Prevalence and genetic diversity analysis of human coronaviruses among cross-border children.

Prevalence and genetic diversity analysis of human coronaviruses among cross-border children.

Abstract

BACKGROUND:

More than a decade after the outbreak of human coronaviruses (HCoVs) SARS in Guangdong province and Hong Kong SAR of China in 2002, there is still no reoccurrence, but the evolution and recombination of the coronaviruses in this region are still unknown. Therefore, surveillance on the prevalence and the virus variation of HCoVs circulation in this region is conducted.

METHODS:

A total of 3298 nasopharyngeal swabs samples were collected from cross-border children (<6 years, crossing border between Southern China and Hong Kong SAR) showing symptoms of respiratory tract infection, such as fever (body temperature > 37.5 °C), from 2014 May to 2015 Dec. Viral nucleic acids were analyzed and sequenced to study the prevalence and genetic diversity of the four human coronaviruses. The statistical significance of the data was evaluated with Fisher chi-square test.

RESULTS:

78 (2.37%; 95%CI 1.8-2.8%) out of 3298 nasopharyngeal swabs specimens were found to be positive for OC43 (36;1.09%), HKU1 (34; 1.03%), NL63 (6; 0.18%) and 229E (2;0.01%). None of SARS or MERS was detected. The HCoVs predominant circulating season was in transition of winter to spring, especially January and February and NL63 detected only in summer and fall. Complex population with an abundant genetic diversity of coronaviruses was circulating and they shared homology with the published strains (99-100%). Besides, phylogenetic evolutionary analysis indicated that OC43 coronaviruses were clustered into three clades (B,D,E), HKU1 clustered into two clades(A,B) and NL63 clustered into two clades(A,B). Moreover, several novel mutations including nucleotides substitution and the insertion of spike of the glycoprotein on the viral surface were discovered.

CONCLUSIONS:

The detection rate and epidemic trend of coronaviruses were stable and no obvious fluctuations were found. The detected coronaviruses shared a conserved gene sequences in S and RdRp. However, mutants of the epidemic strains were detected, suggesting continuous monitoring of the human coronaviruses is in need among cross-border children, who are more likely to get infected and transmit the viruses across the border easily, in addition to the general public.

KEYWORDS:

Cross-border children; Genetic diversity; Human coronaviruses; Molecular epidemiology; Phylogenetic analysis

Jawahar Raina
Read more
Assessment of Nonnucleoside Inhibitors Binding to HIV-1 Reverse Transcriptase Using HYDE Scoring.

Assessment of Nonnucleoside Inhibitors Binding to HIV-1 Reverse Transcriptase Using HYDE Scoring.

Abstract

In this study, 48 inhibitors were docked to 107 allosteric centers of human immunodeficiency virus 1 (HIV-1) reverse transcriptase from the Protein Data Bank (PDB). Based on the average binding scores, quantitative structure-activity relationship (QSAR) equations were constructed in order to elucidate directions of further development in the design of inhibitors. Such developments, informed by structural data, must have a focus on activity against mutated forms of the enzyme, which are the cause of the emergence of multidrug-resistant viral strains. Docking studies employed the HYDE scoring function. Two types of QSARs have been considered: One based on topological descriptors and the other on structural fragments of the inhibitors. Both methods gave similar results, indicating substructures favoring binding to mutated forms of the enzyme.

KEYWORDS:

HIV-1 reverse transcriptase; HYDE; QSAR; docking

Jawahar Raina
Read more
Complete genome analysis of a SARS-like bat coronavirus identified in the Republic of Korea.

Complete genome analysis of a SARS-like bat coronavirus identified in the Republic of Korea.

Abstract

Bats have been widely known as natural reservoir hosts of zoonotic diseases, such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) caused by coronaviruses (CoVs). In the present study, we investigated the whole genomic sequence of a SARS-like bat CoV (16BO133) and found it to be 29,075 nt in length with a 40.9% G+C content. Phylogenetic analysis using amino acid sequences of the ORF 1ab and the spike gene showed that the bat coronavirus strain 16BO133 was grouped with the Beta-CoV lineage B and was closely related to the JTMC15 strain isolated from Rhinolophus ferrumequinum in China. However, 16BO133 was distinctly located in the phylogenetic topology of the human SARS CoV strain (Tor2). Interestingly, 16BO133 showed complete elimination of ORF8 regions induced by a frame shift of the stop codon in ORF7b. The lowest amino acid identity of 16BO133 was identified at the spike region among various ORFs. The spike region of 16BO133 showed 84.7% and 75.2% amino acid identity with Rf1 (SARS-like bat CoV) and Tor2 (human SARS CoV), respectively. In addition, the S gene of 16BO133 was found to contain the amino acid substitution of two critical residues (N479S and T487 V) associated with human infection. In conclusion, we firstly carried out whole genome characterization of the SARS-like bat coronavirus discovered in the Republic of Korea; however, it presumably has no human infectivity. However, continuous surveillance and genomic characterization of coronaviruses from bats are necessary due to potential risks of human infection induced by genetic mutation.

KEYWORDS:

Bat; Frame shift; SARS-like coronavirus; Whole genome; Zoonotic disease

Jawahar Raina
Read more
Epigenetic Promoter DNA Methylation of miR-124 Promotes HIV-1 Tat-Mediated Microglial Activation via MECP2-STAT3 Axis.

Epigenetic Promoter DNA Methylation of miR-124 Promotes HIV-1 Tat-Mediated Microglial Activation via MECP2-STAT3 Axis.

Abstract

The present study demonstrates HIV-1 Tat-mediated epigenetic downregulation of microglial miR-124 and its association with microglial activation. Exposure of mouse primary microglia isolated from newborn pups of either sex to HIV-1 Tat resulted in decreased expression of primary miR-124-1, primary miR-124-2 as well as the mature miR-124. In parallel, HIV-1 Tat exposure to mouse primary microglial cells resulted in increased expression of DNA methylation enzymes, such as DNMT1, DNMT3A, and DNMT3B, which were also accompanied by increased global DNA methylation. Bisulfite-converted genomic DNA sequencing in the HIV-1 Tat-exposed mouse primary microglial cells further confirmed increased DNA methylation of the primary miR-124-1 and primary miR-124-2 promoters. Bioinformatic analyses identified MECP2 as a novel 3'-UTR target of miR-124. This was further validated in mouse primary microglial cells wherein HIV-1 Tat-mediated downregulation of miR-124 resulted in increased expression of MECP2, leading in turn to further repression of miR-124 via the feedback loop. In addition to MECP2, miR-124 also modulated the levels of STAT3 through its binding to the 3'-UTR, leading to microglial activation. Luciferase assays and Ago2 immunoprecipitation determined the direct binding between miR-124 and 3'-UTR of both MECP2 and STAT3. Gene silencing of MECP2 and DNMT1 and overexpression of miR-124 blocked HIV-1 Tat-mediated downregulation of miR-124 and microglial activation. In vitro findings were also confirmed in the basal ganglia of SIV-infected rhesus macaques (both sexes). In summary, our findings demonstrate a novel mechanism of HIV-1 Tat-mediated activation of microglia via downregulation of miR-124, leading ultimately to increased MECP2 and STAT3 signaling.SIGNIFICANCE STATEMENT Despite the effectiveness of combination antiretroviral therapy in controlling viremia, the CNS continues to harbor viral reservoirs. The persistence of low-level virus replication leads to the accumulation of early viral proteins, including HIV-1 Tat protein. Understanding the epigenetic/molecular mechanism(s) by which viral proteins, such as HIV-1 Tat, can activate microglia is thus of paramount importance. This study demonstrated that HIV-1 Tat-mediated DNA methylation of the miR-124 promoter leads to its downregulation with a concomitant upregulation of the MECP2-STAT3-IL6, resulting in microglial activation. These findings reveal an unexplored epigenetic/molecular mechanism(s) underlying HIV-1 Tat-mediated microglial activation, thereby providing a potential target for the development of therapeutics aimed at ameliorating microglial activation and neuroinflammation in the context of HIV-1 infection.

KEYWORDS:

DNA methylation; MECP2; epigenetics; miR-124; microglia; neuroinflammation

Jawahar Raina
Read more
The I22V and L72S substitutions in West Nile virus prM protein promote enhanced prM/E heterodimerisation and nucleocapsid incorporation

The I22V and L72S substitutions in West Nile virus prM protein promote enhanced prM/E heterodimerisation and nucleocapsid incorporation

Abstract

BACKGROUND:

Amino acid substitutions I22V and L72S in the prM protein of West Nile virus Kunjin strain (WNVKUN) were previously shown to enhance virus secretion and virulence, but a mechanism by which this occurred was not determined.

FINDINGS:

Using pulse-chase experiments followed by co-immunoprecipitation with anti-E antibody, we demonstrated that the I22V and L72S substitutions enhanced prM/E heterodimerization for both the E-glycosylated and E-unglycosylated virus. Furthermore, analysis of secreted particles revealed that I22V and L72S substitutions also enhanced nucleocapsid incorporation.

CONCLUSIONS:

We have demonstrated mechanistically that improved secretion of virus particles in the presence of I22V and L72S substitutions was contributed by more efficient prM/E heterodimerization.

Jawahar Raina
Read more
Conserved Role of an N-Linked Glycan on the Surface Antigen of Human Immunodeficiency Virus Type 1 Modulating Virus Sensitivity to Broadly Neutralizing Antibodies against the Receptor and Coreceptor Binding Sites.

Conserved Role of an N-Linked Glycan on the Surface Antigen of Human Immunodeficiency Virus Type 1 Modulating Virus Sensitivity to Broadly Neutralizing Antibodies against the Receptor and Coreceptor Binding Sites.

Abstract

HIV-1 establishes persistent infection in part due to its ability to evade host immune responses. Occlusion by glycans contributes to masking conserved sites that are targets for some broadly neutralizing antibodies (bNAbs). Previous work has shown that removal of a highly conserved potential N-linked glycan (PNLG) site at amino acid residue 197 (N7) on the surface antigen gp120 of HIV-1 increases neutralization sensitivity of the mutant virus to CD4 binding site (CD4bs)-directed antibodies compared to its wild-type (WT) counterpart. However, it is not clear if the role of the N7 glycan is conserved among diverse HIV-1 isolates and if other glycans in the conserved regions of HIV-1 Env display similar functions. In this work, we examined the role of PNLGs in the conserved region of HIV-1 Env, particularly the role of the N7 glycan in a panel of HIV-1 strains representing different clades, tissue origins, coreceptor usages, and neutralization sensitivities. We demonstrate that the absence of the N7 glycan increases the sensitivity of diverse HIV-1 isolates to CD4bs- and V3 loop-directed antibodies, indicating that the N7 glycan plays a conserved role masking these conserved epitopes. However, the effect of the N7 glycan on virus sensitivity to neutralizing antibodies directed against the V2 loop epitope is isolate dependent. These findings indicate that the N7 glycan plays an important and conserved role modulating the structure, stability, or accessibility of bNAb epitopes in the CD4bs and coreceptor binding region, thus representing a potential target for the design of immunogens and therapeutics.

IMPORTANCE:

N-linked glycans on the HIV-1 envelope protein have been postulated to contribute to viral escape from host immune responses. However, the role of specific glycans in the conserved regions of HIV-1 Env in modulating epitope recognition by broadly neutralizing antibodies has not been well defined. We show here that a single N-linked glycan plays a unique and conserved role among conserved glycans on HIV-1 gp120 in modulating the exposure or the stability of the receptor and coreceptor binding site without affecting the integrity of the Env in mediating viral infection or the ability of the mutant gp120 to bind to CD4. The observation that the antigenicity of the receptor and coreceptor binding sites can be modulated by a single glycan indicates that select glycan modification offers a potential strategy for the design of HIV-1 vaccine candidates.

Jawahar Raina
Read more
Characterization of a New Member of Alphacoronavirus with Unique Genomic Features in Rhinolophus Bats.

Characterization of a New Member of Alphacoronavirus with Unique Genomic Features in Rhinolophus Bats.

Abstract

Bats have been identified as a natural reservoir of a variety of coronaviruses (CoVs). Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV (BtCoV/Rh/YN2012) in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3' end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission.

KEYWORDS:

Rhinolophus bat; alphacoronavirus; coronavirus; unique genes

Jawahar Raina
Read more
HSV vector-mediated GAD67 suppresses neuropathic pain induced by perineural HIV gp120 in rats through inhibition of ROS and Wnt5a.

HSV vector-mediated GAD67 suppresses neuropathic pain induced by perineural HIV gp120 in rats through inhibition of ROS and Wnt5a.

Abstract

Human immunodeficiency virus (HIV)-related neuropathic pain is a debilitating chronic condition that is severe and unrelenting. Despite the extensive research, the exact neuropathological mechanisms remain unknown, which hinders our ability to develop effective treatments. Loss of GABAergic tone may have an important role in the neuropathic pain state. Glutamic acid decarboxylase 67 (GAD67) is one of the isoforms that catalyze GABA synthesis. Here, we used recombinant herpes simplex virus (HSV-1) vectors that encode gad1 gene to evaluate the therapeutic potential of GAD67 in peripheral HIV gp120-induced neuropathic pain in rats. We found that (1) subcutaneous inoculation of the HSV vectors expressing GAD67 attenuated mechanical allodynia in the model of HIV gp120-induced neuropathic pain, (2) the anti-allodynic effect of GAD67 was reduced by GABA-A and-B receptors antagonists, (3) HSV vectors expressing GAD67 reversed the lowered GABA-IR expression and (4) the HSV vectors expressing GAD67 suppressed the upregulated mitochondrial superoxide and Wnt5a in the spinal dorsal horn. Taken together, our studies support the concept that recovering GABAergic tone by the HSV vectors may reverse HIV-associated neuropathic pain through suppressing mitochondrial superoxide and Wnt5a. Our studies provide validation of HSV-mediated GAD67 gene therapy in the treatment of HIV-related neuropathic pain.

Jawahar Raina
Read more
Enhancement of the stability of Mycobacterium tuberculosis recombinant antigen expressed in Escherichia coli using cell lysis additives.

Enhancement of the stability of Mycobacterium tuberculosis recombinant antigen expressed in Escherichia coli using cell lysis additives.

Abstract

BACKGROUND:

Mycobacteria tuberculosis (Mtb), the causative agent of tuberculosis, is a slow-growing bacterium. Expression in Escherichia coli is a widely used method for large-scale production of diagnostic antigenic recombinant proteins. Expression of Mtb antigen in E. coli offers a rapid and, inexpensive alternative to conventional protein synthesis from Mtb. The addition of stabilizing additives during cell lysis or storage of Mtb antigenic protein plays a vital role in enhancing antigen stability. In this study, we evaluated the effects of additives on the stability of Mtb antigens expressed in E. coli.

METHODS:

Immunodominant Mtb antigens, i.e., CFP-10, Rv3872, TB7.7, and TB9.7, were cloned, and recombinant proteins overexpressed in E. coli were gradually degraded in a time-dependent manner by incubation at 37 °C. Various stabilizing additives during storage or cell lysis before protein purification were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis.

RESULTS:

CFP-10 and Rv3872 were mainly expressed in soluble form. The degraded form of the expressed protein after incubation at 37 °C was easily observed after 1 week. Increased stability was observed in a solution containing glycine for recombinant CFP-10 and Rv3872. TB9.7 was stable in a solution containing trehalose or mannitol. TB7.7 was stable in a solution containing sucrose, glycine, or polyethylene glycol.

CONCLUSION:

Recombinant Mtb antigen stabilization using chemical additives inhibited protein degradation, leading to increased antigen stability and purification efficiency.

KEYWORDS:

Escherichia coli; Mycobacterium tuberculosis; Recombinant mycobacterial antigen; Stabilizing additives

Jawahar Raina
Read more
Lysosomal Proteases Are a Determinant of Coronavirus Tropism

Lysosomal Proteases Are a Determinant of Coronavirus Tropism

ABSTRACT

Cell entry by coronaviruses involves two principal steps, receptor binding and membrane fusion; the latter requires activation by host proteases, particularly lysosomal proteases. Despite the importance of lysosomal proteases in both coronavirus entry and cell metabolism, the correlation between lysosomal proteases and cell tropism of coronaviruses has not been established. Here, we examined the roles of lysosomal proteases in activating coronavirus surface spike proteins for membrane fusion, using the spike proteins from severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) as the model system. To this end, we controlled the contributions from receptor binding and other host proteases, thereby attributing coronavirus entry solely or mainly to the efficiency of lysosomal proteases in activating coronavirus spike-mediated membrane fusion. Our results showed that lysosomal proteases from bat cells support coronavirus spike-mediated pseudovirus entry and cell-cell fusion more effectively than their counterparts from human cells. Moreover, purified lysosomal extracts from bat cells cleave cell surface-expressed coronavirus spikes more efficiently than their counterparts from human cells. Overall, our study suggests that different lysosomal protease activities from different host species and tissue cells are an important determinant of the species and tissue tropism of coronaviruses.

IMPORTANCE Coronaviruses are capable of colonizing new species, as evidenced by the recent emergence of SARS and MERS coronaviruses; they can also infect multiple tissues in the same species. Lysosomal proteases play critical roles in coronavirus entry by cleaving coronavirus surface spike proteins and activating the fusion of host and viral membranes; they also play critical roles in cell physiology by processing cellular products. How do different lysosomal protease activities from different cells impact coronavirus entry? Here, we controlled the contributions from known factors that function in coronavirus entry so that lysosomal protease activities became the only or the main determinant of coronavirus entry. Using pseudovirus entry, cell-cell fusion, and biochemical assays, we showed that lysosomal proteases from bat cells activate coronavirus spike-mediated membrane fusion more efficiently than their counterparts from human cells. Our study provides the first direct evidence supporting lysosomal proteases as a determinant of the species and tissue tropisms of coronaviruses.

KEYWORDS: coronavirus spike protein, lysosomal proteases, species tropism, tissue tropism
Jawahar Raina
Read more
Development of Monoclonal Antibodies against HIV-1 p24 Protein and Its Application in Colloidal Gold Immunochromatographic Assay for HIV-1 Detection

Development of Monoclonal Antibodies against HIV-1 p24 Protein and Its Application in Colloidal Gold Immunochromatographic Assay for HIV-1 Detection

Abstract

Human immunodeficiency virus type 1 (HIV-1) p24 protein is the most abundant viral protein of HIV-1. This protein is secreted in blood serum at high levels during the early stages of HIV-1 infection, making it a biomarker for early diagnosis. In this study, a colloidal gold immunochromatographic assay (GICA) was established for detecting p24 protein using mouse monoclonal antibodies (mAbs). The HIV-1 p24 protein was expressed in E. coli strain BL21 and the purified protein was used to immunize mice. Stable hybridoma cell lines secreting anti-p24 monoclonal antibodies were obtained after ELISA screening and subcloning by limiting dilution. 34 different capture and labeling mAb pairs were selected by a novel antibody-capture indirect sandwich ELISA and then applied in GICA to detect p24 protein. The GICA method has a limit of detection (LOD) of 25 pg/mL and could detect p24 protein in all 10 positive samples obtained from the National Reference of HIV-1 p24 antigen. Out of 153 negative samples tested, 3 false positives results were obtained. The overall specificity of this test was 98.03%. The good sensitivity and specificity of this method make it a suitable alternative to provide a more convenient and efficient tool for early diagnosis of HIV infection.

Jawahar Raina
Read more
FDA approves first rapid diagnostic test to detect both HIV-1 antigen and HIV-1/2 antibodies

FDA approves first rapid diagnostic test to detect both HIV-1 antigen and HIV-1/2 antibodies

NOW ImmunoDX is proud to introduce there own- HIV-1 p24 antigen test

The U.S. Food and Drug Administration today approved the first rapid Human Immunodeficiency Virus (HIV) test for the simultaneous detection of HIV-1 p24 antigen as well as antibodies to both HIV-1 and HIV-2 in human serum, plasma, and venous or fingerstick whole blood specimens. Approved for use as an aid in the diagnosis of HIV-1 and HIV-2 infection, the Alere Determine HIV-1/2 Ag/Ab Combo test is also the first FDA-approved test that independently distinguishes results for HIV-1 p24 antigen and HIV antibodies in a single test.

Jawahar Raina
Read more
53 results