Virus Growth Research Articles

Application of Irradiation in Food Preservation and Production

The Food irradiation is a tried-and-true technique that's frequently used to improve the quality and the safety of the meat. With the application of this technique, the growth of bacteria, viruses, and parasites is successfully inhibited.

Stimulation of neutral lipid synthesis via viral growth factor signaling and ATP citrate lyase during vaccinia virus infection.

Fatty acid metabolism can provide various products essential for viral infections. How vaccinia virus (VACV), the prototype of poxviruses, modulates fatty acid metabolism is not well understood. Here, we show that VACV infection results in increased neutral lipid droplet synthesis, the organelles that play a crucial role in storing and mobilizing fatty acids for energy production via β-oxidation. Citrate is the first tricarboxylic acid (TCA) cycle intermediate that can be transported to the cytosol to be converted to acetyl-CoA for de novo fatty acid biosynthesis. We found that VACV infection stimulates the S455 phosphorylation of ATP citrate lyase (ACLY), a pivotal enzyme that links citrate metabolism with lipid metabolism. We demonstrate that the inhibition of neutral lipid droplet synthesis and ACLY severely suppresses VACV replication. Remarkably, we found that virus growth factor (VGF)-induced signaling is essential for the VACV-mediated upregulation of ACLY phosphorylation and neutral lipid droplets. Finally, we report that VGF-induced EGFR-Akt pathway and ACLY phosphorylation are important for VACV stimulation of neutral lipid synthesis. These findings identified a new way of rewiring cell metabolism by a virus and a novel function for VGF in the governance of virus-host interactions through the induction of a key enzyme at the crossroads of the TCA cycle and fatty acid metabolism. Our study also provides a mechanism for the role played by VGF and its downstream signaling cascades in the modulation of lipid metabolism in VACV-infected cells.IMPORTANCENeutral lipid droplets are vital players in cellular metabolism. Here, we showed that VACV induces neutral lipid droplet synthesis in infected primary human foreskin fibroblasts and identified the cellular and viral factors needed. We identified VACV encoded growth factor (VGF) as an essential viral factor that induces cellular EGFR-Akt signaling to increase lipid droplets. Interestingly, VACV increases the S455 phosphorylation of ACLY, a key metabolic enzyme that sits at the crossroads of carbohydrate and lipid metabolism in a VGF-EGFR-Akt-dependent manner. We also found that ACLY is vital for VACV-induced lipid droplet synthesis. Our findings identified the modulation of ACLY by a virus and identified it as a potential target for antiviral development against pathogenic poxviruses. Our study also expands the role of growth factor signaling in boosting VACV replication by targeting fatty acid metabolism.

Isolation and characterization of a novel S-gene mutation porcine deltacoronavirus with high pathogenicity from diarrhea piglet in Zhejiang Province, China, 2022.

Porcine deltacoronavirus (PDCoV) is a coronavirus that causes diarrhea in suckling piglets and has the potential for cross-species transmission. Monitoring PDCoV evolution and identifying potential vaccine candidates are crucial due to its high mutation rates in pig populations. In this study, a Chinese PDCoV strain named ZD2022 was successfully isolated from diarrhea piglets in Zhejiang province, followed by genetic evolutionary analysis, assessment of S proteins' biological functions, in vitro cellular adaptation analysis and pathogenicity evaluation. Phylogenetic analyses placed the PDCoV ZD2022 strain within the Southeast Asia Lineage. Sequence analysis revealed 23 mutations in the S protein of ZD2022 compared to most of other Chinese PDCoV strains, including 8 unique mutations (T529I, L579F, Q614H, V709G, S959L, P1010S, V1016F, A1068V). In addition, bioinformatic predictions indicated these mutations impact the hydrophilicity/hydrophobicity, antigenic epitopes and N-glycosylation sites of the ZD2022 S protein. The virus growth curve of ZD2022 showed good cellular adaptation, with peak viral titers of 8.92±0.31 Log10 TCID50/mL in ST cells. Furthermore, ZD2022 exhibited high virulence in suckling piglets, causing severe diarrhea in piglets at 60 hours post-inoculation (hpi) and a mortality rate of 40% (2/5) within 96 hpi. In summary, our findings indicate that the Chinese PDCoV strains continue to mutate, and the novel S gene mutation in strain ZD2022 offers strong cellular adaptation and high pathogenicity, making it a potential candidate strain for vaccine development.

Estimating the impact of social distance policy in mitigating COVID-19 spread with factor-based imputation approach

AbstractWe identify the effectiveness of social distancing policies in reducing the transmission of the COVID-19 spread. We build a model that measures the relative frequency and geographic distribution of the virus growth rate and provides hypothetical infection distribution in the states that enacted the social distancing policies, where we control time-varying, observed and unobserved, state-level heterogeneities. Using panel data on infection and deaths in all US states from February 20 to April 20, 2020, we find that stay-at-home orders and other types of social distancing policies significantly reduced the growth rate of infection and deaths. We show that the effects are time varying and range from the weakest at the beginning of policy intervention to the strongest by the end of our sample period. We also found that social distancing policies were more effective in states with higher income, better education, more white people, more democratic voters, and higher CNN viewership.

Rubella virus assembly requirements and evolutionary relationships with novel rubiviruses.

Rubella virus (RuV) is an enveloped virus that only infects humans, where transplacental infection can cause miscarriage or congenital birth defects. We identified a potential late domain, 278PPAY281, at the C terminus of the E2 envelope protein. However, rather than this domain recruiting the cellular ESCRT machinery as predicted, our data indicate that E2 Y281 promotes a critical interaction of the E2 endodomain with the capsid protein, leading to capsid's localization to the Golgi where virus budding occurs. Revertant analysis demonstrated that two substitutions on the E2 protein could partially rescue virus growth and Cp-Golgi localization. Both residues were found at the corresponding positions in Ruhugu virus E2, supporting the close evolutionary relationship between RuV and Ruhugu virus, a recently discovered rubivirus from bats.

Zika viruses encode 5′ upstream open reading frames affecting infection of human brain cells

Zika virus (ZIKV), an emerging mosquito-borne flavivirus, is associated with congenital neurological complications. Here, we investigate potential pathological correlates of virus gene expression in representative ZIKV strains through RNA sequencing and ribosome profiling. In addition to the single long polyprotein found in all flaviviruses, we identify the translation of unrecognised upstream open reading frames (uORFs) in the genomic 5′ region. In Asian/American strains, ribosomes translate uORF1 and uORF2, whereas in African strains, the two uORFs are fused into one (African uORF). We use reverse genetics to examine the impact on ZIKV fitness of different uORFs mutant viruses. We find that expression of the African uORF and the Asian/American uORF1 modulates virus growth and tropism in human cortical neurons and cerebral organoids, suggesting a potential role in neurotropism. Although the uORFs are expressed in mosquito cells, we do not see a measurable effect on transmission by the mosquito vector in vivo. The discovery of ZIKV uORFs sheds new light on the infection of the human brain cells by this virus and raises the question of their existence in other neurotropic flaviviruses.

The conserved cysteines at position 18, 36, and 49 of Autographa californica multiple nucleopolyhedrovirus VP39 are essential for virus replication.

Autographa californica nucleopolyhedrovirus orf89 (vp39) encodes the major capsid protein VP39. Multiple alignments of protein sequences showed that VP39 has 8 conserved cysteine (Cys) residues. Cysteine residues play an important role in proper function of a protein. To determine the importance of these conserved cysteine residues for virus proliferation, a series of recombinant viruses harboring VP39-Cys mutants were constructed. Viral growth curves and transmission electron microscopy showed that mutation of Cys29, Cys132, Cys169, Cys229, or Cys232 of VP39 to alanine did not affect budded virion production; however, the mutation of Cys18, Cys36, or Cys49 to alanine resulted in interruption of capsid assembly. Co-immunoprecipitation assays showed that mutations of these 8 cysteines individually or simultaneously had no effect on self-association of VP39. Immunofluorescence analysis by confocal microscopy revealed that the subcellular localization of VP39 with mutations in Cys18, Cys36 or Cys49 was exclusively distributed in the cytoplasm of a cell regardless of virus infection or not, while the wild-type VP39 or the VP39 carrying mutations in Cys29, Cys132, Cys169, Cys229, or Cys232 was distributed throughout the cytoplasm and the nucleus. Our results demonstrated that Cys18, Cys36, and Cys49 are essential for the proper localization of VP39, which is a prerequisite for successful nucleocapsid assembly of the virus.

2'-O-methyltransferase-deficient yellow fever virus: Restricted replication in the midgut and secondary tissues of Aedes aegypti mosquitoes severely limits dissemination.

The RNA genome of orthoflaviviruses encodes a methyltransferase within the non-structural protein NS5, which is involved in 2'-O-methylation of the 5'-terminal nucleotide of the viral genome resulting in a cap1 structure. While a 2'-O-unmethylated cap0 structure is recognized in vertebrates by the RNA sensor RIG-I, the cap1 structure allows orthoflaviviruses to evade the vertebrate innate immune system. Here, we analyzed whether the cap0 structure is also recognized in mosquitoes. Replication analyses of 2'-O-methyltransferase deficient yellow fever virus mutants (YFV NS5-E218A) of the vaccine 17D and the wild-type Asibi strain in mosquito cells revealed a distinct downregulation of the cap0 viruses. Interestingly, the level of inhibition differed for various mosquito cells. The most striking difference was found in Aedes albopictus-derived C6/36 cells with YFV-17D cap0 replication being completely blocked. Replication of YFV-Asibi cap0 was also suppressed in mosquito cells but to a lower extent. Analyses using chimeras between YFV-17D and YFV-Asibi suggest that a synergistic effect of several mutations across the viral genome accompanied by a faster initial growth rate of YFV-Asibi cap1 correlates with the lower level of YFV-Asibi cap0 attenuation. Viral growth analyses in Dicer-2 knockout cells demonstrated that Dicer-2 is entirely dispensable for attenuating the YFV cap0 viruses. Translation of a replication-incompetent cap0 reporter YFV-17D genome was reduced in mosquito cells, indicating a cap0 sensing translation regulation mechanism. Further, oral infection of Aedes aegypti mosquitoes resulted in lower infection rates for YFV-Asibi cap0. The latter is related to lower viral loads found in the midguts, which largely diminished dissemination to secondary tissues. After intrathoracic infection, YFV-Asibi cap0 replicated slower and to decreased amounts in secondary tissues compared to YFV-Asibi cap1. These results suggest the existence of an ubiquitously expressed innate antiviral protein recognizing 5'-terminal RNA cap-modifications in mosquitoes, both in the midgut as well as in secondary tissues.

Effect of mutations in GDNV motif of viral hemorrhagic septicemia virus (VHSV) L protein on polymerase activity, viral growth, and in vivo virulence

Most Mononegavirales viruses have a GDNQ motif within the L protein, whereas Novirhabdovirus species feature a GDNV motif. This study examined the function of the GDNV motif within the L protein of viral hemorrhagic septicemia virus (VHSV) by modifying its amino acid composition. Substituting the aspartic acid (D) with valine (V) completely abolished polymerase activity in a minigenome assay. Replacing GDNV with GDNQ showed no significant difference in luciferase activity. Further characterization using reverse genetically engineered recombinant viruses revealed that rVHSV-LGDNQ exhibited an accelerated replication rate and higher virus titer in EPC cells than rVHSV-wild. Olive flounder infected with rVHSV-LGDNQ experienced higher early-stage mortality but lower overall mortality than those infected with rVHSV-wild. These findings suggest that while the GDNQ motif may positively influence VHSV replication speed, it may not confer an overall advantage for the ultimate viral pathogenicity.

Comparative study of the propagation and plaque titration conditions for human coronavirus OC43 as a surrogate for SARS-CoV-2.

The ongoing COVID-19 pandemic is threatening human health globally. The development of effective drugs and vaccines against SARS-CoV-2 is hindered by the limited access to high-biosafety-level facilities. Although human coronavirus (HCoV) OC43, a low-pathogenic endemic human coronavirus, has been used as a surrogate virus for SARS-CoV-2 research, a standard technique for HCoV-OC43 culture and plaque titration has not been established. Our objective was to establish optimized culture and titration protocols for HCoV-OC43. The growth kinetics and permissibility to HCoV-OC43 infection of seven different cell lines were examined concurrently at two different temperatures, 33°C and 37°C. Cell lines exhibiting a cytopathic effect (CPE) were selected for plaque titration. No significant difference in the rate of cell growth was observed at the two temperatures tested. Interestingly, HCoV-OC43 was found not to be a high-temperature-sensitive virus, since it grew well at 37°C. Although RD, LLC-MK2, MRC-5, and HCT-8 cell lines supported virus growth with an obvious cytopathic effect and a high yield of virus after two days of infection, only RD cells were suitable for producing countable plaques. The incubation of the cells with 1.2% low-viscosity Avicel as an overlay medium at 37°C for 4 days appeared to promote clearer and sharper plaque morphology. However, further optimization of the plaque titration protocol is still required due to the continued observation of plaque size variation and hazy zones. We propose a cost-effective protocol for HCoV-OC43 culture and plaque titration that can be implemented at a standard conventional temperature without the need for additional special equipment.

Hemagglutinin and neuraminidase of a non-pathogenic H7N7 avian influenza virus coevolved during the acquisition of intranasal pathogenicity in chickens.

Polybasic amino acid residues at the hemagglutinin (HA) cleavage site are insufficient to induce the highly pathogenic phenotype of avian influenza viruses in chickens. In our previous study, an H7N7 avian influenza virus named "Vac2sub-P0", which is nonpathogenic despite carrying polybasic amino acids at the HA cleavage site, was passaged in chick air sacs, and a virus with high intravenous pathogenicity, Vac2sub-P3, was obtained. Intranasal infection with Vac2sub-P3 resulted in limited lethality in chickens; therefore, in this study, this virus was further passaged in chicken lungs, and the resultant virus, Vac2sub-P3L4, acquired high intranasal pathogenicity. Experimental infection of chickens with recombinant viruses demonstrated that mutations in HA and neuraminidase (NA) found in consecutive passages were responsible for the increased pathogenicity. The HA and NA functions of Vac2sub-P3L4 were compared with those of the parental virus in vitro; the virus growth at 40 °C was faster, the binding affinity to a sialic acid receptor was lower, and the rate of release by NA from the cell surface was lower, suggesting that these changes enabled the virus to replicate efficiently in chickens with high intranasal pathogenicity. This study demonstrates that viruses that are highly pathogenic when administered intranasally require additional adaptations for increased pathogenicity to be highly lethal to intranasally infected chickens.

The Pentamer glycoprotein complex inhibits viral Immediate Early transcription during Human Cytomegalovirus infections

The Pentamer complex of Human Cytomegalovirus (HCMV) consists of the viral glycoproteins gH, gL, UL128, UL130, and UL131 and is incorporated into infectious virions. HCMV strains propagated extensively in vitro in fibroblasts carry UL128, UL130, or UL131 alleles that do not make a functional complex and thus lack Pentamer function. Adding functional Pentamer to such strains decreases virus growth in fibroblasts. Here, we show that the Pentamer inhibits productive HCMV replication in fibroblasts by repressing viral Immediate Early (IE) transcription. We show that ectopic expression of the viral IE1 protein, a target of Pentamer-mediated transcriptional repression, complements the growth defect of a Pentamer-positive virus. Furthermore, we show that the Pentamer also represses viral IE transcription in cell types where HCMV in vitro latency is studied. Finally, we identify UL130 as a functional subunit of the Pentamer for IE transcriptional repression and demonstrate that cyclic AMP Response Element (CRE) and NFkB sites within the Major Immediate Early Promoter that drives IE1 transcription contribute to this repression. We conclude that the HCMV Pentamer represses viral IE transcription.

Modeling suggests SARS-CoV-2 rebound after nirmatrelvir-ritonavir treatment is driven by target cell preservation coupled with incomplete viral clearance.

In a subset of SARS-CoV-2 infected individuals treated with the oral antiviral nirmatrelvir-ritonavir, the virus rebounds following treatment. The mechanisms driving this rebound are not well understood. We used a mathematical model to describe the longitudinal viral load dynamics of 51 individuals treated with nirmatrelvir-ritonavir, 20 of whom rebounded. Target cell preservation, either by a robust innate immune response or initiation of nirmatrelvir-ritonavir near the time of symptom onset, coupled with incomplete viral clearance, appear to be the main factors leading to viral rebound. Moreover, the occurrence of viral rebound is likely influenced by time of treatment initiation relative to the progression of the infection, with earlier treatments leading to a higher chance of rebound. Finally, our model demonstrates that extending the course of nirmatrelvir-ritonavir treatment, in particular to a 10-day regimen, may greatly diminish the risk for rebound in people with mild-to-moderate COVID-19 and who are at high risk of progression to severe disease. Altogether, our results suggest that in some individuals, a standard 5-day course of nirmatrelvir-ritonavir starting around the time of symptom onset may not completely eliminate the virus. Thus, after treatment ends, the virus can rebound if an effective adaptive immune response has not fully developed. These findings on the role of target cell preservation and incomplete viral clearance also offer a possible explanation for viral rebounds following other antiviral treatments for SARS-CoV-2.

AlphaFold2 Modeling and Molecular Dynamics Simulations of the Conformational Ensembles for the SARS-CoV-2 Spike Omicron JN.1, KP.2 and KP.3 Variants: Mutational Profiling of Binding Energetics Reveals Epistatic Drivers of the ACE2 Affinity and Escape Hotspots of Antibody Resistance.

The most recent wave of SARS-CoV-2 Omicron variants descending from BA.2 and BA.2.86 exhibited improved viral growth and fitness due to convergent evolution of functional hotspots. These hotspots operate in tandem to optimize both receptor binding for effective infection and immune evasion efficiency, thereby maintaining overall viral fitness. The lack of molecular details on structure, dynamics and binding energetics of the latest FLiRT and FLuQE variants with the ACE2 receptor and antibodies provides a considerable challenge that is explored in this study. We combined AlphaFold2-based atomistic predictions of structures and conformational ensembles of the SARS-CoV-2 spike complexes with the host receptor ACE2 for the most dominant Omicron variants JN.1, KP.1, KP.2 and KP.3 to examine the mechanisms underlying the role of convergent evolution hotspots in balancing ACE2 binding and antibody evasion. Using the ensemble-based mutational scanning of the spike protein residues and computations of binding affinities, we identified binding energy hotspots and characterized the molecular basis underlying epistatic couplings between convergent mutational hotspots. The results suggested the existence of epistatic interactions between convergent mutational sites at L455, F456, Q493 positions that protect and restore ACE2-binding affinity while conferring beneficial immune escape. To examine immune escape mechanisms, we performed structure-based mutational profiling of the spike protein binding with several classes of antibodies that displayed impaired neutralization against BA.2.86, JN.1, KP.2 and KP.3. The results confirmed the experimental data that JN.1, KP.2 and KP.3 harboring the L455S and F456L mutations can significantly impair the neutralizing activity of class 1 monoclonal antibodies, while the epistatic effects mediated by F456L can facilitate the subsequent convergence of Q493E changes to rescue ACE2 binding. Structural and energetic analysis provided a rationale to the experimental results showing that BD55-5840 and BD55-5514 antibodies that bind to different binding epitopes can retain neutralizing efficacy against all examined variants BA.2.86, JN.1, KP.2 and KP.3. The results support the notion that evolution of Omicron variants may favor emergence of lineages with beneficial combinations of mutations involving mediators of epistatic couplings that control balance of high ACE2 affinity and immune evasion.

Detection of enterovirus RNA in pancreas and lymphoid tissues of organ donors with type 1 diabetes.

The nPOD-Virus group collaboratively applied innovative technologies to detect and sequence viral RNA in pancreas and other tissues from organ donors with type 1 diabetes. These analyses involved the largest number of pancreas samples collected to date. We analysed pancreas, spleen, pancreatic lymph nodes, and duodenum samples from the following donor groups: a) donors with type 1 diabetes (n=71), with (n=35) or without (n=36) insulin-containing islets, (b) donors with single or double islet autoantibody positivity without diabetes (n=22) and c) autoantibody-negative donors without diabetes (control donors) (n=74). Five research laboratories participated in this collaborative effort using approaches for unbiased discovery of RNA viruses (two RNA-Seq platforms), targeted detection of Enterovirus A-D species using RT-PCR, and tests for virus growth in cell-culture. Direct RNA-Seq did not detect virus signal in pancreas samples, whereas RT-PCR detected enterovirus RNA confirmed by sequencing in low amounts in pancreas samples in three of the five donor groups, namely donors with type 1 diabetes with insulin-containing islets, 16% (5/32) donors being positive, donors with single islet autoantibody positivity with 53% (8/15) donors being positive, and non-diabetic donors with 8% (4/49) being enterovirus RNA positive. Detection of enterovirus RNA was significantly more frequent in single islet autoantibody-positive donors compared to donors with type 1 diabetes with insulin-deficient islets (p-value <0.001) and control donors (p-value 0.004). In some donors, pancreatic lymph nodes were also positive. RT-PCR detected enterovirus RNA also in spleen of a small number of donors and virus enrichment in susceptible cell lines before RT-PCR resulted in much higher rate in spleen positivity, particularly in donors with type 1 diabetes. Interestingly, the enterovirus strains detected did not cause a typical lytic infection, possibly reflecting their persistence-prone nature. This was the largest coordinated effort to examine the presence of enterovirus RNA in pancreas of organ donors with type 1 diabetes, using a multitude of assays. These findings are consistent with the notion that both the subjects with type 1 diabetes and those with islet autoantibodies may carry a low-grade enterovirus infection in the pancreas and lymphoid tissues.

Propagation of koi herpesvirus using embryonated chicken eggs: a potential substitute method for fish vaccine production?

Koi herpesvirus disease (KHVD) is caused by a large DNA virus that commonly infects carp, Cyprinus carpio (koi and common carp). KHVD has spread rapidly across the globe and caused high mortality in all ages of common carp and koi. Until now, no effective treatment has been applied to prevent KHV infection impacting the mass production of koi and common carp . An environmentally friendly alternative strategy for controlling fish disease is vaccination. One of the challenges facing conventional viral vaccine production is the requirement for koi or common carp cell cultures, which must be frequently maintained with expensive materials required for virus propagation. This study aims to obtain KHV that has been propagated in embryonated chicken eggs (ECE) to formulate an affordable KHV vaccine for the aquaculture industry. This research consisted of three stages; the first stage was virus inoculation into various parts of eggs (allantoic fluid, chorioallantoic membrane, amniotic fluid, and egg yolk). The second stage was observing viral growth and collecting ECE fluid and membranes. The third stage involved quantitatively determining the viral genomic copy numbers using the quantitative Polymerase Chain Reaction (qPCR) assay. KHV propagation in various parts of ECE resulted in varying viral genomic copy numbers with a high DNA copy number reported in allantoic fluid on the third day after inoculation. Further work is required to monitor virus titer in later passages and optimize methodology for using ECE as the potential alternative to cultured cells as the medium for virus propagation. In the future the system could be developed to produce promising vaccine candidates with more affordable vaccine prices for the Indonesian fish farming industry.

Inhibition of SARS-CoV-2 growth in the lungs of mice by a peptide-conjugated morpholino oligomer targeting viral RNA

Further development of direct-acting antiviral agents against human SARS-CoV-2 infections remains a public health priority. Here, we report that an antisense peptide-conjugated morpholino oligomer (PPMO), named 5’END-2, targeting a highly conserved sequence in the 5’UTR of SARS-CoV-2 genomic RNA, potently suppressed SARS-CoV-2 growth in vitro and in vivo. In Hela-ACE 2 cells, 5’END-2 produced IC50 values of between 40 nM and 1.15 μM in challenges using six genetically disparate strains of SARS-CoV-2, including JN.1. In vivo, using K18-hACE2 mice and the WA-1/2020 virus isolate, two doses of 5’END-2 at 10 mg/kg, administered intranasally on the day before and the day after infection, produced approximately 1.4 log10 virus titer reduction in lung tissue at 3 days post-infection. Under a similar dosing schedule, intratracheal administration of 1.0 to 2.0 mg/kg 5’END-2 produced over 3.5 log10 virus growth suppression in mouse lungs. Electrophoretic mobility shift assays characterized specific binding of 5’END-2 to its complementary target RNA. Further, using reporter constructs containing SARS-CoV-2 5’UTR leader sequence, in an in-cell system, we observed that 5’END-2 could interfere with translation in a sequence-specific manner. The results demonstrate that direct pulmonary delivery of 5’END-2 PPMO is a promising antiviral strategy against SARS-CoV-2 infections and warrants further development.

Evaluation of three alternative methods to the plaque reduction neutralizing assay for measuring neutralizing antibodies to dengue virus serotype 2

BackgroundDengue is a global public health challenge which requires accurate diagnostic methods for surveillance and control. The gold standard for detecting dengue neutralizing antibodies (nAbs) is the plaque reduction neutralization test (PRNT), which is both labor-intensive and time-consuming. This study aims to evaluate three alternative approaches, namely, the MTT-based (or (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) microneutralization assay, the xCELLigence real-time cell analysis (RTCA), and the immuno-plaque assay-focus reduction neutralization test (iPA-FRNT).MethodsTwenty-two residual serum samples were tested for DENV-2 nAbs using all four assays at three neutralization endpoints of 50%, 70% and 90% inhibition in virus growth. For each neutralization endpoint, results were compared using linear regression and correlation analyses. Test performance characteristics were further obtained for iPA-FRNT using 38 additional serum samples.ResultsPositive correlation of DENV-2 neutralization titers for the MTT-based microneutralization assay and the PRNT assay was only observed at the neutralization endpoint of 50% (r = 0.690). In contrast, at all three neutralization end points, a linear trend and positive correlation of DENV-2 neutralization titers for the xCELLigence RTCA and the PRNT assays were observed, yielding strong or very strong correlation (r = 0.829 to 0.967). This was similarly observed for the iPA-FRNT assay (r = 0.821 to 0.916), which also offered the added advantage of measuring neutralizing titers to non-plaque forming viruses.ConclusionThe xCELLigence RTCA and iPA-FRNT assays could serve as suitable alternatives to PRNT for dengue serological testing. The decision to adopt these methods may depend on the laboratory setting, and the utility of additional applications offered by these technologies.

Amplifying the Customer Voice: Unleashing the Power of Influencer Partnerships and Co-creation to Drive Viral Growth

Amplifying the Customer Voice: Unleashing the Power of Influencer Partnerships and Co-creation to Drive Viral Growth

Genetic trans-complementation of L-protease fails to rescue the infectious foot-and-mouth disease virus from the Lbpro defective genome

Outbreaks of the foot-and-mouth disease (FMD) have major economic impact on the global livestock industry by affecting the animal health and product safety. L-protease, a non-structural protein of FMDV, is a papain-like cysteine proteinase involved in viral protein processing as well as cleavage of host proteins for promoting the virus growth. FMDV synthesizes two forms of leader proteinase, Lpro (Labpro and Lbpro), where the deletion of Labpro is lethal and Lbpro deletion is reported to be attenuated. Defective replicons have been used by trans-complementing the deleted gene to produce one time replicating virus; thus, the bio-safety procedure can be compromised in the production units. Attempts are made to rescue of ΔLbproFMDV Asia1 virus by co-expressing the Lbpro protein carried in pcDNA plasmid. Mutant FMDV cDNA, pAsia-ΔLbpro, was constructed by PCR mediated mutagenesis using inverse primers. Transfection of BHK-21 cells with in-vitro transcribed RNA from the constructs failed to produce an infective mutant FMDV. Genetic trans-complementation of the Lbpro, which was done by co-transfecting the pcDNALbpro plasmid DNA along with the pAsia-ΔLbpro RNA in BHK-21 cells also failed to produce viable virus. Expression experiments of reporter genes and indirect immune-fluorescence confirmed the production of the viral proteins in wild type FMDV pAsiaWT; however, it was absent in the pAsia-ΔLbpro indicating that the leaderless virus was unable to produce infectious progeny and infect the cells. Failure to produce virus either by Lbpro deleted mutant clone or by genetic complementation suggests little chance of reversion of the disabled virus with large deletions of FMDV genome.