Efficacy of high-intensity focused ultrasound combined with LNG-IUS for adenomyosis: a systematic review and meta-analysis.

OBJECTIVE: To evaluate the efficacy and safety of high-intensity focused ultrasound (HIFU) combined with the levonorgestrel intrauterine system (LNG-IUS) for adenomyosis. METHODS: We searched PubMed, Embase, Cochrane Library, Web of Science, CNKI, SinoMed, Wanfang, and VIP databases from their inception to Nov 20, 2021 for relevant articles that compared HIFU combined with LNG-IUS vs. HIFU alone in patients with adenomyosis. RevMan5.4 software was used for the data analysis. The primary outcome was changes in volume of the uterine. Secondary outcomes included visual analog scale (VAS) scores for dysmenorrhea, serum CA125 level, recurrence rate, changes in volume of the adenomyotic lesion, menstrual volume scores, and adverse reactions. Data synthesis was conducted using a random-effects model with significant heterogeneity (I2 > 50%), and using a fixed-effects model otherwise. This study is registered on the PROSPERO platform (CRD42021295214). RESULTS: The final analysis included 13 studies, with a total of 1861 patients. Results of analysis revealed that there was no significant difference in uterine volume reduction between the HIFU control group and the HIFU/LNG-IUS group at 3 months after procedure (MD:30.63). Compared with the HIFU control group, the HIFU/LNG-IUS group had more pronounced reduction in uterine volume at 6 (MD:29.04) and 12 months (MD:22.10) after procedure. The HIFU/LNG-IUS group has lower VAS scores for dysmenorrhea than the HIFU control group at 3 (MD:1.68), 6 (MD:1.69), and 12 months (MD:1.30) after procedure. Serum CA125 level in the HIFU/LNG-IUS group decreased more significantly than the HIFU control group at 6 (MD:18.34) and 12 months (MD:18.49) after procedure. The recurrence rate in the HIFU/LNG-IUS group was lower than that in the HIFU control group (RR:0.20). CONCLUSIONS: Compared to HIFU control group, HIFU/LNG-IUS group for the management of adenomyosis had more advantages in alleviating symptoms and decreasing the volumes of the uterine and adenomyotic lesions. However, since the number of the included studies was too small and some of them were not RCT, this conclusion needs to be referenced with caution.

Comparison of immune response to human rhinovirus C and respiratory syncytial virus in highly differentiated human airway epithelial cells.

BACKGROUND: Human rhinovirus C (HRV-C) accounts for a large proportion of HRV-related illnesses, but the immune response to HRV-C infection has not been elucidated. Our objective was to assess the effect of HRV-C on cytokine secretion in human bronchial epithelial (HBE) cells grown at air-liquid interface (ALI) and compare it with that of respiratory syncytial virus (RSV). METHODS: HBE cells were differentiated at ALI culture and the full-length cDNA clones of HRV-C651 and HRV-C15, clinical isolates of HRV-C79 and HRV-C101, and two RSV isolates were inoculated in the HBE cells. The effect of HRV-C on cytokine secretion was assessed and compared with that of RSV. RESULTS: HRV-Cs infect and propagate in fully differentiated HBE cells and significantly increase the secretion of IFN-λ1, CCL5, IP10, IL-6, IL-8, and MCP-1. The virus loads positively correlated with the levels of the cytokines. HRV-C induced lower secretion of CCL5 (P = 0.048), IL-6 (P = 0.016), MCP-1 (P = 0.008), and IL-8 (P = 0.032), and similar secretion of IP10 (P = 0.214) and IFN-λ1 (P = 0.214) when compared with RSV. CONCLUSION: HBE ALI culture system supported HRV-C infection and propagation and HRV-C induced relatively weaker cytokine expression than RSV.

GII.13/21 Noroviruses Recognize Glycans with a Terminal ß-Galactose via an Unconventional Glycan Binding Site.

Human noroviruses (huNoVs) recognize histo-blood group antigens (HBGAs) as host susceptibility factors. GII.13 and GII.21 huNoVs form a unique genetic lineage that emerged from mainstream GII NoVs via development of a new, nonconventional glycan binding site (GBS) that binds Lea antigen. This previous finding raised the question of whether the new GII.13/21 GBS really has such a narrow glycan binding spectrum. In this study, we provide solid phenotypic and structural evidence indicating that this new GBS recognizes a group of glycans with a common terminal ß-galactose (ß-Gal). First, we found that P domain proteins of GII.13/21 huNoVs circulating at different times bound three glycans sharing a common terminal ß-Gal, including Lec, lactose, and mucin core 2. Second, we solved the crystal structures of the GII.13 P dimers in complex with Lec and mucin core 2, which showed that ß-Gal is the major binding saccharide. Third, nonfat milk and lactose blocked the GII.13/21 P domain-glycan binding, which may explain the low prevalence of GII.13/21 viruses. Our data provide new insight into the host interactions and epidemiology of huNoVs, which would help in the control and prevention of NoV-associated diseases.IMPORTANCE Evidence from both phenotypic binding assay and structural study support the observed interactions of human noroviruses (huNoVs) with histo-blood group antigens (HBGAs) as receptors or attachment factors, affecting their host susceptibility. GII.13 and GII.21 genotypes form a unique genetic lineage that differs from the mainstream GII huNoVs in their unconventional glycan binding site. Unlike the previous findings that GII.13/21 genotypes recognize only Lea antigen, we found in this study that they can interact with a group of glycans with a common terminal ß-Gal, including Lec, lactose, and mucin core 2. However, this wide glycan binding spectrum in a unique binding mode of the GII.13/21 huNoVs appears not to increase their prevalence, probably due to the existence of decoy glycan receptors in human gastrointestinal tract limiting their infection. Our findings shed light on the host interaction and epidemiology of huNoVs, which would impact the strategy of huNoV control and prevention.

Glycan binding patterns of human rotavirus P[10] VP8* protein.

BACKGROUND: Rotaviruses (RVs) are a major cause of acute children gastroenteritis. The rotavirus P [10] belongs to P[I] genogroup of group A rotaviruses that mainly infect animals, while the rotavirus P [10] was mainly identified from human infection. The rotavirus P [10] is an unusual genotype and the recognition pattern of cellular receptors remains unclear. METHODS: We expressed and purified the RV P [10] VP8* protein and investigated the saliva and oligosaccharide binding profiles of the protein. A homology model of the P [10] VP8* core protein was built and the superimposition structural analysis of P [10] VP8* protein on P [19] VP8* in complex with mucin core 2 was performed to explore the possible docking structural basis of P [10] VP8* and mucin cores. RESULTS: Our data showed that rotavirus P [10] VP8* protein bound to all ABO secretor and non-secretor saliva. The rotavirus P [10] could bind strongly to mucin core 2 and weakly to mucin core 4. The homology modeling indicated that RV P [10] VP8* binds to mucin core 2 using a potential glycan binding site that is the same to P [19] VP8* belonging to P[II] genogroup. CONCLUSION: Our results suggested an interaction of rotavirus P [10] VP8* protein with mucin core 2 and mucin core 4. These findings offer potential for elucidating the mechanism of RV A host specificity, evolution and epidemiology.

Three-dimensional Culture of Human Airway Epithelium in Matrigel for Evaluation of Human Rhinovirus C and Bocavirus Infections.

OBJECTIVE: Newly identified human rhinovirus C (HRV-C) and human bocavirus (HBoV) cannot propagate in vitro in traditional cell culture models; thus obtaining knowledge about these viruses and developing related vaccines are difficult. Therefore, it is necessary to develop a novel platform for the propagation of these types of viruses. METHODS: A platform for culturing human airway epithelia in a three-dimensional (3D) pattern using Matrigel as scaffold was developed. The features of 3D culture were identified by immunochemical staining and transmission electron microscopy. Nucleic acid levels of HRV-C and HBoV in 3D cells at designated time points were quantitated by real-time polymerase chain reaction (PCR). Levels of cytokines, whose secretion was induced by the viruses, were measured by ELISA. RESULTS: Properties of bronchial-like tissues, such as the expression of biomarkers CK5, ZO-1, and PCK, and the development of cilium-like protuberances indicative of the human respiration tract, were observed in 3D-cultured human airway epithelial (HAE) cultures, but not in monolayer-cultured cells. Nucleic acid levels of HRV-C and HBoV and levels of virus-induced cytokines were also measured using the 3D culture system. CONCLUSION: Our data provide a preliminary indication that the 3D culture model of primary epithelia using a Matrigel scaffold in vitro can be used to propagate HRV-C and HBoV.

The suppression of innate immune response by human rhinovirus C.

Rhinovirus C (RV-C), a newly identified group of human rhinoviruses (RVs), is associated with exacerbation of severe asthma. The type I interferon (IFN) response induced by this virus and the mechanisms of evasion of IFN-mediated innate immunity for RV-C remain unclear. In this study, we constructed a full-length cDNA clone of RV-C (LZ651) from a clinical sample. IFN-ß mRNA and protein levels were not elevated in differentiated Human bronchial epithelial (HBE) cells at the air-liquid interface infected with RV-C, except in the early stage of infection. The ability to attenuate IFN-ß activation was ascribed to 3Cpro of RV-C, and the 40-His site of 3Cpro played an important role. Furthermore, RIG-I was degraded by 3Cpro in a caspase-dependent manner and 3Cpro cleaved MAVS at 148 Q/A, which inhibited IFN signaling. Taken together, our results demonstrate the mechanism by which RV-C circumvents the production of type I IFN in infected cells.

Emergence of human caliciviruses among diarrhea cases in southwest China.

BACKGROUND: Acute diarrhea is one of the most serious problems in global public health that causes considerable morbidity and mortality worldwide. Human caliciviruses (HuCV) including norovirus (NoV, genogroup GI and GII) and sapovirus (SaV), is a leading cause of acute sporadic diarrhea in individuals across all age groups. However, few studies had been conducted clarifying the characteristics of HuCV in diarrhea cases across all age groups in China. Our study was aimed at assessing the HuCV-related diarrhea burden and NoV genotypes distribution in southwest China. METHODS: The study was conducted in four hospitals in Kunming city, Yunnan province, from June 2014 to July 2015. Stool specimens were collected from 1,121 diarrhea cases and 319 healthy controls in outpatient departments. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect NoV (GI, GII) and SaV. Sequencing was applied to confirm the three viral infections and phylogenetic analysis was performed to determine their genotypes. A structured questionnaire was used to record the demographic information and clinical symptoms of subjects. RESULTS: HuCV was detected at an 11.0 % infection rate in 1,121 diarrhea cases and at 3.4 % rate in 319 non-diarrhea subjects (p < 0.0001, OR = 3.5, 95 % CI 1.8-6.5). The prevalence of the NoV genogroup GII and genotype GII.4 in diarrhea cases was significantly higher than that found in healthy controls (p < 0.0001, p = 0.018, respectively). NoV GII (n = 118, 10.5 %) was the most common HuCV subtype in diarrhea cases, followed by SaV (n = 3, 0.3 %) and NoV GI (n = 2, 0.2 %). Of 118 NoV GII strains isolated from diarrhea patients. GII.4 (n = 55, 46.6 %) was the predominant strain, followed by GII.3 (n = 28, 23.7 %), GII.12 (n = 25, 21.2 %), GII.17 (n = 8, 6.8 %), and GII.5 (n = 2, 1.7 %). Of the 55 GII.4 strains, the GII.4 Sydney 2012 variant had absolutely predominant prevalence (n = 52, 94.5 %), followed by the NoV GII.4-2006b variant (n = 3, 5.5 %). The GII.4 Orleans 2009 variant was not found in diarrhea cases of the study. CONCLUSIONS: NoV GII was the major genogroup and GII.4 was the most predominant strain detected in diarrhea patients. The GII.17 is an emergent variant in sporadic diarrhea and might become the predominant strain in diarrhea cases in the near future. Rapid, accurate detection kits need to be developed to help us find and treat NoV-associated diarrhea in clinical settings in a timely manner.

Gene Knockdown in Human Rhinovirus 1B Using 2'-OMe-modified siRNAs Results in the Reactivation of the Interferon Response.

The aim of this study was to investigate the knockdown efficiency of 2'-O-methylated (2'-OMe)-modified small interfering RNAs (siRNAs) on human rhinovirus 1B (HRV1B) replication and the interferon response. Thus, 24 2'-OMe-modified siRNAs were designed to target HRV1B. The RNA levels of HRV1B, Toll-like receptor 3, melanoma differentiation-associated gene 5, retinoic acid inducible gene-I, and interferons were determined in HRV1B-infected HeLa and BEAS-2B epithelial cells transfected with 2'-OMe-modified siRNAs. The results revealed that all 2'-OMe-modified siRNAs interfered with the replication of HRV1B in a cell-specific and transfection efficiency-dependent manner. Viral activation of Toll-like receptor 3, melanoma differentiation-associated gene 5, retinoic acid inducible gene-I, and the interferon response was detected. In conclusion, the 2'-OMe-modified siRNAs used in this study could interfere with HRV1B replication, possibly leading to the reactivation of the interferon response.

Norovirus Infection and Histo-blood Group Antigens in Children Hospitalized with Diarrhea in Lulong and Chenzhou in China.

Norovirus (NoV) is a pathogen that commonly causes viral diarrhea in children. Studies indicate that NoV recognizes human histo-blood group antigens (HBGAs) as cell attachment factors. In order to explore the correlation between of NoV infection and HBGAs, a cross-sectional study was conducted in children less than five years old who were hospitalized with diarrhea in two areas of China between November 2014 and February 2015. Of the paired stool and saliva samples taken from 424 children, NoV was detected in 24 (6%) children, with viral genotypes GII.3 (n=5), GII.4 (n=14), GII.12 (n=1), and GII.17 (n=4). All of the individuals having NoV infection were either secretors (Lea-b+/Lex-y+) or partial secretors (Lea+b+/Lex+y+) except one GII.3 infection of a non-secretor (Lea+b-/Lex+y-). These results suggest that secretor positive is associated with NoV infection, although non-secretors are not absolutely protected from NoV infection.

Cytokine responses and correlations thereof with clinical profiles in children with enterovirus 71 infections.

BACKGROUND: Severe complications associated with EV71 infections caused many infants death. However, the pathogenesis of EV71 infection in the severe cases remained poorly understood. METHODS: In this study we collected plasma and cerebrospinal fluid (CSF) specimens drawn in the acute and/or recovery phases from EV71-infected individuals, and plasma specimens from healthy children served as normal controls. We compared the levels of cytokines and chemokines determined by a Luminex-based cytokine bead array. RESULTS: The plasma levels of IL-1ß and IL-6 were significantly higher in severe and critical cases than in mild patients and normal controls. Higher plasma levels of IL-6, IL-10, and IL-8 were evident in critical than severe cases. The CSF levels of IL-6, IL-8, and IP-10 were higher, and that of RANTES lower (compared to plasma), in severe and critical patients. Significantly lower CSF levels of cytokines and chemokines were recorded in the recovery than the acute phase in severe and critical cases treated with intravenous immunoglobulin (IVIG) and glucocorticoids. Only the CSF levels of IL-6, IP-10, and IL-8 were significantly correlated with white blood cell counts, and absolute neutrophil and monocyte counts, in severe cases. Furthermore, the CSF levels of IL-6 were correlated with temperature in both cases. CONCLUSIONS: These data indicate that a major cytokine response and inflammation, in both plasma and the CNS, are features of disease caused by EV71 infection. Systemic inflammation caused by EV71 infection exacerbated the deterioration of the disease, and resulted in the disease progression to the critical illness stage.

The induction and suppression of type I and type III interferons by human group H rotavirus.

Group H Rotavirus (RVH) is associated with human diarrhea gastroenteritis. The interferon (IFN) response induced by RVH remains unclear. In this study, we first studied the characteristic feature of RVH and found J19 strain of RVH grew less efficiently compared with the G6P1 strain of RVA. Next, we found that infection with the J19 virus resulted in the secretion of IFN-λ1, but not IFN-ß, while both IFN-ß and IFN-λ1 could inhibit J19 replication significantly in Caco-2 cells. NSP1 played an important role in the suppression of type I and type III IFN response, and NSP5 protein significantly inhibited activation of IFN-λ1. J19 NSP1 suppressed the induction of IFN-ß obviously than G6P1 NSP1, while G6P1 NSP1 reduced IFN-λ1 induction to the greatest extent compared with G9P8, Wa, and J19 NSP1s. Our studies reveal the propagation feature of RVH and interferon induction and suppression by group H rotavirus.

Core decompression vs. allogenic non-vascularized bone grafting in patients with osteonecrosis of the femoral head.

Background: Core decompression and allogenic non-vascularized bone grafting are used in the early stage of osteonecrosis of the femoral head for a period. Since the comparison of the core decompression and allogenic non-vascularized bone grafting are less reported, the purpose of our study was to investigate the difference of two procedures in patients with the osteonecrosis of the femoral head. Methods: Between January 2018 and January 2019, 59 patients (64 hips) were divided into core decompression group and non-vascularized bone grafting group according to their procedures. The primary outcomes are visual analog score (VAS) and Harris hip score. Survivorship was analyzed with the collapse of the femoral head or conversion to total hip arthroplasty (THA) as the endpoint. Results: At the final follow-up, two hips underwent THA in the core decompression group and three hips in the allogenic non-vascularized bone grafting group. The radiographic survival rates were 76.9% and 77.3%, respectively, in both groups. The VAS of the core decompression group was 6.08 ± 1.164 and 3.30 ± 1.431 before and 2 years after operation (P < 0.05), respectively. The VAS of the allogenic non-vascularized bone grafting group was 6.00 ± 1.209 and 3.15 ± 1.537 before and 2 years after operation (P < 0.05), respectively. The Harris hip score of the core decompression group was 52.49 ± 6.496 before operation, and 2 years after operation, it increased by 81.14 ± 8.548 (P < 0.05); The Harris hip score of allogenic the non-vascularized bone grafting group was 53.56 ± 5.925 and 81.33 ± 7.243 before and 2 years after operation (P < 0.05), respectively. In the core decompression group, body mass index (BMI) >25 kg/m2 was correlated with the collapse of femoral head or conversion to THA [P < 0.05; 95% confidence interval (CI), 0.006-1.334], and Association Research Circulation Osseous (ARCO) III was correlated with the collapse of femoral head or conversion to THA (P < 0.05; 95% CI, 2.514-809.650). In the allogenic non-vascularized bone grafting group, age, BMI, and ARCO stage were significantly associated with the collapse of femoral head or conversion to THA (P > 0.05). Conclusion: The clinical survival rate of the femoral head in the core decompression group was slightly better than that in the allogenic non-vascularized bone grafting group. There was no significant difference in the radiographic survival rate of the femoral head between the two groups. Both groups can alleviate pain and improve functional of patients, but there was no significant difference in the degree of improvement. In the core decompression group, BMI >25 kg/m2 and ARCO III correlated with the collapse of femoral head or conversion to THA. In the allogenic non-vascularized bone grafting group, no association was found between age, BMI, and ARCO stage and the collapse of femoral head or conversion to THA. Level of evidence: III.

Efficacy of High-Intensity Focused Ultrasound Combined With GnRH-a for Adenomyosis: A Systematic Review and Meta-Analysis.

Objective: High-intensity focused ultrasound (HIFU) is an innovative non-invasive technology used for adenomyosis. Gonadotropin-releasing hormone agonist (GnRH-a) is a hormone commonly used for adenomyosis. We investigated and assessed the efficacy of HIFU combined with GnRH-a for adenomyosis. Methods: For this systematic review and meta-analysis, we searched Pubmed, Cochrane Library, Web of Science, Embase, CNKI, WanFang, and VIP databases for relevant articles published in Chinese or English that compared HIFU combined with GnRH-a vs. HIFU alone in patients with adenomyosis. The last literature search was completed on January 31, 2021. Two reviewers independently assessed study eligibility and assessed risk of bias. Another two reviewers extracted the data. The RevMan5.3 software was used for the data analysis. Changes in volume of the uterine and adenomyotic lesion were defined as the primary outcomes. The secondary outcomes were visual analog scale (VAS) scores for dysmenorrhea, menstrual volume scores, serum CA125 levels, and recurrence rate. This study is registered with PROSPERO (CRD42021234301). Results: Three hundred and ninety potentially relevant articles were screened. Nine studies with data for 766 patients were finally included. Compared with the HIFU alone group, the HIFU combined with GnRH-a group had a higher rate of uterine volume reduction (MD 7.51, 95% CI 5.84-9.17, p < 0.00001), smaller adenomyotic lesion volume (MD 4.11, 95% CI 2.93-5.30, p < 0.00001), lower VAS score for dysmenorrhea (MD 1.27, 95% CI 0.54-2.01, p = 0.0007) and menstrual volume score (MD 0.88, 95% CI 0.73-1.04, p < 0.00001), and lower CA125 level (SMD 0.31, 95% CI 0.05-0.56, p = 0.02) after the procedure. The recurrence rate in the HIFU combined with GnRH-a group was lower than that in the HIFU alone group (RR 0.28, 95% CI 0.10-0.82, p = 0.02). Conclusions: Compared with HIFU treatment alone, HIFU combined with GnRH-a for the treatment of adenomyosis has greater efficacy in decreasing the volumes of the uterine and adenomyotic lesions and alleviating symptoms. However, since the number of the included studies was too small and most of them were written in Chinese, this conclusion needs to be referenced with caution. And the long-term evidence of its efficacy is still insufficient. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/ identifier [CRD42021234].

Single-nucleotide polymorphisms of HLA and Polygonum multiflorum-induced liver injury in the Han Chinese population.

BACKGROUND: Polygonum multiflorum is one of the leading causes of herb-induced liver injury in China. HLA-B*35:01 is reported to be a potential biomarker of Polygonum multiflorum-induced liver injury (PM-DILI). However, little is known about the relationship between single-nucleotide polymorphisms (SNPs) and PM-DILI. AIM: To identify SNPs that indicate susceptibility to PM-DILI. METHODS: We conducted a systematic study enrolling 382 participants from four independent hospitals, including 73 PM-DILI patients, 118 patients with other drug-induced liver injury (other-DILI) and 191 healthy controls. Whole-exome sequencing was performed for 8 PM-DILI patients and 8 healthy controls who were randomly selected from the above subjects. Nineteen SNPs that showed high frequencies in the 8 PM-DILI patients were selected as candidate SNPs and then screened in 65 PM-DILI patients, 118 other-DILI patients and 183 healthy controls using the MassARRAY system. HLA-B high-resolution genotyping was performed for the 73 PM-DILI and 118 other-DILI patients. The Han-MHC database was selected as a population control for HLA-B analysis. P < 6.25 × 10-3 after Bonferroni correction was considered significant. RESULTS: The frequencies of rs111686806 in the HLA-A gene, rs1055348 in the HLA-B gene, and rs202047044 in the HLA-DRB1 gene were significantly higher in the PM-DILI group than in the control group [27.2% vs 11.6%, P = 1.72 × 10-5, odds ratio (OR) = 3.96, 95% confidence interval (CI): 2.21-7.14; 42.5% vs 8.6%, P = 1.72 × 10-19, OR = 13.62, 95%CI: 7.16-25.9; 22.9% vs 8.1%, P = 4.64 × 10-6, OR = 4.1, 95%CI: 2.25-7.47]. Only rs1055348 showed a significantly higher frequency in the PM-DILI group than in the other-DILI group (42.5% vs 13.6%, P = 1.84 × 10-10, OR = 10.06, 95%CI: 5.06-20.0), which suggested that it is a specific risk factor for PM-DILI. rs1055348 may become a tag for HLA-B*35:01 with 100% sensitivity and 97.7% specificity in the PM-DILI group and 100% sensitivity and 98.1% specificity in the other-DILI group. Furthermore, HLA-B*35:01 was confirmed to be associated with PM-DILI with a frequency of 41.1% in the PM-DILI group compared with 11.9% (P = 4.30 × 10-11, OR = 11.11, 95%CI: 5.57-22.19) in the other-DILI group and 2.7% (P = 6.22 × 10-166, OR = 62.62, 95%CI: 35.91-109.20) in the Han-MHC database. CONCLUSION: rs111686806, rs1055348, and rs202047044 are associated with PM-DILI, of which, rs1055348 is specific to PM-DILI. As a tag for HLA-B*35:01, rs1055348 may become an alternative predictive biomarker of PM-DILI.

Susceptibility of human tonsillar epithelial cells to enterovirus 71 with normal cytokine response.

A recent histopathologic study implicated human tonsillar crypt epithelium as an important site for EV71 replication in EV71-caused fatal cases. This study aimed to confirm the susceptibility of human tonsillar epithelium to EV71. Two human tonsillar epithelial cell lines (UT-SCC-60A and UT-SCC-60B) were susceptive to EV71, and PI3K/AKT, p38, ERK1/2, and JNK1/2 signal pathways were activated. Interferon-α, IL-8, IL-1ß, IL-6 and IL-12p40 were induced and regulated by PI3K/AKT, p38, ERK1/2, and JNK1/2 signal pathways. PI3K/AKT pathway activation appeared to suppress the induction of TNF-α, which induced cell survival by inhibiting GSK-3ß. The activation of NF-κB was observed but inhibited by these pathways in EV71 infection. Furthermore, ERK1/2 and JNK1/2 were essential for efficient EV71 replication. Human tonsillar epithelial cells support EV71 replication and display innate antiviral immunity in vitro, indicating that human tonsillar epithelial cells may be novel targets for EV71 infection and replication in vivo.

A novel hepatovirus identified in wild woodchuck Marmota himalayana.

Hepatitis A virus (HAV) is a hepatotropic picornavirus that causes acute liver disease worldwide. Here, we report on the identification of a novel hepatovirus tentatively named Marmota Himalayana hepatovirus (MHHAV) in wild woodchucks (Marmota Himalayana) in China. The genomic and molecular characterization of MHHAV indicated that it is most closely related genetically to HAV. MHHAV has wide tissue distribution but shows tropism for the liver. The virus is morphologically and structurally similar to HAV. The pattern of its codon usage bias is also consistent with that of HAV. Phylogenetic analysis indicated that MHHAV groups with known HAVs but forms an independent branch, and represents a new species in the genus Hepatovirus within the family Picornaviridae. Antigenic site analysis suggested MHHAV has a new antigenic property to other HAVs. Further evolutionary analysis of MHHAV and primate HAVs led to a most recent common ancestor estimate of 1,000 years ago, while the common ancestor of all HAV-related viruses including phopivirus can be traced back to 1800 years ago. The discovery of MHHAV may provide new insights into the origin and evolution of HAV and a model system with which to explore the pathogenesis of HAV infection.

Prevalence of Coxsackievirus A6 and Enterovirus 71 in Hand, Foot and Mouth Disease in Nanjing, China in 2013.

BACKGROUND: Although hand, foot and mouth disease (HFMD) has been strongly associated with enterovirus 71 (EV71), coxsackievirus A16 (CVA16) and other enteroviruses, studies regarding coxsackievirus A6 (CVA6) infection in HFMD are limited. The aim of this study was to identify the major etiological agents causing HFMD in Nanjing in 2013 and explore the clinical and genetic characteristics of the prevalent enterovirus (EV) types in HFMD. METHODS: A total of 394 throat swabs were collected from hospitalized children diagnosed with HFMD from April to July 2013. EVs were detected by reverse transcription polymerase chain reaction of 5' UTR sequences. Genotyping and phylogenetic analysis were based on VP4 sequences. Demographic and clinical data were obtained. RESULTS: Of the specimens, 68.5% (270/394) were positive for EVs. The genotypes and detection rates were CVA6, 30.00% (81/270); EV71, 17.41% (47/270); HRV, 11.11% (30/270); CVA10, 3.33% (9/270); CVA2, 1.11% (3/270); CVA16, 0.74% (2/270); EV68, 0.37% (1/270); echovirus 6, 0.37% (1/270); echovirus 9, 0.37% (1/270), respectively. Patients infected with CVA6 displayed symptoms atypical of HFMD, including larger vesicles on their limbs and buttocks. Phylogenetic analysis revealed 2 genetically distinct CVA6 strains that circulated independently within the region. Patients infected with CVA6 were more likely to have abnormal periphery blood white blood cell and C-reactive protein levels, while EV71 was more likely to infect the central nervous system, as indicated by clinical manifestations and white blood cell analysis of cerebrospinal fluid. CONCLUSIONS: Multiple EV genotypes contributed to HFMD in Nanjing in 2013, and CVA6 was the dominant genotype. The clinical presentation of CVA6 infection differs from that of EV71 infection in HFMD.

Association of Chemotactic Chemokine Ligand 5 Polymorphisms with the Risk of Developing Severe Enterovirus 71 Infection.

Respiratory damage is a main manifestation of severe Enterovirus 71 (EV71) infection. Polymorphisms of -403G/A (rs2107538), -28C/G (rs2280788), and In1.1T/C (rs2280789) in chemotactic chemokine ligand 5 (CCL5) have linked with many respiratory diseases. In this study, we explored the possible correlation of CCL5 polymorphisms with severe EV71 infection. Blood samples were obtained from 87 children hospitalized for EV71 infection. Fifty-seven healthy children were enrolled as asymptomatic controls. Genotype and allele frequencies were analyzed by logistic regression analysis. There were statistically significant differences in polymorphisms of CCL5 -403G/A and In1.1T/C for dominant model (P = 0.016; P = 0.027) and additive model (P = 0.010; P = 0.019) between patients with severe EV71 infection and asymptomatic controls. With ordinal logistic regression model analysis, statistically significant differences were found between polymorphisms of CCL5 (-403G/A) (P = 0.034) with the severity of EV71 infection after adjusting for age. The frequency of A-C-C haplotype was significantly higher in EV71 infection patients than controls (P = 0.032). These results suggest that CCL5 -403G/A and In1.1T/C polymorphisms may contribute to severe EV71 infection and individuals with haplotype of A-C-C may exhibit higher risk of developing severe EV71 infection. These findings may provide insights into pathogenic and protective mechanisms of severe EV71 infection.

Construction of interferon alpha/beta receptor subunit 1 gene knockout Caco-2 cell line based on CRISPR/Cas9 system / 中国生物制品学杂志

@#Objective To knockout interferon alpha/beta receptor subunit 1(IFNAR1) gene in human colorectal adenocarcinoma cells Caco-2 using clustered regularly interspaced short palinmic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system to construct IFNAR1 knockout Caco-2 cell line.Methods The single guide RNA(sgRNA)sequence was designed to specifically recognize the exon region of IFNAR1 gene using CRISPR/Cas9 technology,and the LentiCRISPRv2-IFNAR1-sgRNA recombinant plasmid was constructed.Caco-2 cells were infected with the plasmid packaged by lentivirus and screened by puromycin resistance.The obtained monoclonal cell lines were cultured by limited dilution method,which were verified for the effect of IFNAR1 gene knockout by target gene sequencing and Western blot,and detected for the mRNA levels of CXC chemokine ligand 10(CXCL10)and interferon-stimulatd gene 20(ISG20)in IFNAR1knockout cells by adding exogenous IFNβ.Results Sequencing results of plasmid LentiCRISPRv2-IFNAR1-sgRNA showed that the insertion sites were all located at the sticky end of BsmBⅠenzyme digestion.Two IFNAR1 knockout monoclonal cell lines were obtained.The sequencing results showed that Caco-2-IFNAR1-KO1 had 5 bp deletion in the sixth exon of IFNAR1,and Caco-2-IFNAR1-KO2 had 18 bp deletion and 1 bp insertion in the seventh exon.Compared with wild-type Caco-2 cells,Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells showed no expression of IFNAR1 protein.Compared with no IFNβ stimulation,the mRNA levels of CXCL10 gene(t = 0.566 and 1.268 respectively,P>0.05)and ISG20 gene(t =1.522 and 1.733 respectively,P>0.05)in Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells stimulated by 50 ng/mL IFNβ showed no significant increase.While compared with those of wild-type Caco-2 cells,the mRNA levels of CXCL10gene(t = 6.763 and 6.777 respectively,P<0.05)and ISG20 gene(t = 5.664 and 5.65 respectively,P<0.05)in Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells decreased significantly under the stimulation of 50 ng/mL exogenous IFNβ.Conclusion Caco-2 cell line with IFNAR1 knockout was successfully constructed by using CRISPR/Cas9 technology,and the downstream molecules activated by IFNAR(interferon alpha/beta receptor)in this cell line were obviously inhibited,which provided a powerful tool for further exploration of the innate immune response and replication packaging mechanism of Caco-2 cells after virus infection.