Expression and Purification of His-Tagged Variants of Human Hepatitis A Virus 3C Protease.

BACKGROUND: Protease 3C (3Cpro) is the only protease encoded in the human hepatitis A virus genome and is considered as a potential target for antiviral drugs due to its critical role in the viral life cycle. Additionally, 3Cpro has been identified as a potent inducer of ferroptosis, a newly described type of cell death. Therefore, studying the molecular mechanism of 3Cpro functioning can provide new insights into viral-host interaction and the biological role of ferroptosis. However, such studies require a reliable technique for producing the functionally active recombinant enzyme. OBJECTIVE: Here, we expressed different modified forms of 3Cpro with a hexahistidine tag on the N- or C-terminus to investigate the applicability of immobilized metal Ion affinity chromatography (IMAC) for producing 3Cpro. METHODS: We expressed the proteins in Escherichia coli and purified them using IMAC, followed by gel permeation chromatography. The enzymatic activity of the produced proteins was assayed using a specific chromogenic substrate. RESULTS: Our findings showed that the introduction and position of the hexahistidine tag did not affect the activity of the enzyme. However, the yield of the target protein was highest for the variant with seven C-terminal residues replaced by a hexahistidine sequence. CONCLUSION: We demonstrated the applicability of our approach for producing recombinant, enzymatically active 3Cpro.

Expression, purification, and crystal structure of mpox virus A41 protein.

Mpox is a zoonotic disease that was once endemic in Africa countries caused by mpox virus. However, cases recently have been confirmed in many non-endemic countries outside of Africa. The rapidly increasing number of confirmed mpox cases poses a threat to the international community. In-depth studies of key viral factors are urgently needed, which will inform the design of multiple antiviral agents. Mpox virus A41L gene encodes a secreted protein, A41, that is nonessential for viral replication, but could affect the host response to infection via interacting with chemokines. Here, mpox virus A41 protein was expressed in Sf9 cells, and purified by affinity chromatography followed by gel filtration. Surface plasmon resonance spectroscopy showed that purified A41 binds a certain human chemokine CXCL8 with the equilibrium dissociation constant (KD) being 1.22 × 10-6 M. The crystal structure of mpox virus A41 protein was solved at 1.92 Å. Structural analysis and comparison revealed that mpox virus A41 protein adopts a characteristic ß-sheet topology, showing minor differences with that of vaccinia virus. These preliminary structural and functional studies of A41 protein from mpox virus will help us better understand its role in chemokine subversion, and contributing to the knowledge to viral chemokine binding proteins.

A Novel Wide-Range Freshwater Cyanophage MinS1 Infecting the Harmful Cyanobacterium Microcystis aeruginosa.

Microcystis aeruginosa, as one of the major players in algal bloom, produces microcystins, which are strongly hepatotoxic, endangering human health and damaging the ecological environment. Biological control of the overgrowth of Microcystis with cyanophage has been proposed to be a promising solution for algal bloom. In this study, a novel strain of Microcystis cyanophage, MinS1, was isolated. MinS1 contains an icosahedral head approximately 54 nm in diameter and a 260 nm-long non-contractile tail. The phage genome consists of a linear, double-stranded 49,966 bp DNA molecule, which shares very low homology with known phages in the NCBI database (only 1% of the genome showed weak homology with known phages when analyzed by megablast). The phage contains 75 ORFs, of which 23 ORFs were predicted to code for proteins of known function, 39 ORFs were predicted to code for proteins of unknown function, and 13 ORFs showed no similarity to any protein sequences. Transmission electron microscopy and phylogenetic analysis showed that MinS1 belongs to the family Siphoviridae. Various experiments confirmed that the phage could infect several different orders of cyanobacteria, including Chroococcales, Nostocales, Oscillatoriales, Hormogonales, and Synechococcales, indicating that it has a very broad host range. In addition, MinS1 has no known antibiotic tolerance genes, virulence genes, and tRNAs, and it is tolerant to temperature, pH, UV, and salinity, suggesting that MinS1 has good potential for application as a biological control agent against cyanobacterial blooms. This study expands the diversity and knowledge of cyanophages, and it provides useful information for the development of novel prevention and control measures against cyanobacterial blooms.

Prolonged SARS-CoV-2 Positivity in Immunocompetent Patients: Virus Isolation, Genomic Integrity, and Transmission Risk.

Current guidelines for patient isolation in COVID-19 cases recommend a symptom-based approach, averting the use of control real-time reverse transcription PCR (rRT-PCR) testing. However, we hypothesized that patients with persistently positive results by RT-PCR for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could be potentially infectious for a prolonged time, even if immunocompetent and asymptomatic, which would demand a longer social isolation period than presently recommended. To test this hypothesis, 72 samples from 51 mildly symptomatic immunocompetent patients with long-lasting positive rRT-PCR results for SARS-CoV-2 were tested for their infectiousness in cell culture. The serological response of samples from those patients and virus genomic integrity were also analyzed. Infectious viruses were successfully isolated from 34.38% (22/64) of nasopharynx samples obtained 14 days or longer after symptom onset. Indeed, we observed successful virus isolation up to 128 days. Complete SARS-COV-2 genome integrity was demonstrated, suggesting the presence of replication-competent viruses. No correlation was found between the isolation of infectious viruses and rRT-PCR cycle threshold values or the humoral immune response. These findings call attention to the need to review current isolation guidelines, particularly in scenarios involving high-risk individuals. IMPORTANCE In this study, we evaluated mildly symptomatic immunocompetent patients with long-lasting positive rRT-PCR results for SARS-CoV-2. Infectious viruses were successfully isolated in cell cultures from nasopharynx samples obtained 14 days or longer after symptom onset. Indeed, we observed successful virus isolation for up to 128 days. Moreover, SARS-CoV-2 genome integrity was demonstrated by sequencing, suggesting the presence of replication-competent viruses. These data point out the risk of continuous SARS-CoV-2 transmission from patients with prolonged detection of SARS-CoV-2 in the upper respiratory tract, which has important implications for current precaution guidelines, particularly in settings where vulnerable individuals may be exposed (e.g., nursing homes and hospitals).

Development of a Purified Viral Preparation for Studies of COVID-19 (SARS-CoV-2) Biology.

Different methods for producing bulk quantities of concentrated and purified SARS-CoV-2 for the use as antigens and for the research into COVID-19 biology were tested.

Targeted proteomics as a tool to detect SARS-CoV-2 proteins in clinical specimens.

The rapid, sensitive and specific detection of SARS-CoV-2 is critical in responding to the current COVID-19 outbreak. In this proof-of-concept study, we explored the potential of targeted mass spectrometry (MS) based proteomics for the detection of SARS-CoV-2 proteins in both research samples and clinical specimens. First, we assessed the limit of detection for several SARS-CoV-2 proteins by parallel reaction monitoring (PRM) MS in infected Vero E6 cells. For tryptic peptides of Nucleocapsid protein, the limit of detection was estimated to be in the mid-attomole range (9E-13 g). Next, this PRM methodology was applied to the detection of viral proteins in various COVID-19 patient clinical specimens, such as sputum and nasopharyngeal swabs. SARS-CoV-2 proteins were detected in these samples with high sensitivity in all specimens with PCR Ct values <24 and in several samples with higher CT values. A clear relationship was observed between summed MS peak intensities for SARS-CoV-2 proteins and Ct values reflecting the abundance of viral RNA. Taken together, these results suggest that targeted MS based proteomics may have the potential to be used as an additional tool in COVID-19 diagnostics.

Expression and purification of phage T7 ejection proteins for cryo-EM analysis.

Bacteriophages of the Podoviridae family densely package their genomes into precursor capsids alongside internal virion proteins called ejection proteins. In phage T7 these proteins (gp14, gp15, and gp16) are ejected into the host envelope forming a DNA-ejectosome for genome delivery. Here, we describe the purification and characterization of recombinant gp14, gp15, and gp16. This protocol was used for high-resolution cryo-EM structure analysis of the T7 periplasmic tunnel and can be adapted to study ejection proteins from other phages. For complete details on the use and execution of this protocol, please refer to Swanson et al. (2021).

Characterization of a Novel Phage ΦAb1656-2 and Its Endolysin with Higher Antimicrobial Activity against Multidrug-Resistant Acinetobacter baumannii.

Acinetobacter baumannii is a nosocomial pathogen, which is a problem worldwide due to the emergence of a difficult-to-treat multidrug-resistant A. baumannii (MDRAB). Endolysins are hydrolytic enzymes produced by a bacteriophage that can be used as a potential therapeutic agent for multidrug-resistant bacterial infection in replacing antibiotics. Here, we isolated a novel bacteriophage through prophage induction using mitomycin C from clinical A. baumannii 1656-2. Morphologically, ΦAb1656-2 was identified as a Siphoviridae family bacteriophage, which can infect MDRAB. The whole genome of ΦAb1656-2 was sequenced, and it showed that it is 50.9 kb with a G + C content of 38.6% and 68 putative open reading frames (ORFs). A novel endolysin named AbEndolysin with an N-acetylmuramidase-containing catalytic domain was identified, expressed, and purified from ΦAb1656-2. Recombinant AbEndolysin showed significant antibacterial activity against MDRAB clinical strains without any outer membrane permeabilizer. These results suggest that AbEndolysin could represent a potential antimicrobial agent for treating MDRAB clinical isolates.

Conjugation of Zika virus EDIII with CRM197, 8-arm PEG and mannan for development of an effective Zika virus vaccine.

Zika virus (ZIKV) induces neurological and autoimmune complications such as microcephaly and Guillain-Barre syndrome. Effective vaccines are necessary to prevent the ZIKV infection. E protein of ZIKV is responsible for virus attachment, entry, and fusion. The domain III of E protein (EDIII) contains the neutralizing epitopes and is ideal to act as an antigen for ZIKV vaccine. However, EDIII is poorly immunogenic. CRM197 is a carrier protein and can activate T helper cells for EDIII. Mannan is a ligand of TLR-4 or TLR-2. Eight-arm PEG can link multiple EDIII molecules in one entity. In the present study, EDIII was covalently conjugated with CRM197, 8-arm PEG and mannan to improve the immunogenicity of EDIII. The conjugate (CRM-EDIII-PM) elicited high EDIII-specific antibody titers in the BALB/c mice. Th1-type cytokines (IFN-γ and IL-2) and Th2-type cytokines (IL-5 and IL-10) were secreted at a marked level. Thus, CRM-EDIII-PM could stimulate potent humoral and cellular immune response to EDIII. The serum exposure of CRM-EDIII-PM to the immune system was prolonged. Moreover, CRM-EDIII-PM did not lead to apparent toxicity to the organs. Therefore, CRM-EDIII-PM was expected as a promising vaccine candidate for its ability to induce strong immune responses.

Genome editing in archaeal viruses and endogenous viral protein purification.

Archaea-infecting viruses are morphologically and genomically among the most diverse entities. Unfortunately, they are also fairly understudied due to a lack of efficient genetic tools. Here, we present a detailed protocol for the CRISPR/Cas-based genome editing of the virus SIRV2 infecting the genus Sulfolobus, which could easily be adapted to other archaeal viruses. This protocol also includes the procedure for endogenous viral protein purification and identification, allowing for assessing the molecular mechanisms behind virus life cycle and virus-host interactions. For complete details on the use and execution of this protocol, please refer to Mayo-Muñoz et al. (2018) and Bhoobalan-Chitty et al. (2019).

Isolation of Reconstructed Functional Ribonucleoprotein Complexes of Machupo Virus.

Arenaviruses initiate infection by delivering a transcriptionally competent ribonucleoprotein (RNP) complex into the cytosol of host cells. The arenavirus RNP consists of the large (L) RNA-dependent RNA polymerase (RdRP) bound to a nucleoprotein (NP)-encapsidated genomic RNA (viral RNA [vRNA]) template. During transcription and replication, L must transiently displace RNA-bound NP to allow for template access into the RdRP active site. Concomitant with RNA replication, new subunits of NP must be added to the nascent complementary RNAs (cRNA) as they emerge from the product exit channel of L. Interactions between L and NP thus play a central role in arenavirus gene expression. We developed an approach to purify recombinant functional RNPs from mammalian cells in culture using a synthetic vRNA and affinity-tagged L and NP. Negative-stain electron microscopy of purified RNPs revealed they adopt diverse and flexible structures, like RNPs of other Bunyavirales members. Monodispersed L-NP and trimeric ring-like NP complexes were also obtained in excess of flexible RNPs, suggesting that these heterodimeric structures self-assemble in the absence of suitable RNA templates. This work allows for further biochemical analysis of the interaction between arenavirus L and NP proteins and provides a framework for future high-resolution structural analyses of this replication-associated complex. IMPORTANCE Arenaviruses are rodent-borne pathogens that can cause severe disease in humans. All arenaviruses begin the infection cycle with delivery of the virus replication machinery into the cytoplasm of the host cell. This machinery consists of an RNA-dependent RNA polymerase-which copies the viral genome segments and synthesizes all four viral mRNAs-bound to the two nucleoprotein-encapsidated genomic RNAs. How this complex assembles remains a mystery. Our findings provide direct evidence for the formation of diverse intracellular arenavirus replication complexes using purification strategies for the polymerase, nucleoprotein, and genomic RNA of Machupo virus, which causes Bolivian hemorrhagic fever in humans. We demonstrate that the polymerase and nucleoprotein assemble into higher-order structures within cells, providing a model for the molecular events of arenavirus RNA synthesis. These findings provide a framework for probing the architectures and functions of the arenavirus replication machinery and thus advancing antiviral strategies targeting this essential complex.

Exploring Codon Adjustment Strategies towards Escherichia coli-Based Production of Viral Proteins Encoded by HTH1, a Novel Prophage of the Marine Bacterium Hypnocyclicus thermotrophus.

Marine viral sequence space is immense and presents a promising resource for the discovery of new enzymes interesting for research and biotechnology. However, bottlenecks in the functional annotation of viral genes and soluble heterologous production of proteins hinder access to downstream characterization, subsequently impeding the discovery process. While commonly utilized for the heterologous expression of prokaryotic genes, codon adjustment approaches have not been fully explored for viral genes. Herein, the sequence-based identification of a putative prophage is reported from within the genome of Hypnocyclicus thermotrophus, a Gram-negative, moderately thermophilic bacterium isolated from the Seven Sisters hydrothermal vent field. A prophage-associated gene cluster, consisting of 46 protein coding genes, was identified and given the proposed name Hypnocyclicus thermotrophus phage H1 (HTH1). HTH1 was taxonomically assigned to the viral family Siphoviridae, by lowest common ancestor analysis of its genome and phylogeny analyses based on proteins predicted as holin and DNA polymerase. The gene neighbourhood around the HTH1 lytic cassette was found most similar to viruses infecting Gram-positive bacteria. In the HTH1 lytic cassette, an N-acetylmuramoyl-L-alanine amidase (Amidase_2) with a peptidoglycan binding motif (LysM) was identified. A total of nine genes coding for enzymes putatively related to lysis, nucleic acid modification and of unknown function were subjected to heterologous expression in Escherichia coli. Codon optimization and codon harmonization approaches were applied in parallel to compare their effects on produced proteins. Comparison of protein yields and thermostability demonstrated that codon optimization yielded higher levels of soluble protein, but codon harmonization led to proteins with higher thermostability, implying a higher folding quality. Altogether, our study suggests that both codon optimization and codon harmonization are valuable approaches for successful heterologous expression of viral genes in E. coli, but codon harmonization may be preferable in obtaining recombinant viral proteins of higher folding quality.

Identification of Receptor Binding Proteins in Flagellotropic Agrobacterium Phage 7-7-1.

The rapid discovery of new and diverse bacteriophages has driven the innovation of approaches aimed at detailing interactions with their bacterial hosts. Previous studies on receptor binding proteins (RBPs) mainly relied on their identification in silico and are based on similarities to well-characterized systems. Thus, novel phage RBPs unlike those currently annotated in genomic and proteomic databases remain largely undiscovered. In this study, we employed a screen to identify RBPs in flagellotropic Agrobacterium phage 7-7-1. Flagellotropic phages utilize bacterial flagella as receptors. The screen identified three candidate RBPs, Gp4, Gp102, and Gp44. Homology modelling predicted that Gp4 is a trimeric, tail associated protein with a central ß-barrel, while the structure and function of Gp102 and Gp44 are less obvious. Studies with purified Gp41-247 confirmed its ability to bind and interact with host cells, highlighting the robustness of the RBP screen. We also discovered that Gp41-247 inhibits the growth of host cells in a motility and lipopolysaccharide (LPS) dependent fashion. Hence, our results suggest interactions between Gp41-247, rotating flagellar filaments and host glycans to inhibit host cell growth, which presents an impactful and intriguing focus for future studies.

Molecular evidence suggesting the persistence of residual SARS-CoV-2 and immune responses in the placentas of pregnant patients recovered from COVID-19.

OBJECTIVES: Recent studies have shown the presence of SARS-CoV-2 in the tissues of clinically recovered patients and persistent immune symptoms in discharged patients for up to several months. Pregnant patients were shown to be a high-risk group for COVID-19. Based on these findings, we assessed SARS-CoV-2 nucleic acid and protein retention in the placentas of pregnant women who had fully recovered from COVID-19 and cytokine fluctuations in maternal and foetal tissues. MATERIALS AND METHODS: Remnant SARS-CoV-2 in the term placenta was detected using nucleic acid amplification and immunohistochemical staining of the SARS-CoV-2 protein. The infiltration of CD14+ macrophages into the placental villi was detected by immunostaining. The cytokines in the placenta, maternal plasma, neonatal umbilical cord, cord blood and amniotic fluid specimens at delivery were profiled using the Luminex assay. RESULTS: Residual SARS-CoV-2 nucleic acid and protein were detected in the term placentas of recovered pregnant women. The infiltration of CD14+ macrophages into the placental villi of the recovered pregnant women was higher than that in the controls. Furthermore, the cytokine levels in the placenta, maternal plasma, neonatal umbilical cord, cord blood and amniotic fluid specimens fluctuated significantly. CONCLUSIONS: Our study showed that SARS-CoV-2 nucleic acid (in one patient) and protein (in five patients) were present in the placentas of clinically recovered pregnant patients for more than 3 months after diagnosis. The immune responses induced by the virus may lead to prolonged and persistent symptoms in the maternal plasma, placenta, umbilical cord, cord blood and amniotic fluid.

Identification of protein interactions of grapevine fanleaf virus RNA-dependent RNA polymerase during infection of Nicotiana benthamiana by affinity purification and tandem mass spectrometry.

The RNA-dependent RNA polymerase (1EPol) is involved in replication of grapevine fanleaf virus (GFLV, Nepovirus, Secoviridae) and causes vein clearing symptoms in Nicotiana benthamiana. Information on protein 1EPol interaction with other viral and host proteins is scarce. To study protein 1EPol biology, three GFLV infectious clones, i.e. GHu (a symptomatic wild-type strain), GHu-1EK802G (an asymptomatic GHu mutant) and F13 (an asymptomatic wild-type strain), were engineered with protein 1EPol fused to a V5 epitope tag at the C-terminus. Following Agrobacterium tumefaciens-mediated delivery of GFLV clones in N. benthamiana and protein extraction at seven dpi, when optimal 1EPol:V5 accumulation was detected, two viral and six plant putative interaction partners of V5-tagged protein 1EPol were identified for the three GFLV clones by affinity purification and tandem mass spectrometry. This study provides insights into the protein interactome of 1EPol during GFLV systemic infection in N. benthamiana and lays the foundation for validation work.

Characterization and purification of pentameric chimeric protein particles using asymmetric flow field-flow fractionation coupled with multiple detectors.

Porcine circovirus causes the post-weaning multi-systemic wasting syndrome. Despite the existence of commercial vaccines, the development of more effective and cheaper vaccines is expected. The usage of chimeric antigens allows serological differentiation between naturally infected and vaccinated animals. In this work, recombinant pentameric vaccination protein particles spontaneously assembled from identical subunits-chimeric fusion proteins derived from circovirus capsid antigen Cap and a multimerizing subunit of mouse polyomavirus capsid protein VP1 were purified and characterized using asymmetric flow field-flow fractionation (AF4) coupled with UV and MALS/DLS (multi-angle light scattering/dynamic light scattering) detectors. Various elution profiles were tested, including constant cross-flow and decreasing cross-flow (linearly and exponentially). The optimal sample retention, separation efficiency, and resolution were assessed by the comparison of the hydrodynamic radius (Rh) measured by online DLS with the Rh values calculated from the simplified retention equation according to the AF4 theory. The results show that the use of the combined elution profiles (exponential and constant cross-flow rates) reduces the time of the separation, prevents undesirable sample-membrane interaction, and yields better resolution. Besides, the results show no self-associations of the individual pentameric particles into larger clusters and no sample degradation during the AF4 separation. The Rg/Rh ratios for different fractions are in good correlation with morphological analyses performed by transmission electron microscopy (TEM). Additionally to the online analysis, the individual fractions were subjected to offline analysis, including batch DLS, TEM, and SDS-PAGE, followed by Western blot.

Epidemiological surveillance of SARS-CoV-2 by genome quantification in wastewater applied to a city in the northeast of France: Comparison of ultrafiltration- and protein precipitation-based methods.

The aim of the present study was to develop a simple, sensitive, and specific approach to quantifying the SARS-CoV-2 genome in wastewater and to evaluate this approach as a means of epidemiological surveillance. Twelve wastewater samples were collected from a metropolitan area in north-eastern France during April and May 2020. In addition to the quantification of the SARS-CoV-2 genome, F-specific RNA phages of genogroup II (FRNAPH GGII), naturally present in wastewater, were used as an internal process control for the viral concentration and processing of RT-PCR inhibitors. A concentration method was required to allow the quantification of the SARS-CoV-2 genome over the longest possible period. A procedure combining ultrafiltration, phenol-chloroform-isoamyl alcohol purification, and the additional purification of the RNA extracts was chosen for the quantification of the SARS-CoV-2 genome in 100-mL wastewater samples. At the same time, the COVID-19 outbreak was evaluated through patients from the neighbouring University Hospital of Nancy, France. A regular decrease in the concentration of the SARS-CoV-2 genome from ~104 gc/L to ~102 gc/L of wastewater was observed over the eight weeks of the study, during which the population was placed under lockdown. The SARS-CoV-2 genome was even undetectable during one week in the second half of May and present but non-quantifiable in the last sample (28 May). A concordant circulation in the human community was highlighted by virological diagnosis using respiratory samples, which showed a decrease in the number of COVID-19 cases from 677 to 52 per week over the same period. The environmental surveillance of COVID-19 using a reliable viral quantification procedure to test wastewater is a key approach. The real-time detection of viral genomes can allow us to predict and monitor the circulation of SARS-CoV-2 in clinical settings and survey the entire urban human population.

Composition of HBsAg is predictive of HBsAg loss during treatment in patients with HBeAg-positive chronic hepatitis B.

BACKGROUND & AIMS: During treatment of chronic HBV infections, loss or seroconversion of the HBV surface antigen (HBsAg) is considered a functional cure. HBsAg consists of the large (LHBs), middle (MHBs), and small surface protein (SHBs) and their relative proportions correlate strongly with disease stage. Our aim was to assess the association between HBsAg composition and functional cure during treatment. METHODS: A total of 83 patients were retrospectively analyzed. HBsAg loss was achieved by 17/64 patients during nucleos(t)ide analogue (NA) treatment and 3/19 patients following treatment with pegylated interferon-alfa2a (PEG-IFN) for 48 weeks. Sixty-three patients without HBsAg loss were matched as controls. LHBs, MHBs and SHBs were quantified in sera collected before and during treatment. RESULTS: Before treatment, median MHBs levels were significantly lower in patients with subsequent HBsAg loss than in those without (p = 0.005). During treatment, MHBs and LHBs proportions showed a fast decline in patients with HBsAg loss, but not in patients with HBV e antigen seroconversion only or patients without serologic response. MHBs became undetectable by month 6 of NA treatment in all patients with HBsAg loss, which occurred on average 12.8 ± 8.7 (0-52) months before loss of total HBsAg. Receiver-operating characteristic analyses revealed that the proportion of MHBs was the best early predictor of HBsAg loss before NA treatment (AUC = 0.726, p = 0.019). In patients achieving HBsAg loss with PEG-IFN, the proportions of MHBs and LHBs showed similar kinetics. CONCLUSION: Quantification of HBsAg proteins shows promise as a novel tool to predict early treatment response. These assessments may help optimize individual antiviral treatment, increasing the rates of functional cure in chronically HBV-infected patients. LAY SUMMARY: The hepatitis B surface antigen (HBsAg) is a key serum marker for viral replication. Loss of HBsAg is considered stable remission, which can be achieved with antiviral treatments. We have investigated whether the ratios of the different components of HBsAg, namely the large (LHBs) and medium (MHBs) HBsAg during different treatments are associated with the occurrence of HBsAg loss. We found that LHBs and MHBs decrease earlier than total HBsAg before HBsAg loss and we propose LHBs and MHBs as promising novel biomarker candidates for predicting cure of HBV infection.

Bacteriophages that infect Gram-negative bacteria as source of signal-arrest-release motif lysins.

Treatment of infections caused by multidrug-resistant (MDR) Gram-negative bacteria is challenging, a potential solution for which is the use of bacteriophage-derived lytic enzymes. However, the exogenous action of bacteriophage lysins against Gram-negative bacteria is hindered due to the presence of an impermeable outer membrane in these bacteria. Nevertheless, recent research has demonstrated that some lysins are capable of permeating the outer membrane of Gram-negative bacteria with the help of signal peptides. In the present study, we investigated the genomes of 309 bacteriophages that infect Gram-negative pathogens of clinical interest in order to determine the evolutionary markers of signal peptide-containing lysins. Complete genomes displayed 265 putative lysins, of which 17 (6.41%) contained signal-arrest-release motifs and 41 (15.47%) contained cleavable signal peptides. There was no apparent relationship between host specificity and lysin diversity. Nevertheless, the evolution of lysin genes might not be independent of the rest of the bacteriophage genome once pan-genome clustering and lysin diversity appear to be correlated. In addition, signal peptide- and signal-arrest-release-containing lysins were monophyletically distributed in the protein cladogram, suggesting that the natural selection of holin-independent lysins is divergent. Our study screened 58 (21.89%) out of 265 potential candidates for in vitro experimentation against MDR bacteria.

Cloning, expression and characterization of P1 and NIa proteases from banana bract mosaic virus (BBrMV).

Banana bract mosaic virus (BBrMV) causes the banana bract mosaic disease in banana. It belongs to the genus Potyvirus within the family Potyviridae. To the best of our knowledge apart from BBrMV coat protein gene, there are no reports on cloning, expression and characterization of any other genes from BBrMV. In this study, the BBrMV P1 and NIa protease genes were amplified from BBrMV infected banana plant cultivar Nendran and were cloned into the protein expression vector pET28b. Recombinant plasmids were transferred to BL21-CodonPlus (DE3)-RP cells and the IPTG (Isopropyl ß-d-1-thiogalactopyranoside) induced BBrMV P1 and NIa proteins with molecular weights of 42 and 32 KDa respectively were purified on Ni-NTA resin column under denaturing conditions using 8 M urea. BBrMV P1 and NIa purified proteins were detected by Western blot using anti-histidine antibody. The activity of both P1 and NIa proteases in native form was analyzed through in-gel zymographic assay. The activities of both the proteases were strongly inhibited by PMSF, suggesting that both the proteases are the serine type proteases. Interestingly both the proteases showed a temperature optimum of 50 °C while the pH optimum was 8. Both proteases lost their activity when incubated at 70 °C for 1 h. This is the first report of expression, purification and characterization of BBrMV P1 and NIa proteases.