Role of ribosomal pathways and comorbidity in COVID-19: Insight from SARS-CoV-2 proteins and host proteins interaction network analysis.

The COVID-19 pandemic has become a significant global issue in terms of public health. While it is largely associated with respiratory complications, recent reports indicate that patients also experience neurological symptoms and other health issues. The objective of this study is to examine the network of protein-protein interactions (PPI) between SARS-CoV-2 proteins and human host proteins, pinpoint the central genes within this network implicated in disease pathology, and assess their viability as targets for drug development. The study adopts a network-based approach to construct a network of 29 SARS-CoV-2 proteins interacting with 2896 host proteins, with 176 host genes being identified as interacting genes with all the viral proteins. Gene ontology and pathway analysis of these host proteins revealed their role in biological processes such as translation, mRNA splicing, and ribosomal pathways. We further identified EEF2, RPS3, RPL9, RPS16, and RPL11 as the top 5 most connected hub genes in the disease-causing network, with significant interactions among each other. These hub genes were found to be involved in ribosomal pathways and cytoplasmic translation. Further a disease-gene interaction was also prepared to investigate the role of hub genes in other disorders and to understand the condition of comorbidity in COVID-19 patients. We also identified 13 drug molecules having interactions with all the hub genes, and estradiol emerged as the top potential drug target for the COVID-19 patients. Our study provides valuable insights using the protein-protein interaction network of SARS-CoV-2 proteins with host proteins and highlights the molecular basis of manifestation of COVID-19 and proposes drug for repurposing. As the pandemic continues to evolve, it is anticipated that investigating SARS-CoV-2 proteins will remain a critical area of focus for researchers globally, particularly in addressing potential challenges posed by specific SARS-CoV-2 variants in the future.

An efficient computational protocol for template-based design of peptides that inhibit interactions involving SARS-CoV-2 proteins.

The RNA-dependent RNA polymerase (RdRp) complex of SARS-CoV-2 lies at the core of its replication and transcription processes. The interfaces between holo-RdRp subunits are highly conserved, facilitating the design of inhibitors with high affinity for the interaction interface hotspots. We, therefore, take this as a model protein complex for the application of a structural bioinformatics protocol to design peptides that inhibit RdRp complexation by preferential binding at the interface of its core subunit nonstructural protein, nsp12, with accessory factor nsp7. Here, the interaction hotspots of the nsp7-nsp12 subunit of RdRp, determined from a long molecular dynamics trajectory, are used as a template. A large library of peptide sequences constructed from multiple hotspot motifs of nsp12 is screened in-silico to determine sequences with high geometric complementarity and interaction specificity for the binding interface of nsp7 (target) in the complex. Two lead designed peptides are extensively characterized using orthogonal bioanalytical methods to determine their suitability for inhibition of RdRp complexation. Binding affinity of these peptides to accessory factor nsp7, determined using a surface plasmon resonance (SPR) assay, is slightly better than that of nsp12: dissociation constant of 133nM and 167nM, respectively, compared to 473nM for nsp12. A competitive ELISA is used to quantify inhibition of nsp7-nsp12 complexation, with one of the lead peptides giving an IC50 of 25µM . Cell penetrability and cytotoxicity are characterized using a cargo delivery assay and MTT cytotoxicity assay, respectively. Overall, this work presents a proof-of-concept of an approach for rational discovery of peptide inhibitors of SARS-CoV-2 protein-protein interactions.

In-silico approaches for identification of compounds inhibiting SARS-CoV-2 3CL protease.

The world has witnessed of many pandemic waves of SARS-CoV-2. However, the incidence of SARS-CoV-2 infection has now declined but the novel variant and responsible cases has been observed globally. Most of the world population has received the vaccinations, but the immune response against COVID-19 is not long-lasting, which may cause new outbreaks. A highly efficient pharmaceutical molecule is desperately needed in these circumstances. In the present study, a potent natural compound that could inhibit the 3CL protease protein of SARS-CoV-2 was found with computationally intensive search. This research approach is based on physics-based principles and a machine-learning approach. Deep learning design was applied to the library of natural compounds to rank the potential candidates. This procedure screened 32,484 compounds, and the top five hits based on estimated pIC50 were selected for molecular docking and modeling. This work identified two hit compounds, CMP4 and CMP2, which exhibited strong interaction with the 3CL protease using molecular docking and simulation. These two compounds demonstrated potential interaction with the catalytic residues His41 and Cys154 of the 3CL protease. Their calculated binding free energies to MMGBSA were compared to those of the native 3CL protease inhibitor. Using steered molecular dynamics, the dissociation strength of these complexes was sequentially determined. In conclusion, CMP4 demonstrated strong comparative performance with native inhibitors and was identified as a promising hit candidate. This compound can be applied in-vitro experiment for the validation of its inhibitory activity. Additionally, these methods can be used to identify new binding sites on the enzyme and to design new compounds that target these sites.

Crosstalk between SARS-CoV-2 Infection and Neurological Disorders: A Review.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent responsible for coronavirus disease (COVID-19), is an issue of global concern since March 2020. The respiratory manifestations of COVID-19 have widely been explained in the last couple of months of the pandemic. Initially, the virus was thought to be restricted to the pulmonary system; however, as time progressed and cases increased during the second wave of COVID-19, the virus affected other organs, including the nervous system. The neurological implication of SARS-CoV-2 infection is mounting, as substantiated by various reports, and in the majority of COVID-19 patients with neurological symptoms, the penetration of SARS-CoV-2 in the central nervous system (CNS) is likely. SARS-CoV-2 can enter the nervous system by exploiting the routes of olfactory mucosa, olfactory and sensory nerve endings, or endothelial and nerve tissues, thus crossing the neural-mucosal interface in the olfactory mucosa in the nose. Owing to multifactorial and complex pathogenic mechanisms, COVID-19 adds a large-scale risk to the entire nervous system. A thorough understanding of SARSCoV- 2 neurological damage is still vague; however, our comprehension of the virus is rapidly developing. The present comprehensive review will gain insights and provide neurological dimensions of COVID-19 and their associated anomalies. The review presents the entry routes of SARS-CoV-2 into the CNS to ascertain potential targets in the tissues owing to infection. We also discuss the molecular mechanisms involved, the array of clinical symptoms, and various nervous system diseases following the attack of SARS-CoV-2.

Development of Highly Sensitive Sandwich ELISA for the Early-Phase Diagnosis of Chikungunya Virus Utilizing rE2-E1 Protein.

Introduction: Chikungunya is caused by an alpha virus transmitted to humans by an infected mosquito. Infection is generally considered to be self-limiting and non-critical. Chikungunya infection may be diagnosed by severe joint pain with fever, but it is difficult to diagnose because the symptoms of chikungunya are common to many pathogens, including dengue fever. Diagnosis mainly depends on viral culture, reverse transcriptase polymerase chain reaction (RT-PCR), and IgM ELISA. Early and accurate diagnosis of the virus can be achieved by the application of PCR methods, but the high cost and the need for a thermal cycler restrict the use of such methods. On the other hand, antibody-based IgM ELISA is considered to be inexpensive, but antibodies against chikungunya virus (CHIKV) only develop after 4 days of infection, so it has limited application in the earlier diagnosis of viral infection and the management of patients. Because of these challenges, a simple antigen-based sensitive, specific, and rapid detection method is required for the early and accurate clinical diagnosis of chikungunya. Methods: The amino acid sequence of CHIKV ectodomain E1 and E2 proteins was analyzed using bioinformatics tools to determine the antigenic residues, particularly the B-cell epitopes and their characteristics. Recombinant E2-E1 CHIKV antigen was used for the development of polyclonal antibodies in hamsters and IgG was purified. Serological tests of 96 CHIKV patients were conducted by antigen-capture ELISA using primary antibodies raised against rCHIKV E2-E1 in hamsters and human anti-CHIKV antibodies. Results: We observed high specificity and sensitivity, of 100% and 95.8%, respectively, and these values demonstrate the efficiency of the test as a clinical diagnostic tool. There was no cross-reactivity with samples taken from dengue patients. Discussion: Our simple and sensitive sandwich ELISA for the early-phase detection of CHIKV infection may be used to improve the diagnosis of chikungunya.

Crosstalk between SARS-CoV-2 Infection and Type II Diabetes.

Since the outbreak of coronavirus disease (COVID-19) in Wuhan, China, triggered by severe acute respiratory coronavirus 2 (SARS-CoV-2) in late November 2019, spreading to more than 200 countries of the world, the ensuing pandemic to an enormous loss of lives, mainly the older population with comorbidities, like diabetes, cardiovascular disease, chronic obstructive pulmonary disease, obesity, and hypertension. Amongst these immune-debilitating diseases, SARS-CoV-2 infection is the most common in patients with diabetes due to the absence of a normal active immune system to fight the COVID-19. Recovery of patients having a history of diabetes from COVID-19 encounters several complications, and their management becomes cumbersome. For control of coronavirus, antiviral medications, glucose-lowering agents, and steroids have been carefully evaluated. In the present review, we discuss the crosstalk between SARS-CoV-2 infection and patients with a history of diabetes. We mainly emphasize the molecular factors that are involved in diabetic individuals recently infected by SARS-CoV-2 and developed COVID-19 disease. Lastly, we examine the medications available for the long-term management of diabetic patients with SARS-CoV-2 infection.

miRNAs in SARS-CoV-2 Infection: An Update.

Coronavirus disease-2019 (COVID-19) is a highly infectious disease caused by newly discovered severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since the inception of SARS-CoV-2 in Wuhan, China, the virus has traveled more than 200 countries globally. The role of SARS-CoV-2 in COVID-19 has been thoroughly investigated and reviewed in the last 22 months or so; however, a comprehensive outline of miRNAs in SARS-CoV- 2 infection is still missing. The genetic material of SARS-CoV-2 is a single-stranded RNA molecule nearly 29 kb in size. RNA is composed of numerous sub-constituents RNA is found in the cells in a number of forms. including microRNAs (miRNAs). miRNAs play an essential role in biological processes like apoptosis, cellular metabolism, cell death, cell movement, oncogenesis, intracellular signaling, immunity, and infection. Lately, miRNAs have been involved in SARS-CoV-2 infection, though the clear demonstration of miRNAs in the SARS-CoV-2 infection is not fully elucidated. The present review article summarizes recent findings of miRNAs associated with SARS-CoV-2 infection. We presented various facets of miRNAs. miRNAs as the protagonists in viral infection, the occurrence of miRNA in cellular receptors, expression of miRNAs in multiple diseases, miRNA as a biomarker, and miRNA as a therapeutic tool have been discussed in detail. We also presented the vaccine status available in various countries.

Mucormycosis and COVID-19 pandemic: Clinical and diagnostic approach.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is yet to be controlled worldwide, especially in India. The second wave of coronavirus disease 2019 (COVID-19) led to panic and confusion in India, owing to the overwhelming number of the population that fell prey to this highly infectious virus of recent times. In the second wave of COVID-19, the patients had to fight both the virus and opportunistic infections triggered by fungi and bacteria. Repeated use of steroids, antibiotics, and oxygen masks during the management of severely and critically ill COVID-19 patients nurtured opportunistic infections such as mucormycosis. Despite mucormycosis being a decades-old disease, it has gained notice of its widespread occurrence in COVID-19 patients throughout India. Instances of mucormycosis are usually unearthed in immunocompromised individuals and are caused by the inhalation of filamentous fungi, either from the natural environment or through supportive care units. In the recent outbreak during the second wave of COVID-19 in India, it has been seen to cause secondary infection as it grows along with the treatment of COVID-19. Furthermore, COVID-19 patients with comorbidities such as diabetes were more likely to have the mucormycosis co-infection because of their challenged immune systems' inability to fight it. Despite the hype, mucormycosis still remains neglected and least studied, which is predominantly due to all focus on diagnostics, vaccine, and therapeutic research. In this review, we emphasize mainly on the association of mucormycosis in COVID-19 patients. We also present the molecular mechanism of mucormycosis for a better understanding of the fungal infections in patients who have recently been infected with SARS-CoV-2. Better understanding of fungal pathogens, immediate diagnosis, and management of the infections are crucial in COVID-19 patients, as high mortalities have been recorded in co-infected patients despite recovery from COVID-19.

Dimerization of SARS-CoV-2 nucleocapsid protein affects sensitivity of ELISA based diagnostics of COVID-19.

Nucleocapsid protein (N protein) is the primary antigen of the virus for development of sensitive diagnostic assays of COVID-19. In this paper, we demonstrate the significant impact of dimerization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N-protein on sensitivity of enzyme-linked immunosorbent assay (ELISA) based diagnostics. The expressed purified protein from E. coli is composed of dimeric and monomeric forms, which have been further characterized using biophysical and immunological techniques. Indirect ELISA indicated elevated susceptibility of the dimeric form of the nucleocapsid protein for identification of protein-specific monoclonal antibody as compared to the monomeric form. This finding also confirmed with the modelled structure of monomeric and dimeric nucleocapsid protein via HHPred software and its solvent accessible surface area, which indicates higher stability and antigenicity of the dimeric type as compared to the monomeric form. The sensitivity and specificity of the ELISA at 95% CI are 99.0% (94.5-99.9) and 95.0% (83.0-99.4), respectively, for the highest purified dimeric form of the N protein. As a result, using the highest purified dimeric form will improve the sensitivity of the current nucleocapsid-dependent ELISA for COVID-19 diagnosis, and manufacturers should monitor and maintain the monomer-dimer composition for accurate and robust diagnostics.

Synergistic Effects of Natural Compounds Toward Inhibition of SARS-CoV-2 3CL Protease.

The biggest challenge in medical management and control of the COVID-19 pandemic is the nonavailability of the treatment molecules. While vaccines and other biotherapeutic products for managing COVID-19 have reached the market, a small-molecule cure is yet to be developed. This is relevant because the cost of production, storage, and ease of distribution of a small-molecule drug are significantly more favorable than those of biologics. In this paper, we present a multicompound approach, where two drug molecules are administered concurrently to offer an effective therapy for COVID-19. The co-action of the two compounds, each derived from natural origins, has been demonstrated against the 3CL protease, already recognized as a potential drug target for inhibiting SARS-CoV-2. The pair of compounds pursued in this study are flavonoid and naphthalene scaffold. Individually, they offer ∼30 to 35% inhibition at 10 µM. Comprehensive docking and molecular dynamics simulations elucidate that these compounds exhibit excellent binding in the process, which however quickly deteriorates, and the ligand is separated from the binding site. This suggests that while the ligands initially bind with the protease, they are unable to maintain it for an extended period. However, the simulation showed that a simultaneous docked complex of both the compounds together with the protein boosts the stronger binding for a sufficient time. The enzyme assay exhibited 97 and 85% inhibition activity when both compounds were used together at 100 and 50 µM, respectively. Later, a multiconcentration assay was used to determine the coinhibitory activity, and it was observed that the compounds have ∼20 to 30% inhibition activity even at lower concentrations of 0.5 and 1 µM. Surface plasmon resonance was used to measure the binding of the compounds, and when used together, the compounds had a 10-fold greater binding affinity. Thus, the results demonstrate a synergistic mechanism between the two compounds that enhances the inhibition activity against SARS-CoV-2 3CL protease.

COVID-19 Pandemic and Vaccines Update on Challenges and Resolutions.

The coronavirus disease (COVID-19) is caused by a positive-stranded RNA virus called severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), belonging to the Coronaviridae family. This virus originated in Wuhan City, China, and became the cause of a multiwave pandemic that has killed 3.46 million people worldwide as of May 22, 2021. The havoc intensified with the emergence of SARS-CoV-2 variants (B.1.1.7; Alpha, B.1.351; Beta, P.1; Gamma, B.1.617; Delta, B.1.617.2; Delta-plus, B.1.525; Eta, and B.1.429; Epsilon etc.) due to mutations generated during replication. More variants may emerge to cause additional pandemic waves. The most promising approach for combating viruses and their emerging variants lies in prophylactic vaccines. Several vaccine candidates are being developed using various platforms, including nucleic acids, live attenuated virus, inactivated virus, viral vectors, and protein-based subunit vaccines. In this unprecedented time, 12 vaccines against SARS-CoV-2 have been phased in following WHO approval, 184 are in the preclinical stage, and 100 are in the clinical development process. Many of them are directed to elicit neutralizing antibodies against the viral spike protein (S) to inhibit viral entry through the ACE-2 receptor of host cells. Inactivated vaccines, to the contrary, provide a wide range of viral antigens for immune activation. Being an intracellular pathogen, the cytotoxic CD8+ T Cell (CTL) response remains crucial for all viruses, including SARS-CoV-2, and needs to be explored in detail. In this review, we try to describe and compare approved vaccines against SARS-CoV-2 that are currently being distributed either after phase III clinical trials or for emergency use. We discuss immune responses induced by various candidate vaccine formulations; their benefits, potential limitations, and effectiveness against variants; future challenges, such as antibody-dependent enhancement (ADE); and vaccine safety issues and their possible resolutions. Most of the current vaccines developed against SARS-CoV-2 are showing either promising or compromised efficacy against new variants. Multiple antigen-based vaccines (multivariant vaccines) should be developed on different platforms to tackle future variants. Alternatively, recombinant BCG, containing SARS-CoV-2 multiple antigens, as a live attenuated vaccine should be explored for long-term protection. Irrespective of their efficacy, all vaccines are efficient in providing protection from disease severity. We must insist on vaccine compliance for all age groups and work on vaccine hesitancy globally to achieve herd immunity and, eventually, to curb this pandemic.

COVID-19 and SARS-CoV-2 Variants: Current Challenges and Health Concern.

The ongoing coronavirus disease 2019 (COVID-19) outbreak in Wuhan, China, was triggered and unfolded quickly throughout the globe by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The new virus, transmitted primarily through inhalation or contact with infected droplets, seems very contagious and pathogenic, with an incubation period varying from 2 to 14 days. The epidemic is an ongoing public health problem that challenges the present global health system. A worldwide social and economic stress has been observed. The transitional source of origin and its transport to humans is unknown, but speedy human transportation has been accepted extensively. The typical clinical symptoms of COVID-19 are almost like colds. With case fatality rates varying from 2 to 3 percent, a small number of patients may experience serious health problems or even die. To date, there is a limited number of antiviral agents or vaccines for the treatment of COVID-19. The occurrence and pathogenicity of COVID-19 infection are outlined and comparatively analyzed, given the outbreak's urgency. The recent developments in diagnostics, treatment, and marketed vaccine are discussed to deal with this viral outbreak. Now the scientist is concerned about the appearance of several variants over the globe and the efficacy of the vaccine against these variants. There is a need for consistent monitoring of the virus epidemiology and surveillance of the ongoing variant and related disease severity.

Structural Characterization and Binding Studies of the Ectodomain G Protein of Respiratory Syncytial Virus Reveal the Crucial Role of pH with Possible Implications in Host-Pathogen Interactions.

Respiratory syncytial virus (RSV) is a leading viral pathogen causing acute lower respiratory tract infection in children. The G protein of RSV is involved in attachment with the host cell. It is a neutralizing antigen and thus a vaccine candidate. Heparan sulfate is a type of glycosaminoglycan (GAG) present on the host cell membrane that is involved in attachment with the G protein of RSV. We describe a novel approach for efficient expression and purification of the ectodomain G protein in the prokaryotic system and its biophysical characterization. The native ectodomain G protein was purified using a two-step process by Ni-NTA and DEAE weak anion-exchange chromatography through the supernatant obtained after cell lysis. In addition, the denatured form of the protein was also purified from the solubilized inclusion bodies (IBs) by Ni-NTA affinity chromatography with a higher yield. Dynamic light scattering (DLS) was performed to confirm the homogeneity of the purified protein. The effect of pH on the stability and structure of the purified protein was studied by circular dichroism (CD), fluorescence, and absorbance spectroscopy techniques. Isothermal titration calorimetry (ITC) and microscale thermophoresis (MST) were exploited to demonstrate the interaction of heparan sulfate with the ectodomain G protein. The dynamic light scattering results showed that the purified protein was homogenic and had a well-folded native conformation. Biophysical characterization of the protein revealed that it was stable and had intact secondary and tertiary structures at pH 7.5. CD analysis revealed that the protein showed a loss in the secondary structure at pH values 5.5 and 3.5, while absorbance spectroscopy suggested a stable tertiary structure at pH values 7.5 and 5.5 with a probable aggregation pattern at pH 3.5. This loss in the structure of the ectodomain G protein at low pH can be correlated with its physiological activity. A slight change in pH might play a crucial role in host-pathogen interactions. The fluorescence intensity of the protein decreased on moving toward a lower pH with no spectral shift in emission maxima. In addition, isothermal titration calorimetry and microscale thermophoresis results showed strong binding affinity of the ectodomain G protein with heparan sulfate. The binding of heparan sulfate with protein was probably due to the electrostatic interaction of positively charged amino acid residues of the heparin-binding domain of the protein and the negatively charged group of GAGs. Future studies may involve the development of possible therapeutic agents interacting with the G protein and affecting the overall charge and pH that might hinder the host-pathogen interaction.

Inhibition of Chikungunya Virus Infection by 4-Hydroxy-1-Methyl-3-(3-morpholinopropanoyl)quinoline-2(1H)-one (QVIR) Targeting nsP2 and E2 Proteins.

The re-emergence of Chikungunya virus (CHIKV) infection in humans with no approved antiviral therapies or vaccines is one of the major problems with global significance. In the present investigation, we screened 80 in-house quinoline derivatives for their anti-CHIKV activity by computational techniques and found 4-hydroxy-1-methyl-3-(3-morpholinopropanoyl)quinoline-2(1H)-one (QVIR) to have potential binding affinities with CHIKV nsP2 and E2 glycoproteins. QVIR was evaluated in vitro for its anti-CHIKV potential. QVIR showed strong inhibition of CHIKV infection with an EC50 (50% effective concentration) value of 2.2 ± 0.49 µM without significant cytotoxicity (CC50 > 200 µM) and was chosen for further elucidation of its antiviral mechanism. The infectious viral particle formation was abolished by approximately 72% at a QVIR concentration of 20 µM during infection in the BHK-21 cell line, and the CHIKV RNA synthesis was diminished by 84% for nsP2 as well as 74% for E2, whereas the levels of viral proteins were decreased by 69.9% for nsP2 and 53.9% for E2. Flow cytometry analysis confirmed a huge decline in the expression of viral nsP2 and E2 proteins by 71.84 and 67.7%, respectively. Time of addition experiments indicated that QVIR inhibited viral infection at early and late stages of viral replication cycle, and the optimal inhibition was observed at 16 h post infection. The present study advocates for the first time that QVIR acts as a substantial and potent inhibitor against CHIKV and might be as an auspicious novel drug candidate for the development of therapeutic agents against CHIKV infections.

An in vitro Study on the Role of Hepatitis B Virus X Protein C-Terminal Truncation in Liver Disease Development.

Hepatitis B virus X protein C-terminal 127 amino acid truncation is often found expressed in hepatocellular carcinoma (HCC) tissue samples. The present in vitro study tried to determine the role of this truncation mutant in the hepatitis B-related liver diseases such as fibrosis, cirrhosis, HCC, and metastasis. HBx gene and its 127 amino acid truncation mutant were cloned in mammalian expression vectors and transfected in human hepatoma cell line. Changes in cell growth/proliferation, cell cycle phase distribution, expression of cell cycle regulatory genes, mitochondrial depolarization, and intracellular reactive oxygen species (ROS) level were analyzed. Green fluorescent protein (GFP)-tagged version of HBx and the truncation mutant were also created and the effects of truncation on HBx intracellular expression pattern and localization were studied. Effect of time lapse on protein expression pattern was also analyzed. The truncation mutant of HBx is more efficient in inducing cell proliferation, and causes more ROS production and less mitochondrial depolarization as compared with wild type (wt) HBx. In addition, gene expression is altered in favor of carcinogenesis in the presence of the truncation mutant. Furthermore, mitochondrial perinuclear aggregation is achieved earlier in the presence of the truncation mutant. Therefore, HBx C-terminal 127 amino acid truncation might be playing important roles in the development of hepatitis B-related liver diseases by inducing cell proliferation, altering gene expression, altering mitochondrial potential, inducing mitochondrial clustering and oxidative stress, and changing HBx expression pattern.

Phosphate starvation controls lactose metabolism to produce recombinant protein in Escherichia coli.

Phosphate is one of the major constituents in growth media. It closely regulates central carbon and energy metabolism. Biochemical reactions in central carbon metabolism are known to be regulated by phosphorylation and dephosphorylation of enzymes. Phosphate scarcity can limit microbial productivity. However, microorganisms are evolved to grow in phosphate starvation environments. This study investigates the effect of phosphate-starved response (PSR) stimuli in wild-type and recombinant Escherichia coli cells cultivated in two different substrates, lactose, and glycerol. Phosphate-starved E. coli culture sustained bacterial growth despite the metabolic burden that emanated from recombinant protein expression albeit with altered dynamics of substrate utilisation. Induction of lactose in phosphate-starved culture led to a 2-fold improvement in product titre of rSymlin and a 2.3-fold improvement in product titre of rLTNF as compared with phosphate-unlimited culture. The results obtained in the study are in agreement with the literature to infer that phosphate starvation or limitation can slow down the microbial growth rate in order to produce recombinant proteins. Further, under PSR conditions, gene expression analysis demonstrated that while selected genes (gapdh, pykF, ppsA, icdA) in glycolysis and pentose phosphate pathway (zwf, gnd, talB, tktA) were up-regulated, other genes in lactose (lacY, lacA) and acetate (ackA, pta) pathway were down-regulated. We have demonstrated that cra, crp, phoB, and phoR are involved in the regulation of central carbon metabolism. We propose a novel cross-regulation between lactose metabolism and phosphate starvation. UDP-galactose, a toxic metabolite that is known to cause cell lysis, has been shown to be significantly reduced due to slow uptake of lactose under PSR conditions. Therefore, E. coli employs a decoupling strategy by limiting growth and redirecting metabolic resources to survive and produce recombinant protein under phosphate starvation conditions. KEY POINTS: • Phosphate starvation controls lactose metabolism, which results in less galactose accumulation. • Phosphate starvation modulates metabolic flow of central carbon metabolism. • Product titre improves by 2-fold due to phosphate starvation. • The approach has been successfully applied to production of two different proteins.

Antibody response patterns in chikungunya febrile phase predict protection versus progression to chronic arthritis.

Chikungunya virus (CHIKV) infection causes acute febrile illness in humans, and some of these individuals develop a debilitating chronic arthritis that can persist for months to years for reasons that remain poorly understood. In this study from India, we characterized antibody response patterns in febrile chikungunya patients and further assessed the association of these initial febrile-phase antibody response patterns with protection versus progression to developing chronic arthritis. We found 5 distinct patterns of the antibody responses in the febrile phase: no CHIKV binding or neutralizing (NT) antibodies but PCR positive, IgM alone with no NT activity, IgM alone with NT activity, IgM and IgG without NT activity, and IgM and IgG with NT activity. A 20-month follow-up showed that appearance of NT activity regardless of antibody isotype or appearance of IgG regardless of NT activity during the initial febrile phase was associated with a robust protection against developing chronic arthritis in the future. These findings, while providing potentially novel insights on correlates of protective immunity against chikungunya-induced chronic arthritis, suggest that qualitative differences in the antibody response patterns that have evolved during the febrile phase can serve as biomarkers that allow prediction of protection or progression to chronic arthritis in the future.

Surveillance and genetic characterization of rotavirus strains circulating in four states of North Indian children.

Acute gastroenteritis due to Rotavirus (RV) infection is a major cause of morbidity and mortality in infants and young children worldwide. In India, around 0.1 million death reported annually due to RV illness. So, to assess the disease burden continuous surveillance of the circulating genotypes is needed. This study aimed to ascertain the genetic variance of 429 rotavirus positive specimens observed during the period October 2013-September 2014 at four study centers from North India. Out of 1057 patients enrolled, 1018 stool samples were collected at four centers in four different states of North India. Children aged <5 years who showed the symptoms of severe diarrhea and needed hospitalization were enrolled. The stool samples collected were screened by Enzyme Immuno Assay (EIA), and the RV positive samples were characterized by semi-nested PCR. During the study period October 2013 through September 2014, ~42% patients were found to be rotavirus positive of 1018 collected specimen. In Delhi, Rohtak and Meerut, we observed that Rotavirus is seasonal compared to Tanda (HP). The rate of rotavirus detection was significantly higher among children aged below 2 years, and a total of 21.5% of rotavirus infections comprised children aged <6 months. Genotype G1(48.0%) was predominant and frequently circulating strain whereas G12 (16.8%) and G9 (10.0%) was second and third prevalent strain in the four states of North India. High frequency of G1 genotypes was detected under the age group of 6-11 months which is followed by G12, similarly high rate severe disease was observed due to G1 genotypes followed by P[8], P[6] and G12. The most common types of strains were G1P[8] (27.73% of strains), G12P[6] (13.28%), G9P[4] (7.23%) and G1P[6] (6.75%). The rare strain reported were G1P[9]; P[11] strain was detected in combination with G1, G2, and G12. These data emphasized G12 is the second most predominant strain circulating among Northern Indian children highlights the needs for inclusion in the future polyvalent vaccine to break the burden of rotavirus infection.

BA9 lineage of respiratory syncytial virus from across the globe and its evolutionary dynamics.

Respiratory syncytial virus (RSV) is an important pathogen of global significance. The BA9 is one of the most predominant lineages of the BA genotype of group B RSV that has acquired a 60bp duplication in its G protein gene. We describe the local and global evolutionary dynamics of the second hyper variable region in the C- terminal of the G protein gene of the BA9 lineage. A total of 418 sequences (including 31 study and 387 GenBank strains) from 29 different countries were used for phylogenetic analysis. This analysis showed that the study strains clustered with BA (BA9 and BA8) and SAB4 genotype of group B RSV. We performed time-scaled evolutionary clock analyses using Bayesian Markov chain Monte Carlo methods. We also carried out glycosylation, selection pressure, mutational, entropy and Network analyses of the BA9 lineage. The time to the most recent common ancestor (tMRCA) of the BA genotype and BA9 lineage were estimated to be the years 1995 (95% HPD; 1987-1997) and 2000 (95% HPD; 1998-2001), respectively. The nucleotide substitution rate of the BA genotype [(4.58×10-3 (95% HPD; 3.89-5.29×10-3) substitution/site/year] was slightly faster than the BA9 lineage [4.03×10-3 (95% HPD; 4.65-5.2492×10-3)]. The BA9 lineage was categorized into 3 sub lineages (I, II and III) based on the Bayesian and Network analyses. The local transmission pattern suggested that BA9 is the predominant lineage of BA viruses that has been circulating in India since 2002 though showing fluctuations in its effective population size. The BA9 lineage established its global distribution with report from 23 different countries over the past 16 years. The present study augments our understanding of RSV infection, its epidemiological dynamics warranting steps towards its overall global surveillance.