Our data showed that secretor individuals had a significantly higher risk of infection than nonsecretors. Accordingly, the VP8* protein bound nearly all secretor saliva samples, but not those of nonsecretors, explaining the observed infection of secretor individuals only. This is the first single-outbreak-based investigation showing that P[8] rotavirus infected only secretors. Our investigation also suggests that health education of school students is an important countermeasure against an outbreak of communicable disease.

Prevalence and associated factors of burnout syndrome among healthcare workers in public and private hospitals in Mekelle City, Ethiopia

Introduction: Burnout syndrome, an occupational negative psychosomatic condition, has three components: emotional exhaustion, depersonalization and low personal achievement. This study aimed to assess the prevalence of burnout syndrome and associated factors among public and private healthcare workers in Mekelle City, Tigray, Ethiopia. Methods: A cross-sectional study was conducted among 229 healthcare workers in Mekelle, Kay Kalkidan and Ben Meskerem General Hospitals. An anonymous questionnaire was used for data collection. Pearson Chi-square test and Binary logistic regression analysis were employed. Both tests were conducted at 95% CI with p-value ≤ 0.05 as acceptance area. Result: Overall 109 (47.6%) of respondents had burnout syndrome. Workers in the private hospitals (65.8%) were more at risk compared to those in the public hospital (44.0%). The lower staff/patient ratio in the private hospitals compared with the public hospital might have contributed to the higher prevalence of burnout syndrome. Independent predictor factors were: being female, few years of work experience, working night shifts and long working hours each week. Conclusion: Prevalence of burnout syndrome was high among all respondents but particularly those working in private hospitals. Some socio demographic and occupational factors were also implicated

Characterization of Antigenic Relatedness between GII.4 and GII.17 Noroviruses by Use of Serum Samples from Norovirus-Infected Patients.

A novel GII.17 norovirus variant caused major gastroenteritis epidemics in China in 2014 to 2016. To explore the host immune factors in selection of the emergence of this new variant, we characterized its antigenic relatedness with the GII.4 noroviruses that have dominated in China for decades. Through an enzyme-linked immunosorbent assay (ELISA) and a histo-blood group antigen (HBGA) blocking assay using sera from GII.4 and the GII.17 variant-infected patients, respectively, we observed limited cross-immune reactivity by the ELISA but little reactivity by the HBGA blocking assay between GII.4 norovirus and the new GII.17 variant. Our data suggest that, among other possible factors, GII.4-specific herd immunity had little role in the emergence of the new GII.17 variant. Thus, GII.17 may be an important active antigenic type or immunotype that needs to be considered for future vaccine strategies against human noroviruses.

P[8] and P[4] Rotavirus Infection Associated with Secretor Phenotypes Among Children in South China.

Rotaviruses are known to recognize human histo-blood group antigens (HBGAs) as a host ligand that is believed to play an important role in rotavirus host susceptibility and host range. In this study, paired fecal and saliva samples collected from children with viral gastroenteritis, as well as paired serum and saliva samples collected from the general population in south China were studied to evaluate potential association between rotavirus infections and human HBGA phenotypes. Rotavirus was detected in 75 (28%) of 266 fecal samples and P[8] rotaviruses were found to be the predominant genotype. The HBGA phenotypes of the rotavirus-infected children were determined through their saliva samples. Secretor statuses were found to correlate with the risk of rotavirus infection and all P[8]/P[4] rotavirus infected children were secretors. Accordingly, recombinant VP8* proteins of the P[8]/P[4] rotaviruses bound saliva samples from secretor individuals. Furthermore, correlation between serum P[8]/P[4]-specific IgG and host Lewis and secretor phenotypes has been found among 206 studied serum samples. Our study supported the association between rotavirus infection and the host HBGA phenotypes, which would help further understanding of rotavirus host range and epidemiology.

An outbreak caused by GII.17 norovirus with a wide spectrum of HBGA-associated susceptibility.

During the past norovirus (NoV) epidemic season, a new GII.17 variant emerged as a predominant NoV strain, surpassed the GII.4 NoVs, causing outbreaks of acute gastroenteritis (AGE) in China. Here we report a study of an AGE outbreak in an elementary school in December 2014 caused by the new GII.17 NoV to explore the potential mechanism behind the sudden epidemics of the GII.17 NoV. A total of 276 individuals were sick with typical NoV infection symptoms of vomiting (93.4%), abdominal pain (90.4%), nausea (60.0%), and diarrhea (10.4%) at an attack rate of 5.7-16.9%. Genotyping of the symptomatic patients showed that individuals with a secretor positive status, including those with A, B, and O secretors and Lewis positive blood types, were sensitive to the virus, while the non-secretors and the Lewis negative individual were not. Accordingly, the recombinant capsid P protein of the GII.17 isolate showed a wide binding spectrum to saliva samples of all A, B, and O secretors. Thus, the broad binding spectrum of the new GII.17 variant could explain its widely spread nature in China and surrounding areas in the past two years.

Characterization of severe hand, foot, and mouth disease in Shenzhen, China, 2009-2013.

Hand, foot, and mouth disease (HFMD) is caused by human enteroviruses, especially by enterovirus 71 (EV71) and coxsackievirus A16 (CA16). Patients infected with different enteroviruses show varied clinical symptoms. The aim of this study was to determine whether the etiological spectrum of mild and severe HFMD changed, and the association between pathogens and clinical features. From 2009 to 2013, a total of 2,299 stool or rectal specimens were collected with corresponding patient data. A dynamic view of the etiological spectrum of mild and severe HFMD in Shenzhen city of China was provided. EV71 accounted for the majority proportion of severe HFMD cases and fatalities during 2009-2013. CA16 and EV71 were gradually replaced by coxsackievirus A6 (CA6) as the most common serotype for mild HFMD since 2010. Myoclonic jerk and vomiting were the most frequent severe symptoms. Nervous system complications, including aseptic encephalitis and aseptic meningitis were observed mainly in patients infected by EV71. Among EV71, CA16, CA6, and CA10 infection, fever and pharyngalgia were more likely to develop, vesicles on the hand, foot, elbow, knee and buttock were less likely to develop in patients infected with CA10. Vesicles on the mouth more frequently occurred in the patients with CA6, but less in the patient with EV71. Associations between diverse enterovirus serotypes and various clinical features were discovered in the present study, which may offer further insight into early detection, diagnosis and treatment of HFMD.

Antigenic Relatedness of Norovirus GII.4 Variants Determined by Human Challenge Sera.

The GII.4 noroviruses (NoVs) are a single genotype that is responsible for over 50% of NoV gastroenteritis epidemics worldwide. However, GII.4 NoVs have been found to undergo antigenic drifts, likely selected by host herd immunity, which raises an issue for vaccine strategies against NoVs. We previously characterized GII.4 NoV antigenic variations and found significant levels of antigenic relatedness among different GII.4 variants. Further characterization of the genetic and antigenic relatedness of recent GII.4 variants (2008b and 2010 cluster) was performed in this study. The amino acid sequences of the receptor binding interfaces were highly conserved among all GII.4 variants from the past two decades. Using serum samples from patients enrolled in a GII.4 virus challenge study, significant cross-reactivity between major GII.4 variants from 1998 to 2012 was observed using enzyme-linked immunosorbent assays and HBGA receptor blocking assays. The overall abilities of GII.4 NoVs to bind to the A/B/H HBGAs were maintained while their binding affinities to individual ABH antigens varied. These results highlight the importance of human HBGAs in NoV evolution and how conserved antigenic types impact vaccine development against GII.4 variants.

Inhibition of histo-blood group antigen binding as a novel strategy to block norovirus infections.

Noroviruses (NoVs) are the most important viral pathogens that cause epidemic acute gastroenteritis. NoVs recognize human histo-blood group antigens (HBGAs) as receptors or attachment factors. The elucidation of crystal structures of the HBGA-binding interfaces of a number of human NoVs representing different HBGA binding patterns opens a new strategy for the development of antiviral compounds against NoVs through rational drug design and computer-aided virtual screening methods. In this study, docking simulations and virtual screening were used to identify hit compounds targeting the A and B antigens binding sites on the surface of the capsid P protein of a GII.4 NoV (VA387). Following validation by re-docking of the A and B ligands, these structural models and AutoDock suite of programs were used to screen a large drug-like compound library (derived from ZINC library) for inhibitors blocking GII.4 binding to HBGAs. After screening >2 million compounds using multistage protocol, 160 hit compounds with best predicted binding affinities and representing a number of distinct chemical classes have been selected for subsequent experimental validation. Twenty of the 160 compounds were found to be able to block the VA387 P dimers binding to the A and/or B HBGAs at an IC50<40.0 µM, with top 5 compounds blocking the HBGA binding at an IC50<10.0 µM in both oligosaccharide- and saliva-based blocking assays. Interestingly, 4 of the top-5 compounds shared the basic structure of cyclopenta [a] dimethyl phenanthren, indicating a promising structural template for further improvement by rational design.