Multiplex real-time RT-PCR for detecting chikungunya virus and dengue virus

Objective:To develop diagnostic test for detection chikungunya virus(CHIKV and Dengue virus (DENV) infection.Methods:We have performed a rapid,accurate laboratory confirmative method to simultaneously detect,quantify and differentiate CHIKV and DENV infection by single-step multiplex real-time RT-PCR.Results:The assay’s sensitivity was 97.65%,specificity was 92.59% and accuracy was 95.82%when compared to conventional RT-PCR.Additionally,there was no cross-reaction between CHIKV,DENV,Japanese encephalitis virus,hepatitis C,hepatitis A or hepatitis E virus.Conclusions:This rapid and reliable assay provides a means for simultaneous early diagnosis of CHIKV and DENV in a single-step reaction.

Andrographolide inhibits chikungunya virus infection by up-regulating host innate immune pathways

Objective: To investigate the therapeutic efficacy of andrographolide, a plant derived compound, against chikungunya virus(CHIKV) infection. Methods: Using flow cytometry and immunoblotting assay, in vitro viral protein expression was studied in THP-1 cells line. In Balb/c mouse neonates, viral RNA copy number was determined by real time PCR. Results:The results showed reduced CHIKV protein expression on andrographolide treatment in CHIKV-infected human peripheral blood mononuclear cells, Vero cells and THP-I cell line.In vivo, andrographolide treatment to CHIKV-infected neonates reduced viral RNA copy number. Further. andrographolide also increased cytotoxic T lymphocytes both in vitro and in vivo. Andrographolide also activated host innate immune pathways, viz., protein kinase R.phosphorylated eukaryotic initiation factor 2 α, retinoic acid inducible gene-Ⅰ and interferon regulatory factor 3/7, thereby increasing IFN-a secretion. CHIKV-induced nuclear factor κlight chain enhancer of activated B cells and tumor necrosis factor-a was also reduced on andrographolide treatment. Conclusion: Andrographolide inhibits CHIKV by suppressing viral protein expression and up-regulating host innate immunity and hence could be an effective therapeutic agent against CHIKV infection.

Emerging and re-emerging viruses: A global challenge illustrated by Chikungunya virus outbreaks

In recent decades, the issue of emerging and reemerging infectious diseases, especially those related to viruses, has become an increasingly important area of concern in public health. It is of significance to anticipate future epidemics by accumulating knowledge through appropriate research and by monitoring their emergence using indicators from different sources. The objective is to alert and respond effectively in order to reduce the adverse impact on the general populations. Most of the emerging pathogens in humans originate from known zoonosis. These pathogens have been engaged in long-standing and highly successful interactions with their hosts since their origins are exquisitely adapted to host parasitism. They developed strategies aimed at:(1) maximizing invasion rate;(2) selecting host traits that can reduce their impact on host life span and fertility;(3) ensuring timely replication and survival both within host and between hosts; and(4) facilitating reliable transmission to progeny. In this context, Arboviruses(or ARthropod-BOrne viruses), will represent with certainty a threat for the coming century. The unprecedented epidemic of Chikungunya virus which occurred between 2005 and 2006 in the FrenchReunion Island in the Indian Ocean, followed by several outbreaks in other parts of the world, such as India and Southern Europe, has attracted the attention of medical and state authorities about the risks linked to this re-emerging mosquito-borne virus. This is an excellent model to illustrate the issues we are facing today and to improve how to respond tomorrow.

Surveillance of chikungunya virus in Andhra Pradesh,Southern India

Objective:The study involved survey and screening of areas suspected of chikungunya virus (CHIKV) infection,characterizing the causative agent and identifying the circulating CHIKV genotype.Methods:Acute phase samples were screened by the use of RTPCR using primer set DVRChk-F/DVRChk-R whereas convalescent samples were tested by CHIKV IgM strips. Results:Two hundred and seventy five acute phase samples were screened by RT-PCR.of which 149(54.18%) showed positivity for CHIKV.Later on 192 convalescent phase samples were tested for CHIKV specific antibodies in which 125(65.10%) samples were found to be positive.Four CHIKV strains were selected and subjected to cloning followed by nucleotide sequencing and were submitted to the Genbank DMA database with the Accession numbers(GQ119362,GQ119363, GQl 19364,and FJ225403).The Sequence analysis of "CHIK-Kadapa" strain(GQ119362) with other CHIKV isolates suggested that the present CHIKV strain has(99.23±0.52)%and 100%identity with Central East South African isolates(CESA) at nucleotide and amino acid levels respectively.Two unique non synonymous mutations S168L and D183V were depicted in El gene of the selected strains of the present study.Conclusions:The 14 months survey revealed the circulation of CHIKV in 2008-2009 in Andhra Pradesh and the causative agent is identified to be of Central East South African(CESA) origin.The importance of the non synonymous mutations(S168L and D183V) and their role in the mobility and strength of the El-El and E1-E2 interactions needs further investigations.The study also urges the need for intensifying the epidemiological and entomological surveillance to combat any such CHIKV outbreak in the near future.

Molecular genome tracking of East,Central and South African genotype of Chikungunya virus in South-east Asia between 2006 and 2009

Objective:To understand the epidemiology of the East,Central and South African(ECSA) genotype of Chikungunya virus(CHIKV) in terms of emerging and re-emerging infections,this study has been aimed at investigating the evolutionary parameters,genomic signatures and molecular tracking of the CHIKV ECSA genotype in South-east Asia and coastal areas of the Indian Ocean between 2006 and 2009 by using phylogenetie analysis and the Bayesian Markov Chain Monte Carlo(BMCMC) evolutionary estimation.Methods:Nearly complete genome sequences of 53 CHIKV isolates from all genotypes were subjected to phylogenetie analysis and evolutionary parameter estimation.The amino acids of 67 of ECSA genotype during 2006 to 2009 were compared for finding molecular signature tracking.The ECSA genotype signatures were visualized to find the possible transmission root was projected onto a geographic map.Results: Phylogenetie analysis showed the ECSA genotype was divided into 2 groups.The first group comprises viruses from India and Southeast Asian countries.The second group consists of strains typically circulating in Sri Lanka in 2008.The evolutionary parameters of these groups depicted the time of the most recent common ancestor at approximately 7.5 years ago.The genomic signatures revealed the positions of amino acid variation in each group.Conclusions:The molecular evolution projected onto a geographical map showed the routes of CHIKV transmission from 2006 to 2009.Molecular tracking will assist in understanding transmission routes, epidemiology and molecular evolution of CHIKV.

Cytokine levels in patients with chikungunya virus infection

Objective: To investigate cytokine profile in patients with chikungunya virus (CHIKV) infection. Methods: Twenty eight pairs of serum samples collected from CHIKV infected patients during the outbreak of chikungunya fever in South Thailand in 2008 were obtained. A multiple cytokine assay for detection of 17 cytokines was performed. Results: In the acute stage of CHIKV infection, the patients had significantly higher levels of interleukin-6, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, monocyte chemotactic protein 1 and tumor necrosis factor alpha than the control ( P<0.001, P=0.023, P=0.015, P <0.001 and P=0.024, respectively). When the disease developed to the recovery stage, the patients had significantly lower levels of interleukin-6, granulocyte-macrophage colony-stimulating factor, monocyte chemotactic protein 1 and macrophage inflammatory protein beta than in the acute stage ( P<0.001). Conclusions: This study provides additional information that these cytokines could play roles in pathogenesis of CHIKV infection and could be used as disease biomarkers or drug targets.

Discovery of novel thieno[3,2-b]pyrrole 5-carboxamides as potent inhibitors of Chikungunya virus

Chikungunya fever(CHIKF)is an arboviral disease that typically consists of an acute illness with fever,skin rash,and incapacitating arthralgia.The causative agent of CHIKF is Chikungunya virus(CHIKV),an alphavirus that is transmitted by the Aedes mosquitoes.Despite the re-emergence of CHIKV as an epidemic threat,there is no approved effective anti-viral treatment currently available for CHIKV.In our preliminary studies,selected small molecule inhibitors of arboviruses related to CHIKV were investigated and this led us to identify compounds with thieno[3,2-b]pyrrole scaffold as hits.Building on the discovery of our best hit compounds,5-carboxylic acid thieno[3,2-b]pyrrole 1 and 5-carboxamide thieno[3,2-b]pyrrole 2,the main aim of this study is to optimize their anti-viral activities by synthesizing analogs of thieno[3,2-b]pyrroles 1 and 2 and examine their activities against CHIKV.In these two parallel optimization studies,we synthesized two series of thieno[3,2-b]pyrroles,namely the 5-carboxylic acids and 5-carboxamides that possessed a variety of substituents at N4,C2,C6 or C5positions of the thieno[3,2-b]pyrrole scaffold.These compounds were then examined for their cytotoxicity effects and anti-viral activities using a luminescence-labelled CHIKV infectious clone.The most potent compound in our studies was found in the 5-carboxamide series.The synthesis,biological activity and structure-activity relationship(SAR)will be presented and discussed in detail.

Parsonage-Turner syndrome following chikungunya virus infection:A case report

Rationale: Parsonage-Turner syndrome is a rare syndrome of unknown etiology, affecting mainly the lower motor neurons of the brachial plexus.Chikungunya fever is a mosquito-borne viral disease characterized by acute fever and polyarthritis/polyarthralgia.Patient concerns: A 54-year-old Brazilian male patient who presented with a 2-day history of fever(temperature 38.8 ℃), arthralgia, erythematous rash, diffuse osteomuscular pain and headache, which evolved into left shoulder pain associated with morning stiffness.Diagnosis: Parsonage-Turner syndrome and chikungunya fever.Interventions: Symptomatic treatment(a combination of short-acting dypirone(500 mg every 6 h) and slow-release opioids(tramadol 100 mg every 4 h) and physiotherapy/rehabilitation with improvement.Outcomes: The patient was improved and discharged, remaining with symptomatic treatment and physiotherapy/rehabilitation.Lessons: To the best of our knowledge, there were no reports of Parsonage-Turner syndrome following chikungunya virus infection.Awareness of the possibility of this rare association is important.The present case report highlights the importance of awareness of this association as a new cause of morbidity in patients with chikungunya virus infection.

Modelling the Effects of Vertical Transmission in Mosquito and the Use of Imperfect Vaccine on Chikungunya Virus Transmission Dynamics

In this paper, a deterministic mathematical model for Chikungunya virus (Chikv) transmission and control is developed and analyzed to underscore the effect of vaccinating a proportion of the susceptible human, and vertical transmission in mosquito population. The disease free, and endemic equilibrium states were obtained and the conditions for the local and global stability or otherwise were given. Sensitivity analysis of the effective reproductive number,?Rc?(the number of secondary infections resulting from the introduction of a single infected individual into a population where a proportion is fairly protected) shows that the recruitment rate of susceptible mosquito (ΛM) and the proportion of infectious new births from infected mosquito (β)?are the most sensitive parameters. Bifurcation analysis of the model using center manifold theory reveals that the model undergoes backward bifurcation (coexistence of disease free and endemic equilibrium when Rc?< 1 ). Numerical simulation of the model shows that vaccination of susceptible human population with imperfect vaccine will have a positive impact and that vertical transmission in mosquito population has a negligible effect. To the best of our knowledge, our model is the first to incorporate vaccinated human compartment and vertical transmission in (Chikv) model.

Prediction of promiscuous T cell epitopes in RNA dependent RNA polymerase of Chikungunya virus

Objective: To explore RNA dependent RNA polymerase of Chikungunya virus(CHIKV) and develop T cell based epitopes with high antigenicity and good binding affinity for the human leukocyte antigen(HLA) classes as targets for epitopes based CHIKV vaccine. Methods: In this study we downloaded 371 non-structural protein 4 protein sequences of CHIKV belonging to different regions of the world from the US National Institute of Allergy and Infectious Diseases(NIAID) virus pathogen resource database. All the sequences were aligned by using CLUSTALW software and a consensus sequence was developed by using Uni Pro U Gene Software version 1.2.1. PropredⅠand Propred software were used to predict HLAⅠ and HLAⅡ binding promiscuous epitopes from the consensus sequence of non-structural protein 4 protein. The predicted epitopes were analyzed to determine their antigenicity through Vaxijen server version 2.0. All the HLAⅠ binding epitopes were scanned to determine their immunogenic potential through the Immune Epitope Database(IEDB). All the predicted epitopes of our study were fed to IEDB database to determine whether they had been tested earlier. Results: Twenty two HLA class Ⅱ epitopes and eight HLA classⅠepitopes were predicted. The promiscuous epitopes WMNMEVKII at position 486–494 and VRRLNAVLL at 331–339 were found to bind with 37 and 36 of the 51 HLA class Ⅱ alleles respectively. Epitope MANRSRYQS at position 58–66 and epitopes YQSRKVENM at positions 64–72 were predicted to bind with 12 and 9 HLAⅠI alleles with antigenicity scores of 0.754 9 and 1.013 0 respectively. Epitope YSPPINVRL was predicted to bind 18 HLAⅠ alleles and its antigenicity score was 1.425 9 and immunogenicity score was 0.173 83. This epitope is very useful in the preparation of a universal vaccine against CHIKV infection. Conclusions: Epitopes reported in this study showed promiscuity, antigenicity as well as good binding affinity for the HLA classes. These epitopes will provide the baseline for development of efficacious vaccine for CHIKV.

The Chikungunya virus: An emerging US pathogen

BACKGROUND:The Chikungunya(CHIK)virus was recently reported by the CDC to have spread to the United States.We report an early documented case of CHIK from the state of Pennsylvania after a patient recently returned from Haiti in June of 2014.METHODS:A 39-year-old man presented to the emergency department complaining of fever,fatigue,polyarthralgias and a diffuse rash for two days.Four days before,he returned from a mission trip to Haiti and reported that four of his accompanying friends had also become ill.A CHIK antibody titer was obtained and it was found to be positive.During his hospital stay,he responded well to supportive care,including anti-inflammatories,intravenous hydration and anti-emetics.RESULTS:His condition improved within two days and he was ultimately discharged home.CONCLUSIONS:Manifestations of CHIK can be similar to Dengue fever,which is transmitted by the same species of mosquito,and occasionally as a co-infection.Clinicians should include Chikungunya virus in their differential diagnosis of patients who present with fever,polyarthralgia and rash with a recent history of travel to endemic areas,including those within the United States.

Pathogenesis of the Chikungunya virus and the host immunity response

Along with the running COVID-19 pandemic by the severe acute respiratory syndrome coronavirus 2,the Chikungunya virus is already known as the causative agent of re-emerging Chikungunya fever in many countries after several years of latency;and it’s certainly one of the most important clinical issues possibly due to the lack of appropriate vaccination.Therefore,continuous study and monitoring of this disease outbreak demand attention by the relevant health professionals.Present review has been written in the light of the recently available reports on the Chikungunya virus infection.The genomic structure and its impact on the viral epidemiology,the possible protective immunity,and the infection mitigation strategies have been discussed.It’s already well known that the Chikungunya virus can start infection after getting entrance into the blood circulation through the mosquito bites followed by the dissemination into the major organs like liver,brain,eye,joints and lymph nodes in order to inaugurate the infectivity.Apparently,the occurrence of death is very rare but the extreme fatality and morbidity may occur if the patient has other underlying disease conditions.The molecular aspects of the virus,the site-specific damages caused by the viral infection,and finally,the public awareness of such viral infection as discussed in the current review would help to maintain the public health sustainability especially in the developing countries whereby the knowledge on the required hygiene is poor.

Zika virus is arriving at the American continent

Zika virus is the last arbovirosis arriving at the American continent and it is able to produce severe complications due to its neurotropism, such as microcephaly and it is also associated with Guillain-Barre syndrome(GBS). Presence of Aedes aegypti in many regions of Peru makes it easier to spread and it could be present in population, not being detected because there is a lack of rapid diagnosis methods. It is important to make a quick, effective and combined response to this new threat.

Zika Virus Controversies: Epidemics as a Legacy of Mega Events?

The current knowledge of the Zika Virus epidemic clearly lacks a comprehensive understanding of its determinants and clinical outcomes. Until recently regarded as a “simple” dengue-like infection, it nowadays turned into a real challenge to Public Health around the world. The Zika Epidemic shows a quick spread, affects unprepared health systems, and presents with severe neurological complications of newborns—a concrete threat to pregnancies. This re-emerging infectious disease is a source of deep doubts and harsh debates regarding Public Health and even bioethical issues. Several doubts still remain on how to deal with the various possible transmission ways of the disease, the surge of a generation of thousands of microcephalic newborns (and questions on how to handle them within limited health systems), and severe malformations concentrated so far in Northeastern Brazil. Finally, a debate is raised about how the “Endemic State” of Brazil, deeply merged in a health care crisis for almost 40 years now, currently confronts 3 current epidemics of the arboviruses Dengue, Chikungunya and now Zika, all associated with a lack of basic sanitation. Brazil must deal with these biological threats in the context of the Rio 2016 Olympic Games, since Chikungunya has probably been brought to Brazil during the FIFA Soccer World Cup (2014) and Zika Virus spread is associated with the Soccer Confederations Cup (2013).

Full genomic sequence characterization of the chikungunya virus from an imported case with serum viral concentration below culturable level

Chikungunya virus(CHIKV)infection,responsible for chikungunya fever and occasionally severe symptoms,has emerged as an increasing global health concern following several large-scale outbreaks from Africa,Asia,Europe,and America.Over the past two decades,South and Southeast Asia regions have gradually become hot spots for outbreaks involving multiple CHIKV lineages.In China,most CHIKV infections are imported,making it crucial to trace the origins and transmission routes for effective prevention and control.In January 2024,a case of imported chikungunya fever was confirmed in Guangzhou City,Guangdong Province,China.However,the serum CHIKV viral concentration was too low for cultivation[reverse transcription-polymerase chain reaction(RT-PCR)detection,cycle threshold=32.62].Despite this,we suc-cessfully obtained the viral genome sequence directly from the whole blood sample using an optimized meta-transcriptomic sequencing strategy,achieving a full-length viral genome with an average depth of 54.3x.Further analysis confirmed that the CHIKV virus belonged to the Asian lineage,traced to Timor-Leste,where an endemic CHIKV outbreak had been reported in January 2024,consistent with the patient's travel history.Finally,we analyzed genetic evolutionary trends and amino acid site variations.This study highlights the iden-tification of a CHIKV infection origin using direct whole-blood metatranscriptomic sequencing,a valuable method for rapidly sequencing low viral-load samples.

In silico identification of antiviral compounds for the treatment of chikungunya virus infection:qsar modelling and md simulation analysis

Chikungunya virus(CHIKV),transmitted by arthropods,has gained global recognition for its impact on public health.It has expanded globally,including Africa,Asia,and the Indian subcontinent,and has a helicase protein in its genome that is crucial for its replication.Thus,the study targeted the helicase protein of CHIKV with 745 antiviral compounds using an ML-based QSAR model and molecular docking.Top binders(5279172,78161839,6474310,and 5330286)were selected for MD simulation based on the control(Silvestrol).All compounds had the highest binding scores,with 78161839 showing the most consistent RMSD and the least conformational variation,indicating high stability.It also showed the lowest binding free energy(ΔG¼31.31 kcal/mol),indicating energetically favourable binding.PCA and FEL also depicted the stable complex confirmation of the protein and 78161839 complex during the 100 ns simulation.Overall,this study aimed to identify helicase function antiviral binders that could be experimentally tested for treating CHIKV.

Anti-chikungunya activity of luteolin and apigenin rich fraction from Cynodon dactylon

Objective:To obtain Iuteolin and apigenin rich fraction from the ethanolic extract of Cynodon dactylon(L.)(C.dactylon) Pers and evaluate the fraction's cytotoxicity and anti-Chikungunya potential using Vero cells.Methods:The ethanolic extract of C.dactylon was subjected to silica gel column chromatography to obtain anti-chikungunya virus(CHIKV) fraction.Reverse phase-HPLC and GC-MS studies were carried out to identily the major phytochemicals in the fraction using phylochemical standards.Cytotoxicity and the potential of the fraction against CHIKV were evaluated in vitro using Vero cells.Reduction in viral replication was assessed by reverse transcriptase-polymerase chain reaction(RT-PCR) after treating the viral infected Vero cells with the fraction.Results:Reverse Phase-HPLC and GC-MS studies confirmed the presence of flavonoids,luteolin and apigenin as major phytochemicals in the anti-CHIKV ethanolic fraction of C.dactylon- The fraction was found to exhibit potent viral inhibitory activity(about 98%) at the concentration of 50 μg/mL as observed by reduction in cytopathic effect,and the cytotoxic concentration of the fraction was found to be 250 μg/mL.RT-PCR analyses indicated that the reduction in viral mRNA synthesis in fraction treated infected cells was much higher than the viral infected control cells.Conclusions:Luteolin and apigenin rich ethanolic fraction from C.dactylon can be utilized as a potential therapeutic agent against CHIKV infection as the fraction does not show cytotoxicity while inhibiting the virus.

Extensive evolution analysis of the global chikungunya virus strains revealed the origination of CHIKV epidemics in Pakistan in 2016

Chikungunya virus(CHIKV) is a mosquito-borne virus that causes epidemics widely in the world especially in the tropical and subtropical regions. Phylogenetic analysis has found that the CHIKV lineages were associated with the spatial and temporal distributions, which were related to the virus adaption to the major mosquito species and their distributions. In this study, we reported the complete genome sequences of eight CHIKV isolates from the outbreak in Pakistan last year. Then we reviewed the evolutionary history using extensive phylogenetic analysis, analyzed lineagespecific substitutions in viral proteins, and characterized the spreading pathway of CHIKV strains including the Pakistani strains. The results showed that the Pakistani stains belonged to the ECSA.IOL sub-lineage and derived from India. The genetic properties of the Pakistani strains including the adaptive substitution to vectors were further characterized, and the potential risks from the occurrence of CHIKV infection in Pakistan were discussed. These results provided better understanding of CHIKV evolution and transmission in the world and revealed the possible origination of the CHIKV outbreak and epidemic in Pakistan, which would promote the disease prevention and control in the identified countries and territories with the history of CHIKV infections as well as new regions with potential risk of CHIKV outbreaks.

昆明地区献血人群中寨卡病毒、登革病毒和基孔肯雅病毒感染情况调查

目的了解昆明地区献血人群中寨卡病毒(ZIKV)、登革病毒(DENV)和基孔肯雅病毒(CHIKV)感染的流行情况。方法随机抽取2022年8月-2023年10月昆明地区献血者标本10662份,采用实时荧光定量逆转录聚合酶链反应技术(RT-qPCR)检测ZIKV RNA、DENV RNA和CHIKV RNA。结果10554份标本获得了有效的检测结果,在这次调查中未发现ZIKV、DENV和CHIKV核酸阳性。结论昆明地区无偿献血人群中ZIKV、DENV和CHIKV的感染风险较低,暂时没有必要对昆明地区无偿献血人群进行ZIKV、DENV和CHIKV的常规筛查。

Recent progress on chikungunya virus research

Chikungunya virus(CHIKV) is an arbovirus transmitted by Aedes mosquitos in tropical and subtropical regions across the world. After decades of sporadic outbreaks, it re-emerged in Africa,Asia, India Ocean and America suddenly, causing major regional epidemics recently and becoming a notable global health problem. Infection by CHIKV results in a spectrum of clinical diseases including an acute self-limiting febrile illness in most individuals, a chronic phase of recurrent join pain in a proportion of patients, and long-term arthralgia for months to years for the unfortunate few. No specific anti-viral drugs or licensed vaccines for CHIKV are available so far. A better understanding of virus-host interactions is essential for the development of therapeutics and vaccines. To this end, we reviewed the existing knowledge on CHIKV's epidemiology, clinical presentation, molecular virology, diagnostic approaches, host immune response, vaccine development, and available animal models. Such a comprehensive overview, we believe, will shed lights on the promises and challenges in CHIKV vaccine development.