Babesia gibsoni: Identification, expression, localization, and serological characterization of a Babesia gibsoni 22-kDa protein

Babesia gibsoni causes canine babesiosis. Here, we describe the identification and characterization of a novel gene, BgP22, containing an open reading frame of 621 bp and encoding a 22-kDa protein from B. gibsoni, as a serodiagnostic candidate. The recombinant BgP22 (rBgP22) was expressed and used as an antigen to produce anti-rBgP22 sera in mice. Using these anti-rBgP22 sera, a native 22-kDa protein was recognized by Western blot analysis and observed in the membrane of the parasites by immunofluorescent antibody tests (IFAT). The enzyme-linked immunosorbent assay (ELISA) using the rBgP22 detected specific antibodies to this protein in the sera of dogs experimentally and naturally infected with B. gibsoni in chronic stage. Furthermore, it did not show a cross reaction with the closely related apicomplexan parasites, indicating that the rBgP22 could be used as a diagnostic antigen for a detection of the chronic carrier stages of B. gibsoni infection.     

Molecular characterization and antigenic properties of a novel Babesia gibsoni glutamic acid-rich protein (BgGARP)

Identification and molecular characterization of Babesia gibsoni proteins with potential antigenic properties are crucial for the development and validation of the serodiagnostic method. In this study, we isolated a cDNA clone encoding a novel B. gibsoni 76-kDa protein by immunoscreening of the parasite cDNA library. Computer analysis revealed that the protein presents a glutamic acid-rich region in the C-terminal. Therefore, the protein was designated as B. gibsoni glutamic acid-rich protein (BgGARP). A BLASTp analysis of a translated BgGARP polypeptide demonstrated that the peptide shared a significant homology with a 200-kDa protein of Babesia bigemina and Babesia bovis. A truncated BgGARP cDNA (BgGARPt) encoding a predicted 13-kDa peptide was expressed in Escherichia coli (E. coli), and mouse antisera against the recombinant protein were used to characterize a corresponding native protein. The antiserum against recombinant BgGARPt (rBgGARPt) recognized a 140-kDa protein in the lysate of infected erythrocytes, which was detectable in the cytoplasm of the parasites by confocal microscopic observation. In addition, the specificity and sensitivity of enzyme-linked immunosorbent assay (ELISA) with rBgGARPt were evaluated using B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. Moreover, 107 serum samples from dogs clinically diagnosed with babesiosis were examined using ELISA with rBgGARPt. The results showed that 86 (80.4%) samples were positive by rBgGARPt-ELISA, which was comparable to IFAT and PCR as reference test. Taken together, these results demonstrate that BgGARP is a suitable serodiagnostic antigen for detecting antibodies against B. gibsoni in dogs.     

Recombinant expression of in silico identified Bcell epitope of epsilon toxin of Clostridium perfringens in translational fusion with a carrier protein

Epsilon toxin secreted by Clostridium perfringens types B and D has been directly implicated as the causative agent of fatal enterotoxemia in domestic animals. The aim of the present study is to use in silico approach for identification of B-cell epitope(s) of epsilon toxin, and its expression in fusion with a carrier protein to analyze its potential as vaccine candidate(s). Using different computational analyses and bioinformatics tools, a number of antigenic determinant regions of epsilon toxin were identified. One of the B cell epitopes of epsilon toxin comprising the region (amino acids 40-62) was identified as a promising antigenic determinant. This Etx epitope (Etx_40-62) was cloned and expressed as a translational fusion with B-subunit of heat labile enterotoxin (LTB) of E. coli in a secretory expression system. Similar to the native LTB, the recombinant fusion protein retained the ability to pentamerize and bind to GM₁ ganglioside receptor of LTB. The rLTB.Etx_40-62 could be detected both with anti-Etx and anti-LTB antisera. The rLTB.Etx_40-62 fusion protein thus can be evaluated as a potential vaccine candidate against C. perfringens.

Abbreviations

aa - amino acid(s), Etx - epsilon toxin of Clostridium perfringens, LTB - B-subunit of heat labile enterotoxin of E. coli.     

Computational identification of putative common genomic drug and vaccine targets in Mycoplasma genitalium

Mycoplasma genitalium is an obligate intracellular bacterium that is responsible for several sexually transmitted infections, including non-gonococcal urethritis in men and several inflammatory reproductive tract syndromes in women. Here, we applied subtractive genomics and reverse vaccinology approaches for in silico prediction of potential vaccine and drug targets against five strains of M. genitalium. We identified 403 genes shared by all five strains, from which 104 non-host homologous proteins were selected, comprising of 44 exposed/secreted/membrane proteins and 60 cytoplasmic proteins. Based on the essentiality, functionality, and structure-based binding affinity, we finally predicted 19 (14 novel) putative vaccine and 7 (2 novel) candidate drug targets. The docking analysis showed six molecules from the ZINC database as promising drug candidates against the identified targets. Altogether, both vaccine candidates and drug targets identified here may contribute to the future development of therapeutic strategies to control the spread of M. genitalium worldwide.     

Genetic polymorphisms in the glutamate-rich protein of Plasmodium falciparum field isolates from a malaria-endemic area of Brazil

The genetic diversity displayed by Plasmodium falciparum, the most deadly Plasmodium species, is a significant obstacle for effective malaria vaccine development. In this study, we identified genetic polymorphisms in P. falciparum glutamate-rich protein (GLURP), which is currently being tested in clinical trials as a malaria vaccine candidate, from isolates found circulating in the Brazilian Amazon at variable transmission levels. The study was performed using samples collected in 1993 and 2008 from rural villages situated near Porto Velho, in the state of Rondônia. DNA was extracted from 126 P. falciparum-positive thick blood smears using the phenol-chloroform method and subjected to a nested polymerase chain reaction protocol with specific primers against two immunodominant regions of GLURP, R0 and R2. Only one R0 fragment and four variants of the R2 fragment were detected. No differences were observed between the two time points with regard to the frequencies of the fragment variants. Mixed infections were uncommon. Our results demonstrate conservation of GLURP-R0 and limited polymorphic variation of GLURP-R2 in P. falciparum isolates from individuals living in Porto Velho. This is an important finding, as genetic polymorphisms in B and T-cell epitopes could have implications for the immunological properties of the antigen.     

Artesunate,a potential drug for treatment of Babesia infection

The effects of artesunate, a water-soluble artemisinin derivative, against Babesia species, including Babesia bovis, Babesia gibsoni and Babesia microti were studied. Cultures of B. bovis and B. gibsoni were treated with 0.26, 2.6, 26 and 260 μM artesunate, showing inhibition of parasite growth at concentrations equal to and greater than 2.6 μM artesunate by days 3 post-treatment for B. gibsoni and B. bovis in a dose-dependent manner. Consistent with in vitro experiments, artesunate was effective in the treatment of mice infected with B. microti at doses equal to and greater than 10 mg/kg of body weight on days 8–10 post-infection. Taken together, these results suggest that artesunate could be a potential drug against Babesia infection.     

Sequence and phylogenetic analysis of the thrombospondin-related adhesive protein gene of Babesia gibsoni isolates in dogs in South India

Babesia gibsoni, the causative agent of canine piroplasmosis, is a tick-borne intraerythrocytic protozoan parasite predominantly reported in Asian countries. The present study aimed at genotypic characterization of B. gibsoni isolates prevalent in dogs in Kerala, a southern state of India. Blood samples were collected from 272 dogs in Kerala and B. gibsoni infection was detected by microscopy and polymerase chain reaction (PCR). Molecular confirmation of B. gibsoni parasites was carried out by 18S rRNA nested-PCR, followed by sequencing. Nested-PCR detected a higher percentage of dogs (40.44%) positive for B. gibsoni infection than microscopy where 15.81% dogs were detected positive for infection. Genetic characterization of B. gibsoni isolates (n = 11) prevalent in dogs in the state of Kerala was carried out by PCR amplification and sequencing of the 855 bp thrombospondin-related adhesive protein (TRAP) gene fragment. Phylogenetic analysis of the B. gibsoni TRAP (BgTRAP) gene revealed that B. gibsoni isolates from Kerala formed a distinct cluster with the isolates from north India and Bangladesh, away from other East Asian isolates. Nucleotide analysis of the tandem repeats of BgTRAP gene showed considerable genetic variation among Indian isolates that was shared by B. gibsoni isolates of Bangladesh but not by the isolates of East Asian countries. The results of the present study further confirmed that B. gibsoni parasites in a distinct genetic clade are endemic in dogs in India and Bangladesh. However, elaborate studies are required for better understanding of the genetic diversity of B. gibsoni.     

Closing the empty anti-Babesia gibsoni drug pipeline in vitro using fluorescence-based high throughput screening assay

In the current study, we evaluated the usefulness of a SYBR Green I (SG I) fluorescence assay for evaluation of the inhibitory effects of antibabesial drugs against the in vitro growth of Babesia gibsoni. Linearity and high-throughput screening (HTS) assays exhibited the validity of the SG I fluorescence assay for B. gibsoni parasite when performed at low hematocrits (HCTs) (2.5% and 5%) without daily changing of the medium. Interestingly, 5% HCT showed the highest value of the signal/noise ratio. Of note, there were no significant differences (P > .05) in the IC₅₀s of the commonly used antibabesial drugs (diminazene aceturate and/or imidocarb dipropionate) that calculated by either the SG I fluorescence assay with and without daily medium changing or by the fluorescence and microscopy methods at 2.5% and 5% HCTs. Such results confirmed that both HCTs are valid for mass drug screening against the in vitro growth of B. gibsoni. While the results of the HTS assay add merit to the assay when performed at 5% HCT especially when incubating the plates for 2 h in a dark after adding lysis buffer with SG I stain. Next, nine different drugs were screened to confirm the assay's usefulness. MMV396693, pyronaridine tetraphosphate and nerolidol drugs exhibited the highest effectiveness against the in vitro growth of B. gibsoni, next to diminazene aceturate. In summary, SG I fluorescence assay with 5% HCT without daily changing of the medium for B. gibsoni offers a novel approach for the large-scale screening of huge chemical libraries in in vitro cultures.     

Immunogenicity and growth inhibitory efficacy of the prime-boost immunization regime with DNA followed by recombinant vaccinia virus carrying the P29 gene of Babesia gibsoni in dogs

In recent studies, heterologous prime-boost approaches, employing plasmid DNA and viral vector pathogen-delivering sequences, have been considered an effective protection strategy for intracellular parasite infections. Here, we evaluated the efficacy of such a strategy against the canine Babesia gibsoni infection. The DNA (pCAGGS-P29) and recombinant vaccinia virus (vvP29) both encoding the P29 of B. gibsoni were used in this study. The dogs were immunized 3 times with priming DNA and boosted once with recombinant virus. The dogs immunized with P29 developed a significant level of IgG2 antibody against P29. The response was strongly boosted by the inoculation of vvP29. The peripheral IFN-gamma responses of the dogs immunized with P29 were significantly higher than those of controls after the parasite inoculation. Moreover, the P29 immunized group showed a significantly low level of parasitemia. In conclusion, this study supports the efficacy of a prime-boost strategy for dogs against canine B. gibsoni infection.     

In Vitro Infectivity Assessment by Drug Susceptibility Comparison of Recombinant Leishmania major Expressing Enhanced Green Fluorescent Protein or EGFP-Luciferase Fused Genes with Wild-Type Parasite

Leishmaniasis is a worldwide uncontrolled parasitic disease due to the lack of effective drug and vaccine. To speed up effective drug development, we need powerful methods to rapidly assess drug effectiveness against the intracellular form of Leishmania in high throughput assays. Reporter gene technology has proven to be an excellent tool for drug screening in vitro. The effects of reporter proteins on parasite infectivity should be identified both in vitro and in vivo. In this research, we initially compared the infectivity rate of recombinant Leishmania major expressing stably enhanced green fluorescent protein (EGFP) alone or EGFP-luciferase (EGFP-LUC) with the wild-type strain. Next, we evaluated the sensitivity of these parasites to amphotericin B (AmB) as a standard drug in 2 parasitic phases, promastigote and amastigote. This comparison was made by MTT and nitric oxide (NO) assay and by quantifying the specific signals derived from reporter genes like EGFP intensity and luciferase activity. To study the amastigote form, both B10R and THP-1 macrophage cell lines were infected in the stationary phase and were exposed to AmB at different time points. Our results clearly revealed that the 3 parasite lines had similar in vitro infectivity rates with comparable parasite-induced levels of NO following interferon-γ/lipopolysaccharide induction. Based on our results we proposed the more reporter gene, the faster and more sensitive evaluation of the drug efficiency.     

Characterization of the Babesia gibsoni 12-kDa protein as a potential antigen for the serodiagnosis

A novel gene, BgP12, encoding a 12-kDa protein was identified from Babesia gibsoni. The full-length cDNA of BgP12 contains an open reading frame of 378 bp, corresponding to 126 amino acid (aa) residues consisting of a putative 26 aa signal peptide and a 100 aa mature protein. The recombinant BgP12 (rBgP12) lacking the N-terminal signal peptide was expressed in Escherichia coli as a soluble glutathione S-transferase (GST) fusion protein (rBgP12) that produced an anti-rBgP12 serum in mice after immunization. Using this anti-rBgP12 serum, a native 12-kDa protein in B. gibsoni was recognized by Western blot analysis. Immunofluorescent antibody tests (IFAT) revealed that BgP12 was mainly seen during the ring stage of B. gibsoni trophozoite. An indirect enzyme-linked immunosorbent assay (ELISA) using the rBgP12 detected specific antibodies in the sequential sera of a dog experimentally infected with B. gibsoni beginning 10 days post-infection to 442 days post-infection, even when the dog became chronically infected and showed a low level of parasitemia. Moreover, the antigen did not show cross-reaction with antibodies to the closely related apicomplexan parasites, indicating that the rBgP12 might be an immunodominant antigen for B. gibsoni infection that could be used as a diagnostic antigen for B. gibsoni infection with high specificity and sensitivity.     

Gene Cloning,Expression and Immunogenicity of the Protective Antigen Subolesin in Dermacentor silvarum

Subolesin (4D8), the ortholog of insect akirins, is a highly conserved protective antigen and thus has the potential for development of a broad-spectrum vaccine against ticks and mosquitoes. To date, no protective antigens have been characterized nor tested as candidate vaccines against Dermacentor silvarum bites and transmission of associated pathogens. In this study, we cloned the open reading frame (ORF) of D. silvarum 4D8 cDNA (Ds4D8), which consisted of 498 bp encoding 165 amino acid residues. The results of sequence alignments and phylogenetic analysis demonstrated that D. silvarum 4D8 (Ds4D8) is highly conserved showing more than 81% identity of amino acid sequences with those of other hard ticks. Additionally, Ds4D8 containing restriction sites was ligated into the pET-32(a+) expression vector and the recombinant plasmid was transformed into Escherichia coli rosetta. The recombinant Ds4D8 (rDs4D8) was induced by isopropyl β-D-thiogalactopyranoside (IPTG) and purified using Ni affinity chromatography. The SDS-PAGE results showed that the molecular weight of rDs4D8 was 40 kDa, which was consistent with the expected molecular mass considering 22 kDa histidine-tagged thioredoxin (TRX) protein from the expression vector. Western blot results showed that rabbit anti-D. silvarum serum recognized the expressed rDs4D8, suggesting an immune response against rDs4D8. These results provided the basis for developing a candidate vaccine against D. silvarum ticks and transmission of associated pathogens.     

In vitro screening of compounds from the Food and Drug Administration-approved library identifies anti-Babesia gibsoni activity of idarubicin hydrochloride and vorinostat

Babesia gibsoni is mainly transmitted by hard ticks of the genus Rhipicephalus (R. sanguineus) and Haemaphysalis (H. longicornis), and causes canine babesiosis. Clinical manifestations of B. gibsoni infection include fever, hemoglobinemia, hemoglobinuria, and progressive anemia. Traditional antibabesial therapy, such as imidocarb dipropionate or diminazene aceturate, can only alleviate severe clinical manifestations and cannot eliminate parasites in the host. Food and Drug Administration (FDA)-approved drugs are a solid starting point for researching novel therapy strategies for canine babesiosis. In this work, we screened 640 FDA-approved drugs against the growth of B. gibsoni in vitro. Among them, 13 compounds (at 10 μM) exhibited high growth inhibition (>60%), and two compounds, namely idarubicin hydrochloride (idamycin) and vorinostat, were chosen for further investigation. The half-maximal inhibitory concentration (IC₅₀) values of idamycin and vorinostat were determined to be 0.044 ± 0.008 μM and 0.591 ± 0.107 μM, respectively. Viability results indicated that a concentration of 4 × IC₅₀ of vorinostat prevented the regrowth of treated B. gibsoni, whereas parasites treated with 4 × IC₅₀ concentration of idamycin remained viable. The B. gibsoni parasites treated with vorinostat exhibited degeneration within erythrocytes and merozoites, in contrast to the oval or signet-ring shape of normal B. gibsoni parasites. In conclusion, FDA-approved drugs offer a valuable platform for drug repositioning in antibabesiosis research. Particularly, vorinostat demonstrated promising inhibitory effects against B. gibsoni in vitro, and further studies on vorinostat are necessary to elucidate its mechanism as a novel treatment in infected animal models.     

in silico analyses of metabolic pathway and protein interaction network for identification of next gen therapeutic targets in Chlamydophila pneumoniae

Chlamydophila pneumoniae, the causative agent of chronic obstructive pulmonary disease (COPD), is presently the fifth mortality causing chronic disease in the world. The understanding of disease and treatment options are limited represents a severe concern and a need for better therapeutics. With the advancements in the field of complete genome sequencing and computational approaches development have lead to metabolic pathway analysis and protein-protein interaction network which provides vital evidence to the protein function and has been appropriate to the fields such as systems biology and drug discovery. Protein interaction network analysis allows us to predict the most potential drug targets among large number of the non-homologous proteins involved in the unique metabolic pathway. A computational comparative metabolic pathway analysis of the host H. sapiens and the pathogen C pneumoniae AR39 has been carried out at three level analyses. Firstly, metabolic pathway analysis was performed to identify unique metabolic pathways and non-homologous proteins were identified. Secondly, essentiality of the proteins was checked, where these proteins contribute to the growth and survival of the organism. Finally these proteins were further subjected to predict protein interaction networks. Among the total 65 pathways in the C pneumoniae AR39 genome 10 were identified as the unique metabolic pathways which were not found in the human host, 32 enzymes were predicted as essential and these proteins were considered for protein interaction analysis, later using various criteria''s we have narrowed down to prioritize ribonucleotide-diphosphate reductase subunit beta as a potential drug target which facilitate for the successful entry into drug designing.     

In vitro screening of novel anti-Babesia gibsoni drugs from natural products

Babesia gibsoni is a tick-transmitted intraerythrocytic apicomplexan parasite that causes babesiosis in dogs. Due to the strong side effects and lack of efficacy of current drugs, novel drugs against B. gibsoni are urgently needed. Natural products as a source for new drugs is a good choice for screening drugs against B. gibsoni. The current study focuses on identifying novel potential drugs from natural products against B. gibsoni in vitro. Parasite inhibition was verified using a SYBR green I-based fluorescence assay. A total of 502 natural product compounds were screened for anti-B. gibsoni activity in vitro. Twenty-four compounds showed high growth inhibition (>80%) on B. gibsoni and 5 plant-derived compounds were selected for further study. The half-maximal inhibitory concentration (IC₅₀) values of lycorine (LY), vincristine sulfate (VS), emetine·2HCl (EME), harringtonine (HT) and cephaeline·HBr (CEP) were 784.4 ± 3.3, 643.0 ± 2.8, 253.1 ± 1.4, 23.4 ± 1.2, and 108.1 ± 4.3 nM, respectively. The Madin-Darby canine kidney (MDCK) cell line was used to assess cytotoxicity of hit compounds. All compounds showed minimal toxicity to the MDCK cells. The effects of hit compounds combined with diminazene aceturate (DA) on B. gibsoni were further evaluated in vitro. VS, EME, HT or CEP combined with DA showed synergistic effects against B. gibsoni, whereas LY combined with DA showed an antagonistic effect against B. gibsoni. The results obtained in this study indicate that LY, VS, EME, HT and CEP are promising compounds for B. gibsoni treatment.     

Diversity of Babesia and Theileria species in symptomatic and asymptomatic dogs in Croatia

Babesiosis, the disease caused by tick-borne hematozoan parasites of the genus Babesia, is particularly common in dogs, and is caused by several “large” species of Babesia, as well as by an increasing number of “small” species of Babesia, some of which appear to be more closely related to members of the genus Theileria. In this work, blood samples were collected from 848 randomly selected, asymptomatic dogs and from 81 symptomatic dogs, microscopically positive for Babesia, and characterised by PCR and sequence analysis of a fragment of the ssrRNA gene. A prevalence of 3.42% (29 of 848) was found in asymptomatic dogs and sequence analysis revealed the presence of Babesia canis canis in 20 dogs (69%), Babesia gibsoni in six dogs (21%), Babesia canis vogeli in two dogs (7%) and Theileria annae in one dog (3%). In the group of symptomatic dogs, which were all positive by PCR, B. canis canis was the predominant species (78 dogs, or 96%), followed by single infections with B. canis vogeli, Babesia caballi and Theileria equi. Our study has confirmed that dogs are infected with a wide range of both large and small piroplasm species and subspecies, including B. caballi and T. equi, two parasites usually found in horses. The detection of the pathogenic species B. canis canis and B. gibsoni in asymptomatic dogs indicates that the relationship between parasite species/subspecies and clinical signs of infection in dogs deserves further investigation. Finally, the identities of the tick vectors transmitting T. annae and B. caballi remain to be elucidated.     

Reproduction of Belonolaimus longicaudatus,Meloidogyne javanica,Paratrichodorus minor,and Pratylenchus brachyurus on Pearl Millet (Pennisetum glaucum)

Pearl millet (Pennisetum glaucum) has potential as a grain crop for dryland crop production in the southeastern United States. Whether or not pearl millet will be compatible in rotation with cotton (Gossypium hirsutum), corn (Zea mays), and peanut (Arachis hypogaea) will depend, in part, on its host status for important plant-parasitic nematodes of these crops. The pearl millet hybrid ''TifGrain 102'' is resistant to both Meloidogyne incognita race 3 and M. arenaria race 1; however, its host status for other plant-parasitic nematodes was unknown. In this study, the reproduction of Belonolaimus longicaudatus, Paratrichodorus minor, Pratylenchus brachyurus, and Meloidogyne javanica race 3 on pearl millet (''HGM-100'' and TifGrain 102) was compared relative to cotton, corn, and peanut. Separate greenhouse experiments were conducted for each nematode species. Reproduction of B. longicaudatus was lower on peanut and the two millet hybrids than on cotton and corn. Reproduction of P. minor was lower on peanut and TifGrain 102 than on cotton, corn, and HGM-100. Reproduction of P. brachyurus was lower on both millet hybrids than on cotton, corn, and peanut. Reproduction of M. javanica race 3 was greater on peanut than on the two millet hybrids and corn. Cotton was a nonhost. TifGrain 102 was more resistant than HGM-100 to reproduction of B. longicaudatus, P. minor, and M. javanica. Our results demonstrated that TifGrain 102 was a poor host for B. longicaudatus and P. brachyurus (Rf < 1) and, relative to other crops tested, was less likely to increase densities of P. minor and M. javanica.     

Association of polymorphisms in the interleukin-4 gene with response to hepatitis B vaccine and susceptibility to hepatitis B virus infection: A meta-analysis

Objective

The aim of this meta-analysis is to evaluate the associations between functional polymorphisms in the interleukin-4 (IL4) gene and individuals' responses to hepatitis B vaccine and their susceptibility to hepatitis B virus (HBV) infection.

Methods

A literature search on articles published before December 1st, 2012 was conducted in PubMed, Embase, Web of Science and China BioMedicine (CBM) databases. Crude odds ratios (ORs) with 95% confidence intervals (CIs) were calculated. Statistical analyses were performed using the STATA 12.0 software.

Results

Eight studies were eligible for inclusion in this meta-analysis, including five cross-sectional studies on individual's response to hepatitis B vaccine and three case–control studies on HBV infection risk. The meta-analysis results showed that the T allele of rs2243250, the T allele of rs2070874, and the C allele of rs2227284 in IL4 gene were associated with high responses to hepatitis B vaccine. Further subgroup analysis by ethnicity showed that there was a significant association between IL4 genetic polymorphisms and an individual's responses to hepatitis B vaccine among Asian populations, but similar association was not found among Caucasian populations. However, there was no evidence indicating a correlation between IL4 genetic polymorphism and susceptibility to HBV infection.

Conclusion

Our current meta-analysis suggests that rs2243250, rs2070874 and rs2227284 polymorphisms in IL4 gene may play an important role in determining the response to hepatitis B vaccine, especially among Asian populations. However, further studies are still needed to evaluate the associations between IL4 genetic polymorphisms and HBV infection risk.