Isolation and characterization of emerging Mpox virus from India.

Mpox (previously known as Monkeypox) has recently re-emerged, primarily through human-to-human transmission in non-endemic countries including India. Virus isolation is still considered as the gold standard for diagnosis of viral infections. Here, the qPCR positive skin lesion sample from a patient was inoculated in Vero E6 cell monolayer. Characteristic cytopathic effect exhibiting typical cell rounding and detachment was observed at passage-02. The virus isolation was confirmed by qPCR. The replication kinetics of the isolate was determined that revealed maximum viral titre of log 6.3 PFU/mL at 72 h postinfection. Further, whole genome analysis through next generation sequencing revealed that the Mpox virus (MPXV) isolate is characterized by several unique SNPs and INDELs. Phylogenetically, it belonged to A.2 lineage of clade IIb, forming a close group with all other Indian MPXV along with few from USA, UK, Portugal, Thailand and Nigeria. This study reports the first successful isolation and phenotypic and genotypic characterization of MPXV from India.

One-step single-tube accelerated quantitative nucleoprotein gene-specific reverse transcription loop-mediated isothermal gene amplification (RT-LAMP) assay for rapid, real-time & reliable clinical detection of Ebola virus.

Background & objectives: Due to the absence of specific drugs or vaccines for Ebola virus disease, rapid, sensitive and reliable diagnostic methods are required to control the transmission chain of the disease and for better patient management. Isothermal amplification of nucleic acids has emerged as a promising alternative in which rapid and efficient amplification is achieved at a constant temperature without the thermal cycling required in PCR. Methods: A one-step single-tube accelerated quantitative reverse trascription loop-mediated isothermal amplification (RT-LAMP) assay was developed by targeting the NP gene of 2014 Zaire Ebola virus (ZEBOV). The RT-LAMP assay was found to be specific for ZEBOV, without having any cross-reactivity with related haemorrhagic fever viral agents. Results: The comparative evaluation of Ebola virus NP gene-specific RT-LAMP assay with reverse transcription (RT) - PCR and TaqMan real-time RT-PCR demonstrated that RT-LAMP was 10-1000 folds more sensitive than TaqMan real-time RT-PCR and conventional RT-PCR, respectively, with a detection limit of 1 copy number. In the absence of real-world clinical samples, the feasibility of Ebola virus RT-LAMP assay for clinical diagnosis was evaluated with different body fluids including serum, urine, saliva, semen and stool samples from healthy human volunteers spiked with gamma-irradiated ZEBOV 2014 obtained from Robert Koch Institute, Berlin, Germany, through the European Network for Diagnostics of Imported Viral Diseases. The Ebola virus RT-LAMP assay could correctly be picked up the spiked samples up to 1 copy of viral RNA without having any matrix interference. The monitoring of gene amplification can also be visualized with the naked eye by using SYBR Green I fluorescent dye. Interpretation & conclusions: Thus, due to easy operation without a requirement of sophisticated equipment and skilled personnel, the RT-LAMP assay reported here is a valuable tool as a point-of-care diagnosis for the rapid and real-time detection of Ebola virus in resource-limited healthcare settings of developing countries.

Molecular characterization of B. anthracis isolates from the anthrax outbreak among cattle in Karnataka, India.

BACKGROUND: Anthrax, a zoonotic disease is caused by the Gram positive bacterium Bacillus anthracis. During January 2013, an anthrax outbreak among cattle was reported in Gundlupet Taluk, neighboring Bandipur National Park and tiger reserve, India. The present study aims at the molecular identification and characterization of 12 B. anthracis isolates from this outbreak by 16S rRNA gene sequencing, screening B. anthracis specific prophages and chromosomal markers, protective antigen (pag) gene and canonical single nucleotide polymorphism (canSNP) analysis to subtype the isolates into one of the twelve globally identified clonal sub-lineages of B. anthracis. RESULTS: These isolates had identical 16S rDNA nucleotide sequences with B. anthracis specific dual peaks showing mixed base pair R (G/A) at position 1139 with visual inspection while the automated basecaller software indicated a G. Alternatively the nucleotide A at 1146 position was indicative of the 16S rDNA type 7. Multiple sequence alignment with additional 170 (16S rDNA) sequences of B. cereus sensu lato group from GenBank database revealed 28 new 16S types in addition to eleven 16S types reported earlier. The twelve B. anthracis isolates were found to harbor the four B. anthracis specific prophages (lambdaBa01, lambdaBa02, lambdaBa03, and lambdaBa04) along with its four specific loci markers (dhp 61.183, dhp 77.002, dhp 73.019, and dhp 73.017). The pag gene sequencing identified the isolates as protective antigen (PA) genotype I with phenylalanine-proline-alanine phenotype (FPA phenotype). However, sequence clustering with additional 34 pag sequences from GenBank revealed two additional missense mutations at nucleotide positions 196 bp and 869 bp of the 2294 bp pag sequence among the 5 B. cereus strains with pXO1 like plasmids. The canSNP analysis showed that the isolates belong to A.Br.Aust94 sub-lineage that is distributed geographically in countries of Asia, Africa, Europe and Australia. CONCLUSIONS: The analysis of 16S rDNA sequences reiterated the earlier findings that visual inspection of electropherogram for position 1139 having nucleotide R could be used for B. anthracis identification and not the consensus sequence from base caller. The canSNP results indicated that the anthrax outbreak among cattle was caused by B. anthracis of A.Br.Aust94 sub-lineage.

Dual Aptamer-DNAzyme based colorimetric assay for the detection of AFB1 from food and environmental samples.

In the present study, a colorimetric biosensor strategy is devised in combination with apta-magnetic separation assisted with DNAzyme based colorimetric detection of Aflatoxin B1 (AFB1). The optimized analytical procedures consisted of the capture of AFB1 by biotinylated aptamer conjugated to streptavidin magnetic beads and detection by a colorimetric signal from a DNAzyme modified aptamer in presence hemin and H2O2/TMB (3', 3', 5, 5'- tetramethylbenzidine). The DNA concentration, incubation time, hemin, and NaCl concentrations were evaluated and optimized. The visual optical signal thus generated could determine the presence of AFB1 in the given sample. The selectivity of the method with other mycotoxins was evaluated. The linear range of AFB1 from 0 to 200 ppb was assessed and detected as low as 40 ppb visually. The absorbance of blue color generated by the catalytic reaction was in a linear correlation with AFB1 concentrations and was able to detect as low as 22.6 ppb (LOD). The suitability of the assay for AFB1 quantification in sorghum and natural samples was also evaluated. Thus, the developed assay could be a reliable, inexpensive, alternative tool for possible use as a screening method for aflatoxins and other mycotoxins.

Emergence of human West Nile Virus infection among pediatric population in Madhya Pradesh, India.

Human infections caused by West Nile virus (WNV) mostly remain subclinical and self-limited. However, nearly 20% infected people suffer from febrile illness and very few of them (<1%) may get neuroinvasive illness. Mortality has been reported among children. India somehow has reported very less number of WNV cases in the past. We collected cerebrospinal fluid (CSF) samples from 75 pediatric age group patients clinically suffering from acute encephalitis syndrome. Three of these samples were positive by reverse transcriptase polymerase chain reaction using pan flavivirus primers. On sequencing of the 212 bp long-amplified fragment, it was found to be WNV belonging to lineage 1. This is probably the first report of WNV causing encephalitis from this central part of India.

Cloning, expression & evaluation of potential immunogenic recombinant capsid premembrane protein of West Nile virus.

Background & objectives: West Nile virus (WNV) is a neurotropic flavivirus that has emerged globally as a significant cause of viral encephalitis. The early confirmatory diagnosis of WNV infections is important for timely clinical management and in areas where multiple flaviviruses are endemic. Diagnosis of WNV infection is primarily based on serodiagnosis, followed by virus isolation and identification. The aim of this study was to develop and evaluate a highly sensitive and specific immunoglobulin M (IgM) ELISA using the recombinant CprM protein (rWNV-CprM) for rapid, early and accurate diagnosis of WNV. Methods: The gene coding for the CprM protein of WNV was cloned and expressed in pET 28a vector followed by purification. An indirect IgM microplate ELISA using purified rWNV-CprM protein was optimized having no cross-reactivity with healthy human serum and serum samples obtained from patients with dengue and Japanese encephalitis viruses infection. Results: The comparative evaluation of this rWNV-CprM protein-specific IgM ELISA with plaque reduction neutralization test using 105 blood samples collected from patients suspected to have acute WNV infection revealed 98 per cent concordance with sensitivity and specificity of 100 and 97 per cent, respectively. Interpretation & conclusions: The recombinant CprM protein-based WNV-specific ELISA reported in this study may be useful for rapid screening of large numbers of blood samples in endemic areas during outbreaks.

Oseltamivir-resistant influenza A(H1N1)pdm09 virus associated with high case fatality, India 2015.

Influenza A viruses has been associated with severe global pandemics of high morbidity and mortality with devastating impact on human health and global economy. India witnessed a major outbreak of influenza A(H1N1)pdm09 in 2015. This study comprises detailed investigation of cases died of influenza A(H1N1)pdm09 virus infection during explosive outbreak of 2015, in central part of India. To find out presence of drug resistant virus among patients who died of influenza A(H1N1)pdm09 virus infection and to find out presence of other mutations contributing to the morbidity and mortality. Twenty-two patients having confirmed influenza A(H1N1)pdm09 infection and subsequently died of this infection along with 20 non fatal cases with influenza A(H1N1)pdm09 infection were included in the study. Samples were investigated through RT-PCR/RFLP analysis, followed by nucleotide cycle sequencing of whole NA gene for detection of H275Y amino acid substitution in NA gene responsible for oseltamivir drug resistance. Out of 22 fatal cases, 6 (27.27%) were found to harbor oseltamivir resistant virus strains, whereas the H275Y mutation was not observed among the 20 non fatal cases. Amino acid substitution analysis of complete NA gene revealed V241I, N369K, N386K substitution in all strains playing synergistic role in oseltamivir drug resistance. High morbidity and mortality associated with influenza A(H1N1)pdm09 viruses can be explained by presence of drug resistant strains circulating in this outbreak. Presence of Oseltamivir resistant influenza A(H1N1)pdm09 viruses is a cause of great concern and warrants continuous screening for the circulation of drug resistant strains.

Impact of transmission cycles and vector competence on global expansion and emergence of arboviruses.

Arboviruses are transmitted between arthropod vectors and vertebrate host. Arboviral infection in mosquitoes is initiated when a mosquito feeds on a viremic host. Following ingestion of a viremic blood meal by mosquitoes, virus enters midgut along with the blood, infects and replicates in midgut epithelial cells, and then escapes to the hemocoel, from where it disseminates to various secondary organs including salivary glands. Subsequently, when mosquito bites another host, a new transmission cycle is initiated. The midgut and salivary glands act as anatomical barriers to virus infection and escape. These complex interactions between the virus and vector dictate the vector competence. Thus, vector competence reflects the success in overcoming different barriers within the vector. Along with these, other intrinsic factors like midgut microbiota and immune responses, extrinsic factors like temperature and humidity, and genetic factors like vector genotype and viral genotype have been discussed in this review. Recent advancement on novel molecular tools to study vector competence is also included. Different modes of arboviral transmission like horizontal, vertical, and venereal and how these play role in sustenance and emergence of arboviruses in nature are also discussed. These factors can be exploited to reduce the susceptibility of vectors for the viruses, so as to control arboviral diseases to certain extent.

Evaluation of real-time reverse-transcription loop-mediated isothermal amplification assay for clinical diagnosis of West Nile virus in patients.

Background & objectives: West Nile virus (WNV) is a mosquito-borne flavivirus. The disease can be diagnosed by isolation followed by fluorescent antibody tests, enzyme-linked immunosorbent assay and polymerase chain reaction (PCR) assay. These diagnostic methods are laborious and time-consuming. The present study was aimed to evaluate the real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP) method for rapid, early and accurate diagnosis of WNV. Methods: A one-step single tube accelerated quantitative RT-LAMP assay was evaluated by targeting the Env gene of WNV. The gene amplification was accomplished by incubating the reaction mixture at 63°C for 60 min in both real time turbidimeter as well as routine laboratory water bath/dry heating bath. To rule out contamination issues, proper negative controls, including no template, no primer; and no enzyme, were always kept alongside each run. The RT-LAMP assay was evaluated on 105 clinical samples from individuals having ocular infection. Results: Of the 105 samples tested, 27 were positive for WNV by RT-LAMP assay. The comparative evaluation with conventional RT-PCR revealed 100 per cent accordance with sensitivity and specificity of 100 and 95 per cent, respectively. The specificity of this assay was confirmed with serum samples obtained from patients with dengue and chikungunya. Interpretation & conclusions: The RT-LAMP test seemed to be a sensitive and specific method for rapid detection of WNV infection and would be useful for rapid screening of a large number of clinical samples in endemic areas during outbreaks.

Zika virus: A public health threat.

The resurgence of Zika virus as public health emergency of an international concern with increased incidence of microcephaly has drawn attention of scientific community for its detailed understanding with regard to virus evolution, epidemiology, geographical spread, pathogenesis, etc. The scope of the present review is to discuss the detailed updated information in respect of Zika virus evolution since its inception.

Emergence of influenza A (H1N1)pdm09 genogroup 6B and drug resistant virus, India, January to May 2015.

To investigate the aetiology of the 2015 A(H1N1)pdm09 influenza outbreak in India, 1,083 nasopharyngeal swabs from suspect patients were screened for influenza A(H1N1)pdm09 in the state of Madhya Pradesh. Of 412 positive specimens, six were further characterised by phylogenetic analysis of haemagglutinin (HA) sequences revealing that they belonged to genogroup 6B. A new mutation (E164G) was observed in HA2 of two sequences. Neuraminidase genes in two of 12 isolates from fatal cases on prior oseltamivir treatment harboured the H275Y mutation.

Comparative evaluation of the diagnostic potential of recombinant envelope proteins and native cell culture purified viral antigens of Chikungunya virus.

Despite the fact that Chikungunya resurgence is associated with epidemic of unprecedented magnitude, there are challenges in the field of its clinical diagnosis. However, serological tests in an ELISA format provide a rapid tool for the diagnosis of Chikungunya infection. Indeed, ELISAs based on recombinant proteins hold a great promise as these methods are cost effective and are free from the risk of handling biohazardous material. In this study, the performance of recombinant CHIKV antigens was compared in various ELISA formats for the diagnosis of Chikungunya. Two recombinant antigens derived from the envelope proteins of Chikungunya virus were prepared and evaluated by comparing their competence for detecting circulating antibodies in serum samples of patients infected with CHIKV using MAC-ELISA and indirect IgM-ELISA. The efficacy of the recombinant antigens was also compared with the native antigen. The indirect antibody capture IgM microplate ELISA revealed ≥90% concordance with the native antigen in detecting the CHIKV specific IgM antibodies whereas the recombinant antigen based MAC-ELISA showed 100% specificity. The recombinant antigens used in this study were effective and reliable targets for the diagnosis of CHIKV infection and also provide an alternative for native antigen use which is potentially biohazardous.

Development and comparative evaluation of SYBR Green I-based one-step real-time RT-PCR assay for detection and quantification of West Nile virus in human patients.

The recent outbreaks of West Nile Virus (WNV) in the Northeastern American continents and other regions of the world have made it essential to develop an efficient protocol for surveillance of WN virus. Nucleic acid based techniques like, RT-PCR have the advantage of sensitivity, specificity and rapidity. A one step single tube Env gene specific real-time RT-PCR was developed for early and reliable clinical diagnosis of WNV infection in clinical samples. The applicability of this assay for clinical diagnosis was validated with 105 suspected acute-phase serum and plasma samples from the recent epidemic of mysterious fever in Tamil Nadu, India in 2009-10. The comparative evaluation revealed the higher sensitivity of real-time RT-PCR assay by picking up 4 additional samples with low copy number of template in comparison to conventional RT-PCR. All the real-time positive samples further confirmed by CDC reported TaqMan real-time RT-PCR and quantitative real-time RT-PCR assays for the simultaneous detection of WNV lineage 1 and 2 strains. The quantitation of the viral load samples was done using a standard curve. These findings demonstrated that the assay has the potential usefulness for clinical diagnosis due to detection and quantification of WNV in acute-phase patient serum samples.

Complete genome sequencing and evolutionary analysis of Indian isolates of Dengue virus type 2.

Dengue is the most important arboviral infection of global public health significance. It is now endemic in most parts of the South East Asia including India. Though Dengue virus type 2 (DENV-2) is predominantly associated with major outbreaks in India, complete genome information of Indian DENV-2 is not available. In this study, the full-length genome of five DENV-2 isolates (four from 2001 to 2011 and one from 1960), from different parts of India was determined. The complete genome of the Indian DENV-2 was found to be 10,670 bases long with an open reading frame coding for 3391 amino acids. The recent Indian DENV-2 (2001-2011) revealed a nucleotide sequence identity of around 90% and 97% with an older Indian DENV-2 (1960) and closely related Sri Lankan and Chinese DENV-2 respectively. Presence of unique amino acid residues and non-conservative substitutions in critical amino acid residues of major structural and non-structural proteins was observed in recent Indian DENV-2. Selection pressure analysis revealed positive selection in few amino acid sites of the genes encoding for structural and non-structural proteins. The molecular phylogenetic analysis based on comparison of both complete coding region and envelope protein gene with globally diverse DENV-2 viruses classified the recent Indian isolates into a unique South Asian clade within Cosmopolitan genotype. A shift of genotype from American to Cosmopolitan in 1970s characterized the evolution of DENV-2 in India. Present study is the first report on complete genome characterization of emerging DENV-2 isolates from India and highlights the circulation of a unique clade in South Asia.

Discovery of the first insect nidovirus, a missing evolutionary link in the emergence of the largest RNA virus genomes.

Nidoviruses with large genomes (26.3-31.7 kb; 'large nidoviruses'), including Coronaviridae and Roniviridae, are the most complex positive-sense single-stranded RNA (ssRNA+) viruses. Based on genome size, they are far separated from all other ssRNA+ viruses (below 19.6 kb), including the distantly related Arteriviridae (12.7-15.7 kb; 'small nidoviruses'). Exceptionally for ssRNA+ viruses, large nidoviruses encode a 3'-5'exoribonuclease (ExoN) that was implicated in controlling RNA replication fidelity. Its acquisition may have given rise to the ancestor of large nidoviruses, a hypothesis for which we here provide evolutionary support using comparative genomics involving the newly discovered first insect-borne nidovirus. This Nam Dinh virus (NDiV), named after a Vietnamese province, was isolated from mosquitoes and is yet to be linked to any pathology. The genome of this enveloped 60-80 nm virus is 20,192 nt and has a nidovirus-like polycistronic organization including two large, partially overlapping open reading frames (ORF) 1a and 1b followed by several smaller 3'-proximal ORFs. Peptide sequencing assigned three virion proteins to ORFs 2a, 2b, and 3, which are expressed from two 3'-coterminal subgenomic RNAs. The NDiV ORF1a/ORF1b frameshifting signal and various replicative proteins were tentatively mapped to canonical positions in the nidovirus genome. They include six nidovirus-wide conserved replicase domains, as well as the ExoN and 2'-O-methyltransferase that are specific to large nidoviruses. NDiV ORF1b also encodes a putative N7-methyltransferase, identified in a subset of large nidoviruses, but not the uridylate-specific endonuclease that - in deviation from the current paradigm - is present exclusively in the currently known vertebrate nidoviruses. Rooted phylogenetic inference by Bayesian and Maximum Likelihood methods indicates that NDiV clusters with roniviruses and that its branch diverged from large nidoviruses early after they split from small nidoviruses. Together these characteristics identify NDiV as the prototype of a new nidovirus family and a missing link in the transition from small to large nidoviruses.

Molecular diagnosis and ocular imaging of West Nile virus retinitis and neuroretinitis.

PURPOSE: To describe the ocular features of West Nile virus (WNV) infection proven by serology and molecular diagnostic techniques. DESIGN: Prospective case series. PARTICIPANTS: Fifty-two patients who presented to the uveitis clinic with ocular inflammatory signs and history of fever preceding ocular symptoms between January 2010 and January 2012 were enrolled for laboratory diagnosis. Serum samples were collected from 30 healthy controls from the same geographic area. METHODS: Patients were tested for all endemic infectious diseases that can cause ocular inflammation by serology or molecular diagnostics. When patients had positive antibodies for WNV, serum/plasma samples were tested by real-time reverse transcription (RT) polymerase chain reaction (PCR) and RT loop-mediated isothermal gene amplification assays. The PCR product was subjected to nucleotide sequencing. Fundus fluorescence angiography (FFA), optical coherence tomography (OCT), and indocyanine green angiography were performed. Visual prognosis was analyzed. MAIN OUTCOME MEASURES: Clinical signs (retinitis, neuroretinitis, and choroiditis) and ocular complications (decrease in vision). RESULTS: A total of 37 of 52 patients (71%) showed positive results for at least 2 laboratory tests for WNV. Fundus examination revealed discrete, superficial, white retinitis; arteritis; phlebitis; and retinal hemorrhages with or without macular star. The FFA revealed areas of retinal inflammation with indistinct borders, vascular and optic disc leakage, vessel wall staining, or capillary nonperfusion. Indocyanine green angiography confirmed choroidal inflammation in 1 of the patients who was diabetic. The OCT scan of the macula revealed inner retinal layer edema in active inflammation and retinal atrophy in late stage. At the final visit, 43% of patients had visual acuity better than 6/12. CONCLUSIONS: In addition to previously reported clinical signs, retinitis, neuroretinitis, and retinal vasculitis were seen in this population. Atrophy of the inner retinal layer was seen on OCT after resolution of inflammation. Visual prognosis was good in patients with focal retinitis and poor in patients with occlusive vasculitis. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Development & evaluation of biotinylated DNA probe for clinical diagnosis of chikungunya infection in patients' acute phase serum & CSF samples.

BACKGROUND & OBJECTIVES: The resurgence of chikungunya virus (CHIKV) in the Indian Ocean Islands and India has drawn worldwide attention due to its explosive nature, high morbidity and complex clinico-pathological manifestations. The early confirmatory diagnosis of CHIKV is essential for management as well as control of unprecedented epidemics. The present study describes the development and evaluation of a highly sensitive and specific E1 structural gene specific biotinylated DNA probe for detection of chikungunya virus in clinical samples using a dot blot format. METHODS: The complementary DNA (cDNA) of CHIKV was spotted on to nylon membrane. The membrane was subjected to prehybridization and hybridization and developed using a colour development solution containing DAB chromogen. RESULTS: The CHIKV E1 specific DNA probe was highly sensitive detecting picogram levels of target nucleic acid. The comparative evaluation with SYBR Green I based real-time RT-PCR revealed 99 per cent accordance with a sensitivity and specificity of 99 and 98 per cent, respectively. The specificity of this assay was further confirmed through cross-reaction studies with confirmed dengue and Japanese encephalitis (JE) patient serum samples along with infected culture supernatant of Ross River and Saint Louis encephalitis and plasmid DNA of O'Nyong Nyong, Semlinki forest and Sindbis viruses. INTERPRETATION & CONCLUSION: The DNA probe reported in this study may be useful for specific, sensitive and confirmatory clinical diagnosis of chikungunya infection in acute phase human patient serum and CSF samples. This assay can also be used in the laboratory for quantification of viral antigen in cell culture supernatant for research purpose.

Molecular and virological investigation of a focal chikungunya outbreak in northern India.

Chikungunya (CHIK) fever is one of the most important arboviral infections of medical significance. The objective of the present study is to identify and characterize the etiology of a focal febrile arthritis outbreak from Gwalior, northern India, during October-November 2010. A detailed virological (isolation) and molecular (end-point RT-PCR, quantitative RT-PCR, and nucleotide sequencing) investigation of this outbreak was carried out by collecting and studying 52 clinical samples and 15 mosquito pools from the affected region. The investigation revealed the presence of CHIK viral RNA in 29% of clinical samples and 13% mosquito pool by RT-PCR. The quantification of CHIK viral RNA in samples varied from 10(2.50) to 10(6.67) copies/mL, as demonstrated through quantitative RT-PCR. In addition, six CHIK viruses were isolated from RT-PCR positive samples. The nucleotide sequences of partial E1 gene of five representative CHIK viruses were deciphered, which revealed that all the viral strains from this outbreak belong to the recently emerging ECS African genotype. Identification of Chikungunya virus ECSA African genotype as the etiology of the present outbreak confirms the continued circulation of the novel genotype, since 2006, in India. The identification of CHIK virus in Aedes aegypti also confirmed it as the major vector in northern India.

Identification of 2,4-Diaminoquinazoline Derivative as a Potential Small-Molecule Inhibitor against Chikungunya and Ross River Viruses.

Alphaviruses are serious zoonotic threats responsible for significant morbidity, causing arthritis or encephalitis. So far, no licensed drugs or vaccines are available to combat alphaviral infections. About 300,000 chikungunya virus (CHIKV) infections have been reported in 2023, with more than 300 deaths, including reports of a few cases in the USA as well. The discovery and development of small-molecule drugs have been revolutionized over the last decade. Here, we employed a cell-based screening approach using a series of in-house small-molecule libraries to test for their ability to inhibit CHIKV replication. DCR 137, a quinazoline derivative, was found to be the most potent inhibitor of CHIKV replication in our screening assay. Both, the cytopathic effect, and immunofluorescence of infected cells were reduced in a dose-dependent manner with DCR 137 post-treatment. Most importantly, DCR 137 was more protective than the traditional ribavirin drug and reduced CHIKV plaque-forming units by several log units. CHIKV-E2 protein levels were also reduced in a dose-dependent manner. Further, DCR 137 was probed for its antiviral activity against another alphavirus, the Ross River virus, which revealed effective inhibition of viral replication. These results led to the identification of a potential quinazoline candidate for future optimization that might act as a pan-alphavirus inhibitor.

Utility of IgM ELISA, TaqMan real-time PCR, reverse transcription PCR, and RT-LAMP assay for the diagnosis of Chikungunya fever.

Chikungunya fever a re-emerging infection with expanding geographical boundaries, can mimic symptoms of other infections like dengue, malaria which makes the definitive diagnosis of the infection important. The present study compares the utility of four laboratory diagnostic methods viz. IgM capture ELISA, an in house reverse transcription PCR for the diagnosis of Chikungunya fever, TaqMan real-time PCR, and a one step reverse transcription-loop mediated isothermal amplification assay (RT-LAMP). Out of the 70 serum samples tested, 29 (41%) were positive for Chikungunya IgM antibody by ELISA and 50 (71%) samples were positive by one of the three molecular assays. CHIKV specific nucleic acid was detected in 33/70 (47%) by reverse transcription PCR, 46/70 (66%) by TaqMan real-time PCR, and 43/70 (62%) by RT-LAMP assay. A majority of the samples (62/70; 89%) were positive by at least one of the four assays used in the study. The molecular assays were more sensitive for diagnosis in the early stages of illness (2-5 days post onset) when antibodies were not detectable. In the later stages of illness, the IgM ELISA is a more sensitive diagnostic test. In conclusion we recommend that the IgM ELISA be used as an initial screening test followed one of the molecular assays in samples that are collected in the early phase of illness and negative for CHIKV IgM antibodies. Such as approach would enable rapid confirmation of the diagnosis and implementation of public health measures especially during outbreaks.