Rapid assessment of SARS-CoV-2-evolved variants using virus-like particles.

Efforts to determine why new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants demonstrate improved fitness have been limited to analyzing mutations in the spike (S) protein with the use of S-pseudotyped particles. In this study, we show that SARS-CoV-2 virus-like particles (SC2-VLPs) can package and deliver exogenous transcripts, enabling analysis of mutations within all structural proteins and at multiple steps in the viral life cycle. In SC2-VLPs, four nucleocapsid (N) mutations found universally in more-transmissible variants independently increased messenger RNA delivery and expression ~10-fold, and in a reverse genetics model, the serine-202→arginine (S202R) and arginine-203→methionine (R203M) mutations each produced >50 times as much virus. SC2-VLPs provide a platform for rapid testing of viral variants outside of a biosafety level 3 setting and demonstrate N mutations and particle assembly to be mechanisms that could explain the increased spread of variants, including B.1.617.2 (Delta, which contains the R203M mutation).

Optimized qRT-PCR Approach for the Detection of Intra- and Extra-Cellular SARS-CoV-2 RNAs.

The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Meanwhile, increased demand for testing has led to a shortage of reagents and supplies and compromised the performance of diagnostic laboratories in many countries. Both the World Health Organization (WHO) and the Center for Disease Control and Prevention (CDC) recommend multi-step RT-PCR assays using multiple primer and probe pairs, which might complicate the interpretation of the test results, especially for borderline cases. In this study, we describe an alternative RT-PCR approach for the detection of SARS-CoV-2 RNA that can be used for the probe-based detection of clinical isolates in diagnostics as well as in research labs using a low-cost SYBR green method. For the evaluation, we used samples from patients with confirmed SARS-CoV-2 infections and performed RT-PCR assays along with successive dilutions of RNA standards to determine the limit of detection. We identified an M-gene binding primer and probe pair highly suitable for the quantitative detection of SARS-CoV-2 RNA for diagnostic and research purposes.

Assessment of a novel recombinant vesicular stomatitis virus with triple mutations in its matrix protein as a vaccine for pigs.

Vesicular stomatitis virus (VSV) causes a serious vesicular disease responsible for economic losses in the livestock industry. Currently, there are no suitable vaccines to prevent VSV infection. Although the structural matrix (M) protein of VSV has been shown to be a virulence factor in rodent models, its role in the pathogenicity of VSV infection in livestock species is unknown. We hypothesized that VSV with mutations in the M protein represents a novel live attenuated vaccine candidate. To test this, we introduced mutations into VSV M protein using reverse genetics and assessed their attenuation both in vitro and in pigs, an important natural host of VSV. A recombinant VSV with a triple amino acid mutation in M protein (VSVMT) demonstrated a significantly reduced ability to inhibit the type I interferon (IFN) signaling pathway and to shutoff host gene expression compared to WT-VSV and a mutant virus with a single amino acid deletion (VSVΔM51). Inoculation of pigs with VSVMT induced no apparent vesicular lesions but stimulated virus-neutralizing antibodies and animals were protected against virulent VSV challenge infection. These data demonstrate that the M protein is an important virulence factor for VSV in swine and VSVMT represents a novel vaccine candidate for VSV infections in pigs.

Molecular phylogeny and evolutionary dynamics of matrix gene of avian influenza viruses in China.

In China, several subtype avian influenza viruses consistently circulate in poultry. Numerous studies have focused on the evolution of the hemagglutinin gene; however, studies on the evolution of the matrix (M) gene are limited. In this study, a large-scale phylogenetic analysis of M gene sequences of avian influenza viruses isolated in China revealed that the M gene has evolved into six different lineages denoted as I-VI. The majority of lineages I and IV were isolated in terrestrial birds, while the majority of lineages II, III, V and VI were isolated in aquatic birds. Lineage I included 148 H9N2 subtype viruses (72.2%), lineage II comprised of 63 H6 subtype viruses (100%), and lineage IV included 157 H5 subtype viruses (97.5%). The mean substitution rates of different lineages ranged from 1.32×10(-3) (lineage III) to 3.64×10(-3) (lineage IV) substitutions per site per year. According to the most recent common ancestor of all lineages, lineage III was the oldest lineage, formed in 1981 or even earlier. And lineage V was the most recent, established around the year 2000. Selective pressure on M2 was stronger than that on M1. The strongest selection pressure was observed in lineage IV. In addition, site-by-site analyses of each lineage identified 8 positive selection sites, all in M2. Most of the sites (5 out of 8) were located in the extracellular domain, which is an antigen for vaccine development. The positive selection sites (amino acid positions 66, 82 and 97) are likely associated with virus budding. This study enhanced our knowledge of M gene evolution of avian influenza viruses, and is expected to improve the early detection of new viruses and lead to vaccine development.

Critical assessment of influenza VLP production in Sf9 and HEK293 expression systems.

BACKGROUND: Each year, influenza is responsible for hundreds of thousand cases of illness and deaths worldwide. Due to the virus' fast mutation rate, the World Health Organization (WHO) is constantly on alert to rapidly respond to emerging pandemic strains. Although anti-viral therapies exist, the most proficient way to stop the spread of disease is through vaccination. The majority of influenza vaccines on the market are produced in embryonic hen's eggs and are composed of purified viral antigens from inactivated whole virus. This manufacturing system, however, is limited in its production capacity. Cell culture produced vaccines have been proposed for their potential to overcome the problems associated with egg-based production. Virus-like particles (VLPs) of influenza virus are promising candidate vaccines under consideration by both academic and industry researchers. METHODS: In this study, VLPs were produced in HEK293 suspension cells using the Bacmam transduction system and Sf9 cells using the baculovirus infection system. The proposed systems were assessed for their ability to produce influenza VLPs composed of Hemagglutinin (HA), Neuraminidase (NA) and Matrix Protein (M1) and compared through the lens of bioprocessing by highlighting baseline production yields and bioactivity. VLPs from both systems were characterized using available influenza quantification techniques, such as single radial immunodiffusion assay (SRID), HA assay, western blot and negative staining transmission electron microscopy (NSTEM) to quantify total particles. RESULTS: For the HEK293 production system, VLPs were found to be associated with the cell pellet in addition to those released in the supernatant. Sf9 cells produced 35 times more VLPs than HEK293 cells. Sf9-VLPs had higher total HA activity and were generally more homogeneous in morphology and size. However, Sf9 VLP samples contained 20 times more baculovirus than VLPs, whereas 293 VLPs were produced along with vesicles. CONCLUSIONS: This study highlights key production hurdles that must be overcome in both expression platforms, namely the presence of contaminants and the ensuing quantification challenges, and brings up the question of what truly constitutes an influenza VLP candidate vaccine.

DCE-MRI assessment of the effect of Epstein-Barr virus-encoded latent membrane protein-1 targeted DNAzyme on tumor vasculature in patients with nasopharyngeal carcinomas.

BACKGROUND: EBV-encoded latent membrane protein 1 (EBV-LMP1) is an important oncogenic protein for nasopharyngeal carcinoma (NPC) and has been shown to engage a plethora of signaling pathways. Correspondingly, an LMP1-targeted DNAzyme was found to inhibit the growth of NPC cells both in vivo and in vitro by suppressing cell proliferation and inducing apoptosis. However, it remains unknown whether an LMP1-targeted DNAzyme would affect the vasculature of NPC. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) has been applied in the clinical trials of anti-angiogenic drugs for more than ten years, and Ktrans has been recommended as a primary endpoint. Therefore, the objective of the current study was to use DCE-MRI to longitudinally study the effect of an EBV-LMP1-targeted DNAzyme on the vasculature of patients with NPC. METHODS: Twenty-four patients were randomly divided into two groups: a combined treatment group (radiotherapy + LMP1-targeted DNAzyme) and a radiotherapy alone group (radiotherapy + normal saline). DCE-MRI scans were conducted 1 ~ 2 days before radiotherapy (Pre-RT), during radiotherapy (RT 50 Gy), upon completion of radiotherapy (RT 70 Gy), and three months after radiotherapy (3 months post-RT). Parameters of vascular permeability and intra- and extravascular volumes were subsequently obtained (e.g., Ktrans, kep, ve) using nordicICE software. RESULTS: Both Ktrans and kep values for NPC tumor tissues decreased for both groups after treatment. Moreover, a statistically significant difference in Ktrans values at the pre-therapy and post-therapy timepoints emerged earlier for the combined treatment group (RT 50 Gy, P =0.045) compared to the radiotherapy alone group (3 months post-RT, P = 0.032). For the kep values, the downward trend observed for both the combined treatment group and the radiotherapy alone group were similar. In contrast, ve values for all of the tumor tissues increased following therapy. CONCLUSIONS: The EBV-LMP1-targeted DNAzyme that was tested was found to accelerate the decline of Ktrans values for patients with NPC. Correspondingly, the LMP1-targeted DNAzyme treatments were found to affect the angiogenesis and microvascular permeability of NPC. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01449942. Registered 6 October 2011.

Cost-effective method for the preparation of uniformly labeled myristoylated proteins for NMR measurements.

Nuclear magnetic resonance (NMR) is a powerful technique for solving protein structures or studying their interactions. However, it requires molecules labeled with NMR sensitive isotopes like carbon (13)C and nitrogen (15)N. The recombinant expression of labeled proteins is simple to perform but requires quite expensive chemicals. When there is a need for special labeled chemicals, like uniformly (13)C-labeled myristic acid, the price significantly rises. Here we describe a cost-effective method for the recombinant expression of uniformly labeled myristoylated proteins in Escherichia coli demonstrated on the production of Mason-Pfizer monkey virus matrix protein. We used the ability of E. coli to naturally synthetize myristic acid. When grown in isotopically labeled medium the myristic acid will be labelled as well. Bacteria were co-transfected with plasmid carrying gene for yeast N-myristoyltransferase which ensures myristoylation of expressed protein. This process provided 1.8mg of the myristoylated, doubly labeled ((13)C/(15)N)M-PMV matrix protein from 1L of (15)N/(13)C labeled M9 medium. The price represents approximately 50% cost reduction of conventional method using commercially available [U-(13)C]myristic acid.

A microbial platform for rapid and low-cost virus-like particle and capsomere vaccines.

Studies on a platform technology able to deliver low-cost viral capsomeres and virus-like particles are described. The technology involves expression of the VP1 structural protein from murine polyomavirus (MuPyV) in Escherichia coli, followed by purification using scaleable units and optional cell-free VLP assembly. Two insertion sites on the surface of MuPyV VP1 are exploited for the presentation of the M2e antigen from influenza and the J8 peptide from Group A Streptococcus (GAS). Results from testing on mice following subcutaneous administration demonstrate that VLPs are self adjuvating, that adding adjuvant to VLPs provides no significant benefit in terms of antibody titre, and that adjuvanted capsomeres induce an antibody titre comparable to VLPs but superior to unadjuvanted capsomere formulations. Antibodies raised against GAS J8 peptide following immunization with chimeric J8-VP1 VLPs are bactericidal against a GAS reference strain. E. coli is easily and widely cultivated, and well understood, and delivers unparalleled volumetric productivity in industrial bioreactors. Indeed, recent results demonstrate that MuPyV VP1 can be produced in bioreactors at multi-gram-per-litre levels. The platform technology described here therefore has the potential to deliver safe and efficacious vaccine, quickly and cost effectively, at distributed manufacturing sites including those in less developed countries. Additionally, the unique advantages of VLPs including their stability on freeze drying, and the potential for intradermal and intranasal administration, suggest this technology may be suited to numerous diseases where adequate response requires large-scale and low-cost vaccine manufacture, in a way that is rapidly adaptable to temporal or geographical variation in pathogen molecular composition.

The C-terminal helical domain of dengue virus precursor membrane protein is involved in virus assembly and entry.

The role of the α-helical domain (MH) of dengue virus (DENV) precursor membrane protein in replication was investigated by site-directed mutagenesis. Proline substitutions of three residues (120, 123 and 127) at the C-terminus, but not those at the N-terminus of MH domain, reduced the virus-like particles of DENV1, DENV2 and DENV4 detected in supernatants. In a DENV2 replicon trans-packaging system, these three mutations suppressed particles detected; two of them (I123P and V127P) also affected viral entry. In the context of DENV2 genome-length RNA, all three mutations reduced virion assembly and virus spreading in cell culture. Analysis of revertants showed that mutation A120P could partially support viral infection cycle; in contrast, mutations I123P and V127P were lethal, and adaptations of I123P→I123L and V127P→V127L were required to restore the viral infection cycle. These findings demonstrate that the C-terminus of the MH domain is involved in both assembly and entry of DENV.

MChip, a low density microarray, differentiates among seasonal human H1N1, North American swine H1N1, and the 2009 pandemic H1N1.

BACKGROUND: The MChip uses data from the hybridization of amplified viral RNA to 15 distinct oligonucleotides that target the influenza A matrix (M) gene segment. An artificial neural network (ANN) automates the interpretation of subtle differences in fluorescence intensity patterns from the microarray. The complete process from clinical specimen to identification including amplification of viral RNA can be completed in <8 hours for under US$10. OBJECTIVES: The work presented here represents an effort to expand and test the capabilities of the MChip to differentiate influenza A/H1N1 of various species origin. METHODS: The MChip ANN was trained to recognize fluorescence image patterns of a variety of known influenza A viruses, including examples of human H1N1, human H3N2, swine H1N1, 2009 pandemic influenza A H1N1, and a wide variety of avian, equine, canine, and swine influenza viruses. Robustness of the MChip ANN was evaluated using 296 blinded isolates. RESULTS: Training of the ANN was expanded by the addition of 71 well-characterized influenza A isolates and yielded relatively high accuracy (little misclassification) in distinguishing unique H1N1 strains: nine human A/H1N1 (88·9% correct), 35 human A/H3N2 (97·1% correct), 31 North American swine A/H1N1 (80·6% correct), 14 2009 pandemic A/H1N1 (87·7% correct), and 23 negative samples (91·3% correct). Genetic diversity among the swine H1N1 isolates may have contributed to the lower success rate for these viruses. CONCLUSIONS: The current study demonstrates the MChip has the capability to differentiate the genetic variations among influenza viruses with appropriate ANN training. Further selective enrichment of the ANN will improve its ability to rapidly and reliably characterize influenza viruses of unknown origin.

A catalytically and genetically optimized beta-lactamase-matrix based assay for sensitive, specific, and higher throughput analysis of native henipavirus entry characteristics.

Nipah virus (NiV) and Hendra virus (HeV) are the only paramyxoviruses requiring Biosafety Level 4 (BSL-4) containment. Thus, study of henipavirus entry at less than BSL-4 conditions necessitates the use of cell-cell fusion or pseudotyped reporter virus assays. Yet, these surrogate assays may not fully emulate the biological properties unique to the virus being studied. Thus, we developed a henipaviral entry assay based on a beta-lactamase-Nipah Matrix (betala-M) fusion protein. We first codon-optimized the bacterial betala and the NiV-M genes to ensure efficient expression in mammalian cells. The betala-M construct was able to bud and form virus-like particles (VLPs) that morphologically resembled paramyxoviruses. betala-M efficiently incorporated both NiV and HeV fusion and attachment glycoproteins. Entry of these VLPs was detected by cytosolic delivery of betala-M, resulting in enzymatic and fluorescent conversion of the pre-loaded CCF2-AM substrate. Soluble henipavirus receptors (ephrinB2) or antibodies against the F and/or G proteins blocked VLP entry. Additionally, a Y105W mutation engineered into the catalytic site of betala increased the sensitivity of our betala-M based infection assays by 2-fold. In toto, these methods will provide a more biologically relevant assay for studying henipavirus entry at less than BSL-4 conditions.

Epstein-Barr virus (EBV)-encoded RNA: automated in-situ hybridization (ISH) compared with manual ISH and immunohistochemistry for detection of EBV in pediatric lymphoproliferative disorders.

Detection of Epstein-Barr virus (EBV) may be achieved by various methods, including EBV-encoded RNA (EBER) in-situ hybridization (ISH) and immunohistochemistry (IHC) for latent membrane protein (LMP-1). We compared novel automated ISH and IHC techniques in pediatric lymphoproliferative disorders with results obtained by manual ISH. Thirty-seven pediatric cases previously studied by manual EBER ISH (including 18 EBER-positive, 15 EBER-negative, and 4 EBER-equivocal cases) were used for the study. Automated EBER ISH and automated LMP-1 IHC were performed using the BondMax autostainer and prediluted EBER probe and EBV cell surface 1 to 4 at 1:50 dilution, respectively. Results of each of the automated techniques for EBV detection were compared with results by manual EBER ISH. Compared with manual EBER ISH as the gold standard, automated ISH had a sensitivity and specificity of 94% and 69%, respectively, accuracy of 83%, positive predictive value (PPV) of 79%, and negative predictive value (NPV) of 90%. Automated IHC had a sensitivity of 44%, specificity of 93%, accuracy of 67%, PPV of 88%, and NPV of 59%. Automated ISH and IHC correlated significantly (P < 0.045). Automated ISH is useful for diagnosis of EBV-related pediatric neoplasms, being easy to perform and interpret and requiring only the technologist's time to set up and having a high sensitivity and NPV The automated IHC protocol is of too low sensitivity for routine use, although results show high specificity and PPV.

Assessment of CMV load in solid organ transplant recipients by pp65 antigenemia and real-time quantitative DNA PCR assay: correlation with pp67 RNA detection.

After bone marrow (BM) or solid-organ (SO) transplantation viremic Cytomegalovirus (CMV) infection is observed frequently. Quantitative assay of CMV in blood helps the management of this clinical condition. In the present report, 83 samples from 39 solid organ recipients, three CMV assays were compared simultaneously for the first time: the Nuclisens CMV pp67 assay (nucleic acid sequence-based amplification, NASBA), an "in-house" quantitative real-time PCR assay (TaqMan) for CMV DNA, and pp65 antigenemia. The relation between CMV DNA and pp65 antigenemia, the quantitative assays, was evaluated on a larger group including 251 blood samples from 118 solid organ recipients. Real-time PCR provided the best results; > or =130 CMV DNA copies/2 x 10(5) peripheral blood leukocytes (PBLs) predicted > or =1 pp65 antigen positive (Ag+) cell/2 x 10(5) PBLs. By taking pp65 antigenemia as the "gold standard," the sensitivity of CMV DNA quantitation and of the pp67 RNA assay were 0.95 and 0.20, respectively, while the corresponding specificity values were 0.50 and 0.93. When real-time PCR was considered as the "gold standard," the sensitivity and specificity of the pp65 antigenemia were 0.65 and 0.91, respectively. Among the three tests examined, the sensitivity of the pp67 RNA assay was the lowest. On the other hand, the pp67 RNA assay was highly specific and effective in pinpointing high viremia patients. The present report, by providing predictive values for all three diagnostic profiles, DNA load, antigenemia, and pp67RNA, is a contribution for validation of real-time PCR as a new standard for quantitative assessment of CMV viremia in clinical settings.

Prevalence of Epstein-Barr viral sequences and EBV LMP1 oncogene deletions in Burkitt's lymphoma from Pakistan: epidemiological correlations.

To investigate the potential relationship of socioeconomic status with the prevalence of Epstein-Barr virus (EBV) and to understand the significance of del-LMP-1 within EBV+ cases of Burkitt's lymphoma (BL), we studied 10 cases of BL, 30 cases of diffuse large cell lymphoma (DLCL) arising in nonimmunocompromised patients, and 30 reactive tonsillar biopsy specimens from Pakistan. Each lymphoma was analyzed for EBV by EBER1 RNA in situ hybridization (EBV-RISH). Cases showing hybridization signal within neoplastic cells and all reactive tonsillar tissues were analyzed for EBV strain type by EBNA-2 polymerase chain reaction (PCR) and for the presence of a del-LMP-1 by PCR. Eight of 10 (80%) of BL were EBV+, each containing EBV strain A and a wild-type LMP-1 gene. In contrast, only 4 of 30 DLCL (13%) cases were EBV positive (three strain A, one strain B), each containing a wild-type LMP-1 gene. Fifteen of 30 tonsillar biopsy specimens contained EBV, all of which were strain A and wild-type for LMP1. The prevalence of EBV in BL from Pakistan is slightly lower than in BL in endemic regions, but significantly higher than in BL in North America. EBV positivity probably reflects the socioeconomic status of the patient population and age at seroconversion. The absence of del-LMP-1 within all EBV+ BL cases is consistent with the view that del-LMP-1 is not involved in the pathogenesis of BL, and the presence of del-LMP-1 in EBV+ cases of BL reported in other studies may likely reflect the prevalence of a viral strain containing the 30-bp deletion within the respective population studied.