Human arbovirus infections in Burundi: results of a seroepidemiologic survey, 1980-1982

A serological survey on 623 human sera was conducted in Burundi in 1980-1982, in order to evaluate the frequency of arboviral antibodies in the inhabitants of the three main areas: lowlands, central plateau and mountainous ridge. The results show a rather high activity of arboviruses, mainly in the lowlands (34.2% of inhabitants with antibodies). Chikungunya virus seems to be the most active arbovirus; the activity of Flavivirus is moderate; no trace of activity of yellow fever or West Nile viruses was found; Bunyavirus antibodies (particularly against Ilesha virus) were also detected.     

Les arbovirus ont aussi leur « rêve américain »

Some arboviruses that originated in the Old World have been introduced by humans into the American continent. The first of them was the yellow fever virus, coming from the West African coast with slaves in the 17th–19th centuries, followed by dengue viruses, which were always prevalent within the Americas. Next was theWest Nile virus, introduced in New York in 1999, that spread in only a few years over the whole continent. Then, Chikungunya virus arrived on Saint Martin Island in 2013 after its outbreak in Polynesia; it is now widespread in the Caribbean Islands and on the American continent from the United States to Brazil. Finally, Zika virus, already active in Asia and in the South Pacific region, was introduced in Brazil and spread between the southern part of United States and south Brazil. These unexpected emergences are the consequence of the generalization of transoceanic trading; so, it is humans who are truly responsible for such transportation of viruses from the African and Asian continents. The mechanisms of virus establishment in unusual ecosystems have to be analyzed in order to understand the conditions for the circulation of the viruses, which supposes an adaptation to new hosts and vectors that are sometimes local species (like Culex vectors of West Nile virus) but mainly previously introduced mosquitoes (like Aedes aegypti and/or Aedes albopictus). Over time, all these vectors developed a strong anthropophily and, most of them, a remarkable adaptation to urban environment; hence, these arboviruses can disseminate both in rural and urban context. This type of arboviral emergences will certainly continue in the following years and we must imperatively develop preventive strategies by detecting virus mutations with capacity for emergence, enhancing the sensibility and rapidity of epidemiological surveillance, and becoming ready to face such events that cause a truly international health crisis.     

Anti-arbovirus antibodies positive serum screening in population of Saratov

Seroepidemiological studies revealed the existence of a population stratum immune to West Nile and Sindbis viruses in the Saratov region. Screening of 218 sera showed that the stratum immune to arboviruses increased from 1998 to 2000. The incidence of antibodies to Batai and Sindbis viruses was the highest. Activation of natural foci of these arboviruses can lead to exacerbation of the epidemic situation in the region.     

Application of enzyme immunoassay on infected cells (EIA-IC) for arboviruses

Comparative titrations of alpha-, flavi- and Bunyamwera viruses were made by EIA-IC and according to cytopathic effect (CPE). Specific enzymatic reactions appeared earlier and in higher titres than CPE. The titres of dengue type 1, Mayaro, Powassan and Langat viruses measured by EIA-IC were comparable to those measured by intracerebral inoculation of mice. The cross-reactivity testing of EIA-IC among alphaviruses (Chikungunya, Sindbis and Mayaro), flaviviruses (Japanese encephalitis, Murray valley encephalitis, Kunjin, West Nile, yellow fever and louping ill, Powassan, Langat) and Bunyamwera arboviruses using polyclonal immune ascitic fluids confirmed the high specificity of EIA-IC. Homologous reactions mostly showed higher titres than heterologous ones. No cross-reactivity was seen between alpha-, flavi- and bunyaviruses, among the three alphaviruses, between mosquito-borne and tick-borne flaviviruses, or between JE complex and YF viruses. However, a cross-reactivity to different extent was observed among the four JE complex viruses and among louping ill, Powassan and Langat viruses. The results of EIA-IC cross tests showed that this method can distinguish togavirus group- or species-specific antigens, more precisely than conventional ELISA.     

Polykaryocytosis induced by certain arboviruses in monolayers of BHK-21-528 cells

Multinucleated giant cell formation in a clone of BHK-21 cells, BHK-21-528, was tested with certain arboviruses. Eleven out of 19 viruses tested, Chikungunya, Getah, Sagiyama, Sindbis, Western equine encephalitis, St. Louis encephalitis, Japanese encephalitis, West Nile, Bunyamwera, Germiston and California encephalitis virus induced cell fusion after infection. All cases where giant cells were observed during the experiments, involved the B type cell fusion (fusion from within).     

Nucleotide sequence and deduced amino acid sequence of the structural proteins of dengue type 2 virus, Jamaica genotype

The nucleotide sequence of the 5'-terminal 2469 bases of dengue 2 (Jamaica genotype) virus has been determined and the encoded proteins compared with those of yellow fever and West Nile viruses, which belong to different flavivirus serogroups. The cDNA clone which was sequenced contains a 5'-noncoding region of 96 nucleotides followed by a single open reading frame coding for the structural proteins 5'-C-prM(M)-E-3' and the beginning of the NS1 nonstructural protein. The amino acid sequence homology between the structural polyprotein precursor of dengue 2 virus and those of yellow fever and West Nile viruses is 36.5 and 42%, respectively. The dengue virus structural proteins are similar in size and composition to those of the other flaviviruses. The basic capsid protein and the membrane and envelope proteins have hydrophobic regions at their C termini. The dengue 2 capsid C, membrane M, and envelope E proteins share 13, 36, and 43% homology, respectively, with the cognate proteins of yellow fever virus, and 33, 32, and 47% homology with the cognate proteins of West Nile virus. All 6 cysteine residues in the dengue 2 premembrane protein and all 12 cysteine residues in the dengue 2 envelope protein are conserved in the cognate proteins of yellow fever and West Nile viruses.     

Hepatitis C and arboviral antibodies in the island populations of mauritius and Rodrigues

A serological survey for antibodies to hepatitis C virus (HCV), dengue viruses (DEN), West Nile virus (WN), and sindbis virus (SIN) was carried out in sera of selected groups of the population of the Islands of Mauritius (n = 449) and Rodrigues (n = 115), Indian Ocean. 8.3% of 564 sera were positive for anti-HCV. In Mauritius, 2.1% of sera of healthy individuals were found with anti-HCV. The highest prevalence was found in sexually transmitted disease (STD) patients and prison inmates with 46.2% and 43.8%, respectively. None of the sera from blood donors sampled from Rodrigues Island had anti-HCV. Antibodies to arbo-viruses were detected in sera of individuals from both islands. Anti-DEN IgG was detected in 3.8% of sera from Mauritius and 0.9% from Rodrigues. Anti-WN IgG was detected in 2.2% of sera from Mauritius and 0.9% from Rodrigues. All sera from Rodrigues were without anti-SIN IgG, 1.1% of those from Mauritius were positive. This suggests that arboviruses occur on these islands. © 1994 Wiley-Liss, Inc.     

Small wild mammals and arboviruses in Italy

In 1980 and 1981, sera of 256 small wild mammals (rodentia, insectivora, carnivora) were collected in Piemonte and Southern Italy. They were then tested for antibody against 12 arboviruses by haemagglutination inhibition and complement fixation tests. In the North of the country, 42.8% of sera were found positive against Tahyna or Sicilian Sandfly fever viruses. In the Southern provinces, 44.3% of sera reacted with Bhanja, West Nile or other flaviviruses, Tahyna, Sicilian Sandfly fever and Arumowot viruses. These results lead to suspect the possible role of some small mammals (muridae, Clethrionomys glareolus, Talpa caeca, Talpa romana) in circulating these arboviruses in Italy.     

DNA microarray technique for detection and identification of seven flaviviruses pathogenic for man

A flavivirus microarray was developed for detection and identification of yellow fever (YF), West Nile, Japanese encephalitis (JE), and the dengue 1-4 viruses, which are causing severe human disease all over the world. The microarray was based on 500-nucleotide probe fragments from five different parts of the seven viral genomes. A low-stringent amplification method targeting the corresponding regions of the viral genomic RNA was developed and combined with hybridization to the microarray for detection and identification. For distinction of the generated virus-specific fluorescence-patterns a fitting analysis procedure was adapted. The method was verified as functional for all seven flaviviruses and the strategy for the amplification, combined with the long probes, provided a high tolerance for smaller genetic variability, most suitable for these rapidly changing RNA viruses. A potentially high detection and identification capacity was proven on diverged strains of West Nile and dengue viruses. The lower limit for detection was equivalent, or better, when compared to routinely used RT-PCR methods. The performance of the method was verified on human patient samples containing dengue viruses, or normal human serum spiked with YF or JE viruses. The results demonstrated the ability of the flavivirus microarray to screen simultaneously a sample for several viruses in parallel, in combination with a good lower limit of detection.     

Study of combined foci of mosquito-transmitted arbovirus infections

A total of 5227 serum specimens from humans, horses and swine collected in the seasons of 1968--1976 in 15 administrative areas of the Primorskiy Kray were examined with antigens of a number of mosquito-borne arboviruses: Japanese encephalitis (JE), West Nile (WN), Getah, and Sindbis. Both independent and combined circulation of these viruses in the region was established. Sindbis virus was found to be circulating separately most frequently, West Nile virus the least frequently. According to the results of the serological analysis, the conditions for combined circulation are most closely related for JE and Getah, and JE and WN viruses. The interpretation of the results of examinations of the sera reacting simultaneously with JE and WN virus is most difficult because of close antigenic relationship of these viruses. A set of methods for serological differentiation of bivalent sera to JE and WN viruses and the criteria for the assessment of the results are recommended.     

Simultaneous detection and quantitation of Chikungunya, Dengue and West Nile viruses by multiplex RT-PCR assays and Dengue virus typing using High Resolution Melting

Chikungunya (CHIKV), Dengue (DENV) and West Nile (WNV) viruses are arthropod-borne viruses that are able to emerge or re-emerge in many regions due to climatic changes and increase in travel. Since these viruses produce similar clinical signs it is important for physicians and epidemiologists to differentiate them rapidly. A molecular method was developed for their detection and quantitation in plasma samples and a DENV typing technique were developed. The method consisted in performing two multiplex real-time one-step RT-PCR assays, to detect and quantify the three viruses. Both assays were conducted in a single run, from a single RNA extract containing a unique coextracted and coamplified composite internal control. The quantitation results were close to the best detection thresholds obtained with simplex RT-PCR techniques. The differentiation of DENV types was performed using a High Resolution Melting technique. The assays enable the early diagnosis of the three arboviruses during viremia, including cases of coinfection. The method is rapid, specific and highly sensitive with a potential for clinical diagnosis and epidemiological surveillance. A DENV positive sample can be typed conveniently using the High Resolution Melting technique using the same apparatus.     

Expansion des arboviroses et gestion du risque transfusionnel : exemple des virus West Nile,de la dengue et du chikungunya

Arbovirus infections are increasing in prevalence worldwide. This presents new risks for blood transfusion. This article describes the epidemiology and surveillance of West Nile Virus, dengue and chikungunya and their role in the risk management of transfusions. Arboviruses are RNA viruses and very adaptable by nature. The majority of arbovirus infections are zoonoses. The risk of transmission is multifactorial and concerns the virus, vectors, animal reservoirs, the environment and human behaviour. In recent years, West Nile Virus has become established and widespread in North America, the number of cases of dengue worldwide has increased dramatically, and major epidemics of chikungunya have occurred in the Indian Ocean and Asia. The transmission of dengue and chikungunya is demonstrated in temperate zones. All arboviruses are potentially transmissible by transfusion due to their capacity to induce an asymptomatic viremic phase. The risk of West Nile Virus transmission via transfusion is recognised and prevention measures are well established. The risk of transmission via transfusion of dengue and chikungunya is real but difficult to quantify and the optimum prevention strategy is currently the subject of research. Access to up-to-date epidemiological data is an essential aid to decision-making, especially for donors returning from endemic areas to Europe. The challenge is to define and implement appropriate measures in unpredictable situations.     

Du donneur au receveur : particularités de la chaîne transfusionnelle dans les DOM

Besides specific organisational requirements, the transfusionnal chain in French ultra-marine areas has specificities related to the epidemiology of infectious diseases and to population characteristics. We focus on some of these sociodemographic and medical peculiarities: the challenge of autosufficiency in relation to demographic trends; epidemiologic risks associated to emergent viruses such as dengue and Chikungunya, and the strategies that had been implemented to face last outbreaks; inappropriate selection criteria for eligibility to blood donation (biologic characteristics of Afro-Caribbeans not taken into account for the low hemoglobin deferral threshold; absence of guidelines for the screening of hemoglobinopathies AS/AC, present in 8% of the target population); specific indications for transfusion, such as platelet use in dengue fever or RBC transfusion in sickle cell disease. Due to the high polymorphism of erythrocyte antigens in Afro-Caribbeans, intra-ethnic transfusion facilitates compatibility for common antigens, but is responsible for the emergence of allo-antibodies difficult to identify in the absence of specific antisera or panels; molecular typing of erythrocyte antigens would allow detection of those patients at risk for immunization, expressing variant antigens or lacking high frequency antigens, as well as the characterization of RBC expressing immunogenic so called low frequency antigens. In an era of periodic emergence of new viruses in Europe (dengue, Chikungunya, West Nile virus…) and with the spreading of diseases with high transfusionnal requirements, such as sickle cell disease, ultra-marine services represent laboratories for the study of future trends and problems in transfusion medicine.     

Monitoring and improving the sensitivity of dengue nested RT-PCR used in longitudinal surveillance in Thailand

BackgroundAFRIMS longitudinal dengue surveillance in Thailand depends on the nested RT-PCR and the dengue IgM/IgG ELISA.ObjectiveTo examine and improve the sensitivity of the nested RT-PCR using a panel of archived samples collected during dengue surveillance.Study designA retrospective analysis of 16,454 dengue IgM/IgG ELISA positive cases collected between 2000 and 2013 was done to investigate the sensitivity of the nested RT-PCR. From these cases, 318 acute serum specimens or extracted RNA, previously found to be negative by the nested RT-PCR, were tested using TaqMan real-time RT-PCR (TaqMan rRT-PCR). To improve the sensitivity of nested RT-PCR, we designed a new primer based on nucleotide sequences from contemporary strains found to be positive by the TaqMan rRT-PCR. Sensitivity of the new nested PCR was calculated using a panel of 87 samples collected during 2011–2013.Results and conclusionThe percentage of dengue IgM/IgG ELISA positive cases that were negative by the nested RT-PCR varied from 17% to 42% for all serotypes depending on the year. Using TaqMan rRT-PCR, dengue RNA was detected in 194 (61%) of the 318 acute sera or extracted RNA previously found to be negative by the nested RT-PCR. The newly designed DENV-1 specific primer increased the sensitivity of DENV-1 detection by the nested RT-PCR from 48% to 88%, and of all 4 serotypes from 73% to 87%. These findings demonstrate the impact of genetic diversity and signal erosion on the sensitivity of PCR-based methods.     

The interaction of flavivirus M protein with light chain Tctex-1 of human dynein plays a role in late stages of virus replication

The role of the membrane protein (prM/M) in flavivirus life cycle remains unclear. Here, we identified a cellular interactor to the 40-residue-long ectodomain of prM/M (ectoM) using a yeast two-hybrid screen against a human cDNA library and GST pull-down assays. We showed that dynein light chain Tctex-1 interacts with the ectoM of dengue 1-4, West Nile, and Japanese encephalitis flaviviruses. No interaction was found with yellow fever and tick-borne flaviviruses. This interaction is highly specific since a single amino-acid change in the ectoM abrogates the interaction with Tctex-1. To understand the role of this interaction, silencing of Tctex-1 using siRNA was performed prior to infection. A significant decrease in progeny production was observed for dengue and West Nile viruses. Silencing Tctex-1 inhibited the production of recombinant dengue subviral particles (RSPs). Thus Tctex-1 may play a role in late stages of viral replication through its interaction with the membrane protein.     

Molecular and serological techniques to detect co-circulation of DENV,ZIKV and CHIKV in suspected dengue-like syndrome patients

BackgroundArboviruses are important emerging viruses worldwide. The signs and symptoms of Zika virus (ZIKV) infection are similar to those presented by infections with dengue virus (DENV) and chikungunya virus (CHIKV). Furthermore, diagnosis of ZIKV infection is particularly challenging in dengue endemic regions and with co-circulation of DENV, CHIKV, and ZIKV, making diagnosis based solely on clinical and epidemiological data unreliable. As these three viral infections share similar clinical manifestations, differential diagnosis is crucial.ObjectivesIn this study, diagnoses of ZIKV, CHIKV and DENV infections were investigated in 30 patients with suspected dengue fever residing in the area of co-circulation of these three arboviruses.Study designThe study included whole blood and/or serum samples obtained from 30 patients with suspected dengue fever. All patients were tested for DENV infection as well as for CHIKV and ZIKV infections. Assays for detecting anti-DENV IgM and DENV RNA by semi-nested RT-PCR and ZIKV and CHIKV RNA by real-time RT-PCR were performed.ResultsDENV RNA was not detectable in any of the clinical samples, whereas ZIKV RNA was detectable in 17 samples (56.7%). Co-infection by ZIKV and CHIKV was documented in one case. Of the 17 ZIKV-positive individuals, 8 showed reactivity for anti-DENV IgM, which suggested recent DENV infection, cross-reactivity or co-infection.ConclusionOur findings confirm that accurate laboratory testing is of paramount importance for differential diagnosis in areas of simultaneous transmission of different arboviruses with similar clinical presentations.     

Arboviroses in the region of Nosy-Bé, Madagascar. Serologic and entomologic data

Since 1977, the Pasteur Institute of Madagascar has been studying, during six surveys, the arboviruses of Nosy-Be area, in the north-west of Madagascar. 47.2% out of 271 human sera and 11.3% out of 151 sera of Lemurs, tested for antibodies to 16 arboviruses by the haemagglutination inhibition test, are positive. The results show an important prevalence of Flaviviruses. West Nile and Dengue 1 viruses were probably circulating some years before the surveys. Antibodies against Sindbis and Rift Valley Fever viruses, were found only in few subjects. Bunyamwera and California groups of virus are absent. The rate of positive Lemurs is weak, particularly in Lemur macaco macaco. Flaviviruses are the most frequent. 12,262 haematophagous diptera (11,965 Culicidae belonging to 40 species) were caught. Aedes aegypti and Aedes albopictus are both present. Arbovirus isolation attempts from 394 mosquito pools failed; only Mengo virus was isolated from four pools of Eretmapodites quinquevittatus and one pool of Aedes (Skusea) sp.     

DNA probe array for the simultaneous identification of herpesviruses, enteroviruses, and flaviviruses

Viral infections of the central nervous system (CNS) are caused by a variety of viruses, namely, herpesviruses, enteroviruses, and flaviviruses. The similar clinical signs provoked by these viruses make the diagnosis difficult. We report on the simultaneous detection of these major CNS pathogens using amplification by PCR and detection of amplified products using DNA microarray technology. Consensus primers were used for the amplification of all members of each genus. Sequences specific for the identification of each virus species were selected from the sequence alignments of each target gene and were synthesized on a high-density microarray. The amplified products were pooled, labeled, and cleaved, followed by hybridization on a single array. This method was successfully used to identify herpesviruses, namely, herpes simplex virus type 1 (HSV-1), HSV-2, and cytomegalovirus; all serotypes of human enteroviruses; and five flaviviruses (West Nile virus, dengue viruses, and Langat virus). This approach, which used highly conserved consensus primers for amplification and specific sequences for identification, would be extremely useful for the detection of variants and would probably help solve some unexplained cases of encephalitis. The analytical sensitivity of the method was shown to be 500 genome equivalents ml(-1) for HSV-1, 0.3 50% tissue culture infectious doses (TCID50s) ml(-1) for the enterovirus coxsackievirus A9, and 200 TCID50s ml(-1) for West Nile virus. The clinical sensitivity of this method must now be evaluated.     

Challenges towards serologic diagnostics of emerging arboviruses

BackgroundAppropriate laboratory diagnostics for emerging arboviruses are key for patient management, surveillance and intervention, including molecular tests and serological tests detecting viral antigen or virus-specific antibodies.ObjectivesWe provide an overview of the challenges towards serological testing for the most important emerging arboviruses, including Zika, dengue and chikungunya viruses.SourcesWe retrieved a data set on performance of commercially available antibody- and antigen-detecting tests from 89 peer-reviewed articles conducting a systematic literature research in PubMed.ContentWe identified commonly used antibody- and antigen-detecting tests and analysed their overall performance. We discuss how timing of serological testing and the use of paired samples from acute and convalescent phases of infection are crucial to optimize diagnostic sensitivity and specificity. We then exemplify how serological diagnostics are challenged by the patient's infection history through the ‘original antigenic sin’ and cross-reactive antibodies in the context of global co-circulation of antigenically related viruses. We highlight how individual infection histories with different arboviruses and with other pathogens such as herpes viruses and Plasmodia can produce inaccurate test results. We show that rapid tests for antibody and antigen detection in point-of-care settings have a significantly lower sensitivity compared with laboratory-based tests such as ELISA. We show that the performance of antibody- and antigen-detecting tests varies greatly between tropical regions of endemic transmission and non-endemic regions. Finally, we highlight that test sensitivity and specificity have to be equilibrated carefully and frequently either of them must be prioritized over the other, depending on disease prevalence and intended use of tests.ImplicationsFor reliable serological diagnostics, it is essential to be aware of inherent test limitations. Although multiplexed testing and testing of convalescence samples can improve diagnostic performance, global spread of (re-)emerging viruses requires careful implementation and evaluation of serological testing and unambiguous results may not always be achievable.     

Emergence and re-emergence of viral diseases of the central nervous system

Neurologic disease is a major cause of disability in resource-poor countries and a substantial portion of this disease is due to infections of the CNS. A wide variety of emerging and re-emerging viruses contribute to this disease burden. New emerging infections are commonly due to RNA viruses that have expanded their geographic range, spread from animal reservoirs or acquired new neurovirulence properties. Mosquito-borne viruses with expanding ranges include West Nile virus, Japanese encephalitis virus and Chikungunya virus. Zoonotic viruses that have recently crossed into humans to cause neurologic disease include the bat henipaviruses Nipah and Hendra, as well as the primate-derived human immunodeficiency virus. Viruses adapt to new hosts, or to cause more severe disease, by changing their genomes through reassortment (e.g. influenza virus), mutation (essentially all RNA viruses) and recombination (e.g. vaccine strains of poliovirus). Viruses that appear to have recently become more neurovirulent include West Nile virus, enterovirus 71 and possibly Chikungunya virus. In addition to these newer challenges, rabies, polio and measles all remain important causes of neurologic disease despite good vaccines and global efforts toward control. Control of human rabies depends on elimination of rabies in domestic dogs through regular vaccination. Poliovirus eradication is challenged by the ability of the live attenuated vaccine strains to revert to virulence during the prolonged period of gastrointestinal replication. Measles elimination depends on delivery of two doses of live virus vaccine to a high enough proportion of the population to maintain herd immunity for this highly infectious virus.