Lung-specific MCEMP1 functions as an adaptor for KIT to promote SCF-mediated mast cell proliferation.

Lung mast cells are important in host defense, and excessive proliferation or activation of these cells can cause chronic inflammatory disorders like asthma. Two parallel pathways induced by KIT-stem cell factor (SCF) and FcεRI-immunoglobulin E interactions are critical for the proliferation and activation of mast cells, respectively. Here, we report that mast cell-expressed membrane protein1 (MCEMP1), a lung-specific surface protein, functions as an adaptor for KIT, which promotes SCF-mediated mast cell proliferation. MCEMP1 elicits intracellular signaling through its cytoplasmic immunoreceptor tyrosine-based activation motif and forms a complex with KIT to enhance its autophosphorylation and activation. Consequently, MCEMP1 deficiency impairs SCF-induced peritoneal mast cell proliferation in vitro and lung mast cell expansion in vivo. Mcemp1-deficient mice exhibit reduced airway inflammation and lung impairment in chronic asthma mouse models. This study shows lung-specific MCEMP1 as an adaptor for KIT to facilitate SCF-mediated mast cell proliferation.

Mass migration to Europe: an opportunity for elimination of hepatitis B virus?

People from low-to-middle income countries have been migrating to western Europe on a large scale in recent years. Data indicate that the number of first-time asylum applications by non-EU members increased from 290 000 in 2011 to more than 1·3 million in 2015. During the peak period of migration, The Global Health Sector Strategy on Viral Hepatitis was adopted by WHO. Viral hepatitis, and particularly hepatitis B virus (HBV), is an important disease because of its high prevalence and associated mortality. In some cases, HBV can be carried by refugees arriving from regions of high and intermediate prevalence. Refugees with HBV might not show clinical symptoms and not be diagnosed in destination countries with a low prevalence, where screening is not regularly done. Although transmission to the host population is low, dedicated surveillance and tailored public health policies are required. It is important to note that some of the countries that receive many migrants do not have a universal HBV vaccination programme. In this Viewpoint, we argue that the current large-scale movement from regions with high or intermediate HBV prevalence should be taken as an opportunity to achieve viral hepatitis elimination targets, by establishing a well prepared infrastructure for HBV screening, vaccination, and treatment.

Genomic Diversity of Hepatitis B Virus Infection Associated With Fulminant Hepatitis B Development.

CONTEXT: After five decades of Hepatitis B Virus (HBV) vaccine discovery, HBV is still a major public health problem. Due to the high genetic diversity of HBV and selective pressure of the host immune system, intra-host evolution of this virus in different clinical manifestations is a hot topic of research. HBV infection causes a range of clinical manifestations from acute to chronic infection, cirrhosis and hepatocellular carcinoma. Among all forms of HBV infection manifestations, fulminant hepatitis B infection possesses the highest fatality rate. Almost 1% of the acutely infected patients develop fulminant hepatitis B, in which the mortality rate is around 70%. EVIDENCE ACQUISITION: All published papers deposited in Genbank, on the topic of fulminant hepatitis were reviewed and their virological aspects were investigated. In this review, we highlight the genomic diversity of HBV reported from patients with fulminant HBV infection. RESULTS: The most commonly detected diversities affect regulatory motifs of HBV in the core and S region, indicating that these alterations may convert the virus to an aggressive strain. Moreover, mutations at T-cell and B-cell epitopes located in pre-S1 and pre-S2 proteins may lead to an immune evasion of the virus, likely favoring a more severe clinical course of infection. Furthermore, point and frame shift mutations in the core region increase the viral replication of HBV and help virus to evade from immune system and guarantee its persistence. CONCLUSIONS: Fulminant hepatitis B is associated with distinct mutational patterns of HBV, underlining that genomic diversity of the virus is an important factor determining its pathogenicity.

Molecular identification of hepatitis B virus genotypes/subgenotypes: revised classification hurdles and updated resolutions.

The clinical course of infections with the hepatitis B virus (HBV) substantially varies between individuals, as a consequence of a complex interplay between viral, host, environmental and other factors. Due to the high genetic variability of HBV, the virus can be categorized into different HBV genotypes and subgenotypes, which considerably differ with respect to geographical distribution, transmission routes, disease progression, responses to antiviral therapy or vaccination, and clinical outcome measures such as cirrhosis or hepatocellular carcinoma. However, HBV (sub)genotyping has caused some controversies in the past due to misclassifications and incorrect interpretations of different genotyping methods. Thus, an accurate, holistic and dynamic classification system is essential. In this review article, we aimed at highlighting potential pitfalls in genetic and phylogenetic analyses of HBV and suggest novel terms for HBV classification. Analyzing full-length genome sequences when classifying genotypes and subgenotypes is the foremost prerequisite of this classification system. Careful attention must be paid to all aspects of phylogenetic analysis, such as bootstrapping values and meeting the necessary thresholds for (sub)genotyping. Quasi-subgenotype refers to subgenotypes that were incorrectly suggested to be novel. As many of these strains were misclassified due to genetic differences resulting from recombination, we propose the term "recombino-subgenotype". Moreover, immigration is an important confounding facet of global HBV distribution and substantially changes the geographic pattern of HBV (sub)genotypes. We therefore suggest the term "immigro-subgenotype" to distinguish exotic (sub)genotypes from native ones. We are strongly convinced that applying these two proposed terms in HBV classification will help harmonize this rapidly progressing field and allow for improved prophylaxis, diagnosis and treatment.

UVRAG is required for virus entry through combinatorial interaction with the class C-Vps complex and SNAREs.

Enveloped viruses exploit the endomembrane system to enter host cells. Through a cascade of membrane-trafficking events, virus-bearing vesicles fuse with acidic endosomes and/or lysosomes mediated by SNAREs triggering viral fusion. However, the molecular mechanisms underlying this process remain elusive. Here, we found that UV-radiation resistance-associated gene (UVRAG), an autophagic tumor suppressor, is required for the entry of the prototypic negative-strand RNA virus, including influenza A virus and vesicular stomatitis virus, by a mechanism independent of IFN and autophagy. UVRAG mediates viral endocytic transport and membrane penetration through interactions with the class C vacuolar protein sorting (C-Vps) tethering complex and endosomal glutamine-containing SNAREs [syntaxin 7 (STX7), STX8, and vesicle transport through t-SNARE homolog 1B (Vti1b)], leading to the assembly of a fusogenic trans-SNARE complex involving vesicle-associated membrane protein (VAMP8), but not VAMP7. Indeed, UVRAG stimulates VAMP8 translocation to virus-bearing endosomes. Inhibition of VAMP8, but not VAMP7, significantly reduces viral entry. Our data indicate that UVRAG, in concert with C-Vps, regulates viral entry by assembling a specific fusogenic SNARE complex. Thus, UVRAG governs downstream viral entry, highlighting an important pathway capable of potential antiviral therapeutics.

The antiviral effector IFITM3 disrupts intracellular cholesterol homeostasis to block viral entry.

Vesicle-membrane-protein-associated protein A (VAPA) and oxysterol-binding protein (OSBP) regulate intracellular cholesterol homeostasis, which is required for many virus infections. During entry, viruses or virus-containing vesicles can fuse with endosomal membranes to mediate the cytosolic release of virions, and alterations in endosomal cholesterol can inhibit this invasion step. We show that the antiviral effector protein interferon-inducible transmembrane protein 3 (IFITM3) interacts with VAPA and prevents its association with OSBP, thereby disrupting intracellular cholesterol homeostasis and inhibiting viral entry. By altering VAPA-OSBP function, IFITM3 induces a marked accumulation of cholesterol in multivesicular bodies and late endosomes, which inhibits the fusion of intraluminal virion-containing vesicles with endosomal membranes and thereby blocks virus release into the cytosol. Consequently, ectopic expression or depletion of the VAPA gene profoundly affects IFITM3-mediated inhibition of viral entry. Thus, IFITM3 disrupts intracellular cholesterol homeostasis to block viral entry, further underscoring the importance of cholesterol in virus infection.

Differential impact of immune escape mutations G145R and P120T on the replication of lamivudine-resistant hepatitis B virus e antigen-positive and -negative strains.

Immune escape variants of the hepatitis B virus (HBV) represent an emerging clinical challenge, because they can be associated with vaccine escape, HBV reactivation, and failure of diagnostic tests. Recent data suggest a preferential selection of immune escape mutants in distinct peripheral blood leukocyte compartments of infected individuals. We therefore systematically analyzed the functional impact of the most prevalent immune escape variants, the sG145R and sP120T mutants, on the viral replication efficacy and antiviral drug susceptibility of common treatment-associated mutants with resistance to lamivudine (LAM) and/or HBeAg negativity. Replication-competent HBV strains with sG145R or sP120T and LAM resistance (rtM204I or rtL180M/rtM204V) were generated on an HBeAg-positive and an HBeAg-negative background with precore (PC) and basal core promoter (BCP) mutants. The sG145R mutation strongly reduced HBsAg levels and was able to fully restore the impaired replication of LAM-resistant HBV mutants to the levels of wild-type HBV, and PC or BCP mutations further enhanced viral replication. Although the sP120T substitution also impaired HBsAg secretion, it did not enhance the replication of LAM-resistant clones. However, the concomitant occurrence of HBeAg negativity (PC/BCP), sP120T, and LAM resistance resulted in the restoration of replication to levels of wild-type HBV. In all clones with combined immune escape and LAM resistance mutations, the nucleotide analogues adefovir and tenofovir remained effective in suppressing viral replication in vitro. These findings reveal the differential impact of immune escape variants on the replication and drug susceptibility of complex HBV mutants, supporting the need of close surveillance and treatment adjustment in response to the selection of distinct mutational patterns.

Prevalence, viral replication efficiency and antiviral drug susceptibility of rtQ215 polymerase mutations within the hepatitis B virus genome.

BACKGROUND/AIMS: The rtQ215S mutation in the hepatitis B virus (HBV) polymerase has been described as a secondary mutation associated with resistance to lamivudine (LAM) and adefovir (ADV). We aimed at assessing the prevalence of substitutions at rtQ215 of the HBV polymerase and determining the molecular and functional consequences using phenotypic analyses in vitro. METHODS: The polymerase region was directly sequenced in HBV isolates from a cohort of 249 HBV genotype D-infected patients from Iran (174 males/75 females, 194 treatment-nai ve/ 55 LAM-treated). Replication-competent HBV constructs containing the naturally occurring rtQ215H, rtQ215P and rtQ215S mutations were generated, and compared to wild-type, LAM- (rtM204I, rtL180M/rtM204V) and ADV-resistant (rtN236T) clones. RESULTS: In an Iranian cohort of 249 HBV infected patients, 14.5% (36/249) showed mutations in the rtQ215 locus, namely 6.8% rtQ215S, 3.6% rtQ215P and 4.1% rtQ215H. The frequency of rtQ215 substitutions was higher in LAM-treated than treatment-nai ve patients (25% vs. 11%), but not associated with clinical complications. In phenotypic assays, rtQ215S, rtQ215P and rtQ215H constructs showed equivalent levels of viral replication as wild-type HBV, whereas LAM- and ADV-resistant mutants had significantly impaired replicative capacities. Furthermore, rtQ215S, rtQ215P and rtQ215H harbouring constructs remained susceptible towards treatment with LAM or ADV in vitro. CONCLUSIONS: Our study reveals that rtQ215 substitutions in the HBV polymerase frequently occur in chronic hepatitis B, even without exogenous selection pressures. As these substitutions do neither impair the viral replication efficiency nor susceptibility to LAM or ADV in vitro, rtQ215 substitutions likely represent background polymorphisms rather than resistance mutations with clinical implications.

The rtA194T polymerase mutation impacts viral replication and susceptibility to tenofovir in hepatitis B e antigen-positive and hepatitis B e antigen-negative hepatitis B virus strains.

UNLABELLED: Tenofovir is a new effective treatment option for patients with chronic hepatitis B, but could be potentially hampered by mutations in the hepatitis B virus (HBV) polymerase conferring drug resistance. Drug resistance may occur preferentially if long-term administration is required, for example, in patients with hepatitis B e antigen (HBeAg)-negative HBV infection bearing precore (PC) and basal core promoter (BCP) mutations. The rtA194T polymerase mutation has been found in HBV/HIV coinfected patients during tenofovir treatment and may be associated with tenofovir resistance. We generated replication-competent HBV constructs harboring rtA194T alone or in addition to lamivudine (LAM) resistance (rt180M + rtM204V), PC mutations, and BCP mutations and assessed their replicative capacity after transient transfection in human hepatoma cells. The rtA194T polymerase mutation alone or in conjunction with LAM resistance reduced the replication efficiency as compared with wild-type (WT) HBV. In contrast, combination of rtA194T (+/- LAM resistance) with HBeAg-negative PC or BCP mutants increased the replication capacity of the drug-resistant polymerase mutants, thereby restoring the viral replication to similar levels as WT clones. Clones harboring rtA194T showed partial resistance to tenofovir in vitro and also to LAM but remained susceptible to telbivudine and entecavir. CONCLUSION: The rtA194T polymerase mutation is associated with partial tenofovir drug resistance and negatively impacts replication competence of HBV constructs. Viral replication, however, can be restored to WT levels, if these polymerase mutations occur together with precore or basic core promoter substitutions as found in HBeAg-negative hepatitis B. Patients with HBeAg-negative chronic HBV infection may therefore be at particular risk when developing drug resistance to tenofovir. Telbivudine or entecavir should be considered as effective alternative treatment options for these patients.

Molecular analysis of an HBsAg-negative hepatitis B virus mutant selected in a tenofovir-treated HIV-hepatitis B virus co-infected patient.

The molecular analysis performed in an HIV-hepatitis B virus (HBV) coinfected patient revealed selection of an unusual HBV polymerase mutation (rtV191I) during tenofovir-containing therapy, conferring simultaneously immune escape by HBsAg negativity and resistance to lamivudine but not tenofovir. Phenotypic analysis revealed impaired replicative capacity of mutants, which could be restored by concomitant precore or basal core promoter mutations (HBe-antigen-negativity). HBV mutants carrying drug and vaccine resistance may represent a considerable individual risk and public health concern.

Clinical, virologic and phylogenetic features of hepatitis B infection in Iranian patients.

AIM: To characterize the clinical, serologic and virologic features of hepatitis B virus (HBV) infection in Iranian patients with different stages of liver disease. METHODS: Sixty two patients comprising of 12 inactive carriers, 30 chronic hepatitis patients, 13 patients with liver cirrhosis and 7 patients with hepatocellular carcinoma (HCC) were enrolled in the study. The HBV S, C and basal core promoter (BCP) regions were amplified and sequenced, and the clinical, serologic, phylogenetic and virologic characteristics were investigated. RESULTS: The study group consisted of 16 HBeAg-positive and 46 HBeAg-negative patients. Anti-HBe-positive patients were older and had higher levels of ALT, ASL and bilirubin compared to HBeAg-positive patients. Phylogenetic analysis revealed that all patients were infected with genotype D (mostly ayw2). The G1896A precore (PC) mutant was detected in 58.1% patients. HBeAg-negative patients showed a higher rate of PC mutant compared to HBeAg-positive patients (c2 = 9.682, P = 0.003). The majority of patients with HCC were HBeAg-negative and were infected with PC mutant variants. There was no significant difference in the occurrence of BCP mutation between the two groups, while the rate of BCP plus PC mutants was higher in HBeAg-negative patients (c2 = 4.308, P = 0.04). In the HBV S region, the genetic variability was low, and the marked substitution was P120T/S, with a rate of 9.7% (n = 6). CONCLUSION: In conclusion, HBV/D is the predominant genotype in Iran, and the nucleotide variability in the BCP and PC regions may play a role in HBV disease outcome in HBeAg-negative patients.

Hepatitis B virus (HBV) genotype and YMDD motif mutation profile among patients infected with HBV and untreated with lamivudine.

OBJECTIVES: A few reports exist on hepatitis B virus (HBV) genotype distribution in Iran; however the sample sizes of these studies are insufficient. The first objective of this study was to determine the HBV genotype distribution with a large sample size (147 specimens). The second objective was to determine the incidence of the lamivudine-resistant YMDD mutant profile among HBV-infected patients not treated with lamivudine; some studies have reported that YMDD mutants are detectable even before antiviral treatment. METHODS: We used two cost-effective PCR-based methods that have been developed in-house: gap-PCR and artificially created restriction site-PCR (ACRS-PCR). Also, 11 samples were randomly selected and bi-directionally sequenced and subjected to phylogenetic analysis. RESULTS: Gap-PCR results revealed genotype D of HBV in all patients, and ACRS-PCR results disclosed the absence of mutation within the YMDD motif before antiviral therapy in the study population. Phylogenetic analysis supported the former genotyping results with the segregation of all Iranian HBV isolates in the genotype D branch with a high bootstrap value (99%, 1000 replicates). CONCLUSIONS: The present study using two cost-effective methods showed that genotype D of HBV is dominant among Iranian HBV-infected subjects, and HBV lamivudine-resistant strains do not exist naturally among Iranian patients not treated with lamivudine.

A serological screening assay of human immunodeficiency virus type 1 antibodies based on recombinant protein p24-gp41 as a fusion protein expressed in Escherichia coli.

The objective of this study was expression of a recombinant fusion protein p24-gp41 to gain a proper folding pattern of the proteins which could be recognized by specific antibodies against human immunodeficiency virus type 1 (HIV-1) for development of a reliable serodiagnostic kit. Serodiagnostic method using enzyme-linked immunosorbent assay (ELISA) with the expressed recombinant fusion protein p24-gp41 was carried out to test the sensitivity and specificity of the protein using human sera and various reference panels from Boston Biomedica Inc. (BBI). The level of the expression was determined to be 30% and the final recovery from fermentation and purification process was calculated as 80 mg/L with more than 98% purity. The developed ELISA assay was demonstrated to have 100 and 99.5% sensitivity and specificity, respectively, detecting anti-HIV-1 antibody using 900 positive and 10,000 negative human sera. The developed assay showed reliable results in comparison with other reference HIV ELISA kits using various BBI panels as well. In conclusion, the recombinant fusion protein p24-gp41 was expressed and used to develop a serodiagnostic kit for screening of the HIV-1 with high sensitivity (100%) and specificity (99.5%) which could be useful for screening large groups of blood donors.

Complete genomic sequence and phylogenetic relatedness of hepatitis B virus isolates from Iran.

Hepatitis B virus (HBV) is one of the main etiological agents of acute and chronic liver disease that is still a major public health problem in the world. Numerous HBV isolates have grouped into eight genotypes, A to H, based on the complete genome sequence. To date, no study has been carried out on the complete HBV genome sequence in Iran. The objective of this study was to investigate the complete genome sequence organization and phylogenetic analysis of the five HBV strains, which obtained from Iranian chronic infected patients. Results showed that Iranian strains were closely related to each other, with 97-100% nucleotide similarity. Phylogenetic analysis based on the complete genome sequences and the precore/core gene sequences revealed that all strains were of genotype D, sub-genotype D1 with bootstrap value 100 and 99%, respectively. The S gene encoded Arg122, Pro127, and Lys160 corresponding to subtype ayw2. Iranian HBV isolates had closely related with Turkish HBV strains. All strains had a nucleotide length of 3,182 base pair (bp) except IR-P4 strain, with a 3,185 bp in length and with a unique Phe89 insertion in the X gene. The intragenotypic divergence of the complete genome sequence of Iranian strains was 1.8% and the intergenotypic in genotype D was 3.8% and with the other genotypes was 7.9-15.4%. In conclusion, this study revealed that the HBV genotype D, sub-genotype D1, subtype ayw2 dominates in the Iranian infected patients. A single Phe89 insertion in the X gene of the one Iranian strain with an unforeseen length of 3185 bp was identified.

Study of a polymerase chain reaction-based method for detection of herpes simplex virus type 1 DNA among Iranian patients with ocular herpetic keratitis infection.

PURPOSE: To study the presence of the herpes simplex virus type 1 (HSV-1) glycoprotein D gene in tear films of Iranian patients with herpetic keratitis. METHODS: Twenty-five tear film and eye swab specimens from 25 herpetic keratitis patients and 10 specimens from 10 healthy volunteers were collected in the Farabi Eye Hospital, Tehran, Iran. HSV-1 DNA was detected by using the nested polymerase chain reaction (nPCR) method. Viral isolation was done using conventional viral techniques. A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was used for confirmation of positive cytopathic effect cell culture. The results of a diagnosis by an ophthalmologist team were compared with those of nPCR. RESULTS: HSV-1 DNA was identified in tear films of 88% (23/25) of suspected herpetic keratitis patients. All healthy controls (100%) had negative PCR results. HSV-1 was isolated in cell culture and confirmed by ELISA in 12% (3/25) of herpetic keratitis patients who had epithelial keratitis. The kappa value showed a high level of agreement between ophthalmologist team diagnosis and the PCR results (kappa = 0.86, P < 0.0001). CONCLUSIONS: nPCR is a sensitive, rapid, and powerful tool for detection of HSV-1 DNA in tear films of ocular herpetic keratitis patients and can serve as a supplemental method for diagnosis of herpetic keratitis infection.