An alphavirus vector overcomes the presence of neutralizing antibodies and elevated numbers of Tregs to induce immune responses in humans with advanced cancer.

Therapeutic anticancer vaccines are designed to boost patients' immune responses to tumors. One approach is to use a viral vector to deliver antigen to in situ DCs, which then activate tumor-specific T cell and antibody responses. However, vector-specific neutralizing antibodies and suppressive cell populations such as Tregs remain great challenges to the efficacy of this approach. We report here that an alphavirus vector, packaged in virus-like replicon particles (VRP) and capable of efficiently infecting DCs, could be repeatedly administered to patients with metastatic cancer expressing the tumor antigen carcinoembryonic antigen (CEA) and that it overcame high titers of neutralizing antibodies and elevated Treg levels to induce clinically relevant CEA-specific T cell and antibody responses. The CEA-specific antibodies mediated antibody-dependent cellular cytotoxicity against tumor cells from human colorectal cancer metastases. In addition, patients with CEA-specific T cell responses exhibited longer overall survival. These data suggest that VRP-based vectors can overcome the presence of neutralizing antibodies to break tolerance to self antigen and may be clinically useful for immunotherapy in the setting of tumor-induced immunosuppression.

Understanding the alphaviruses: Recent research on important emerging pathogens and progress towards their control

The alphaviruses were amongst the first arboviruses to be isolated, characterized and assigned a taxonomic status. They are globally very widespread, infecting a large variety of terrestrial animals, insects and even fish, and circulate both in the sylvatic and urban/peri-urban environment, causing considerable human morbidity and mortality. Nevertheless, despite their obvious importance as pathogens, there are currently no effective antiviral drugs with which to treat humans or animals infected by any of these viruses. The EU-supported project—VIZIER (Comparative Structural Genomics of Viral Enzymes Involved in Replication, FP6 Project: 2004-511960) was instigated with an ultimate view of contributing to the development of antiviral therapies for RNA viruses, including the alphaviruses [Coutard, B., Gorbalenya, A.E., Snijder, E.J., Leontovich, A.M., Poupon, A., De Lamballerie, X., Charrel, R., Gould, E.A., Gunther, S., Norder, H., Klempa, B., Bourhy, H., Rohayemj, J., L’hermite, E., Nordlund, P., Stuart, D.I., Owens, R.J., Grimes, J.M., Tuckerm, P.A., Bolognesi, M., Mattevi, A., Coll, M., Jones, T.A., Åqvist, J., Unger, T., Hilgenfeld, R., Bricogne, G., Neyts, J., La Colla, P., Puerstinger, G., Gonzalez, J.P., Leroy, E., Cambillau, C., Romette, J.L., Canard, B., 2008. The VIZIER project: preparedness against pathogenic RNA viruses. Antiviral Res. 78, 37–46]. This review highlights some of the major features of alphaviruses that have been investigated during recent years. After describing their classification, epidemiology and evolutionary history and the expanding geographic distribution of Chikungunya virus, we review progress in understanding the structure and function of alphavirus replicative enzymes achieved under the VIZIER programme and the development of new disease control strategies.

Chronic pain associated with the Chikungunya Fever: long lasting burden of an acute illness

Abstract Background Chikungunya virus (CHIKV) is responsible for major epidemics worldwide. Autochthonous cases were recently reported in several European countries. Acute infection is thought to be monophasic. However reports on chronic pain related to CHIKV infection have been made. In particular, the fact that many of these patients do not respond well to usual analgesics suggests that the nature of chronic pain may be not only nociceptive but also neuropathic. Neuropathic pain syndromes require specific treatment and the identification of neuropathic characteristics (NC) in a pain syndrome is a major step towards pain control. Methods We carried out a cross-sectional study at the end of the major two-wave outbreak lasting 17 months in Réunion Island. We assessed pain in 106 patients seeking general practitioners with confirmed infection with the CHIK virus, and evaluated its impact on quality of life (QoL). Results The mean intensity of pain on the visual-analogical scale (VAS) was 5.8 ± 2.1, and its mean duration was 89 ± 2 days. Fifty-six patients fulfilled the definition of chronic pain. Pain had NC in 18.9% according to the DN4 questionnaire. Conversely, about two thirds (65%) of patients with NC had chronic pain. The average pain intensity was similar between patients with or without NC (6.0 ± 1.7 vs 6.1 ± 2.0). However, the total score of the Short Form-McGill Pain Questionnaire (SF-MPQ)(15.5 ± 5.2 vs 11.6 ± 5.2; p < 0.01) and both the affective (18.8 ± 6.2 vs 13.4 ± 6.7; p < 0.01) and sensory subscores (34.3 ± 10.7 vs 25.0 ± 9.9; p < 0.01) were significantly higher in patients with NC. The mean pain interference in life activities calculated from the Brief Pain Inventory (BPI) was significantly higher in patients with chronic pain than in patients without it (6.8 ± 1.9 vs 5.9 ± 1.9, p < 0.05). This score was also significantly higher in patients with NC than in those without such a feature (7.2 ± 1.5 vs 6.1 ± 1.9, p < 0.05). Conclusions There exists a specific chronic pain condition associated to CHIKV. Pain with NC seems to be associated with more aggressive clinical picture, more intense impact in QoL and more challenging pharmacological treatment.

Alphavirus-based expression vectors: strategies and applications.

Alphaviruses are positive-strand RNA viruses that can mediate efficient cytoplasmic gene expression in insect and vertebrate cells. Through recombinant DNA technology, the alphavirus RNA replication machinery has been engineered for high-level expression of heterologous RNAs and proteins. Amplification of replication-competent alpha-virus RNAs (replicons) can be initiated by RNA or DNA transfection and a variety of packaging systems have been developed for producing high titers of infectious viral particles. Although normally cytocidal for vertebrate cells, variants with adaptive mutations allowing noncytopathic replication have been isolated from persistently infected cultures or selected using a dominant selectable marker. Such mutations have been mapped and used to create new alphavirus vectors for noncytopathic gene expression in mammalian cells. These vectors allow long-term expression at moderate levels and complement previous vectors designed for short-term high-level expression. Besides their use for a growing number of basic research applications, recombinant alphavirus RNA replicons may also facilitate genetic vaccination and transient gene therapy.

Bc1-2 protects mice against fatal alphavirus encephalitis.

Virus-induced apoptosis has been well characterized in vitro, but the role of apoptosis in viral pathogenesis is not well understood. The suicide of a cell in response to viral infection is postulated to be an important host defense for the organism, leading to a reduction in its total viral burden. However, virus-induced death of nonregenerating cells in the central nervous system may be detrimental to the host. Therefore, to investigate the role of apoptosis in the pathogenesis of fatal encephalitis, we constructed a recombinant alphavirus chimera that expresses the antiapoptotic gene, bcl-2, in virally infected neural cells. Infection of neonatal mice with the alphavirus chimera expressing human bcl-2 [Sindbis virus (SIN)/bcl-2] resulted in a significantly lower mortality rate (7.5%) as compared with infection with control chimeric viruses containing a chloramphenicol acetyltransferase (CAT) reporter gene (SIN/CAT) (78.1%) or bcl-2 containing a premature stop codon (SIN/bcl-2stop) (72.1%) (P < 0.001). Viral titers were reduced 5-fold 1 day after infection and 10-fold 6 days after infection in the brains of SIN/bcl-2-infected mice as compared to SIN/CAT or SIN/bcl-2stop-infected mice. In situ end labeling to detect apoptotic nuclei demonstrated a reduction in the number of foci of apoptotic cells in the brains of mice infected with SIN/bcl-2 as compared with SIN/bcl-2stop. The reduction in apoptosis was associated with a reduction in the number of foci of cells expressing alphavirus RNA. Thus, the antiapoptotic gene, bcl-2, suppresses viral replication and protects against a lethal viral disease, suggesting an interaction between cellular genetic control of viral replication and cell death.

A atuação do enfermeiro no combate à dengue e a febre chikungunya: estudo comparativo nos municípios de Parnamirim e Santa Cruz/RN

The research aimed to analyze the role of nurses in the Family Health Strategy (FHS) in the control of Dengue and Chikungunya fever in the cities of Parnamirim and Santa Cruz. It is exploratory and descriptive nature of research, with a qualitative approach, which was developed with nurses of the Family Health Strategy in the municipalities of Parnamirim and Santa Cruz. All research process followed the ethical standards laid out research in Resolution No. 466/2012 of the National Health Council about research involving human beings. Data collection was performed using a semi-structured questionnaire with open and closed questions, organized into three parts: the profile of respondents, knowledge of the disease (transmission, prevention, control) and description of practices. The actions performed by nurses are punctual performed when there is growing disease of the number of cases in order to try to reduce the number of cases. The principal methodology used for them is to talk and guidelines resulting from campaigns and home visits, using leaflets and social networks. Regarding knowledge of nurses in the city of Santa Cruz on Dengue Fever and Chikungunya, is clearly some gaps. Nursing professionals know recognize a suspected case of dengue, but get confused when trying to explain to the Chikungunya fever, exhibiting the same symptoms of dengue. Although everybody had participated in a training on Chikungunya Fever and Dengue, a very limited knowledge of nurses regarding the clinical management. It is suggested that further studies are developed on the ground a few nurses have accepted to participate in the study, so that we can identify strategies, interventions, activities and nursing actions that are consistent with the reality of working in favor of coping Aedes epidemics aegypti that have positive impact on reducing the infestation rates and may be suitable and applied in other regions.

A atuação do enfermeiro no combate à dengue e a febre chikungunya: estudo comparativo nos municípios de Parnamirim e Santa Cruz/RN

The research aimed to analyze the role of nurses in the Family Health Strategy (FHS) in the control of Dengue and Chikungunya fever in the cities of Parnamirim and Santa Cruz. It is exploratory and descriptive nature of research, with a qualitative approach, which was developed with nurses of the Family Health Strategy in the municipalities of Parnamirim and Santa Cruz. All research process followed the ethical standards laid out research in Resolution No. 466/2012 of the National Health Council about research involving human beings. Data collection was performed using a semi-structured questionnaire with open and closed questions, organized into three parts: the profile of respondents, knowledge of the disease (transmission, prevention, control) and description of practices. The actions performed by nurses are punctual performed when there is growing disease of the number of cases in order to try to reduce the number of cases. The principal methodology used for them is to talk and guidelines resulting from campaigns and home visits, using leaflets and social networks. Regarding knowledge of nurses in the city of Santa Cruz on Dengue Fever and Chikungunya, is clearly some gaps. Nursing professionals know recognize a suspected case of dengue, but get confused when trying to explain to the Chikungunya fever, exhibiting the same symptoms of dengue. Although everybody had participated in a training on Chikungunya Fever and Dengue, a very limited knowledge of nurses regarding the clinical management. It is suggested that further studies are developed on the ground a few nurses have accepted to participate in the study, so that we can identify strategies, interventions, activities and nursing actions that are consistent with the reality of working in favor of coping Aedes epidemics aegypti that have positive impact on reducing the infestation rates and may be suitable and applied in other regions.

Phylogenetic analyses of chikungunya virus among travelers in Rio de Janeiro, Brazil, 2014-2015

Chikungunya virus (CHIKV) is a mosquito-borne pathogen that emerged in Brazil by late 2014. In the country, two CHIKV foci characterized by the East/Central/South Africa and Asian genotypes, were established in North and Northeast regions. We characterized, by phylogenetic analyses of full and partial genomes, CHIKV from Rio de Janeiro state (2014-2015). These CHIKV strains belong to the Asian genotype, which is the determinant of the current Northern Brazilian focus, even though the genome sequence presents particular single nucleotide variations. This study provides the first genetic characterisation of CHIKV in Rio de Janeiro and highlights the potential impact of human mobility in the spread of an arthropod-borne virus.

2016 Chikungunya Virus Cases

After the bite of an infected mosquito, onset of illness occurs usually between 4 and 8 days, but can range from 2 to 12 days. Chikungunya is characterized by an abrupt onset of fever frequently accompanied by joint pain. Other common signs and symptoms include muscle pain, headache, nausea, fatigue and rash. The joint pain is often very debilitating, but usually lasts for a few days or may be prolonged to weeks. Hence the virus can cause acute, subacute, or chronic disease. Most patients recover fully, but in some cases joint pain may persist for several months, or even years. Occasional cases of eye, neurological, and heart complications have been reported, as well as gastrointestinal complaints. Serious complications are not common, but in older people, the disease can contribute to the cause of death. Often symptoms in infected individuals are mild and the infection may go unrecognized, or be misdiagnosed in areas where dengue occurs. Co-infections of dengue and chikungunya can occur.

Molecular evolutionary dynamics of Ross River virus and implications for vaccine efficacy

Ross River virus (RRV) is a mosquito-borne member of the genus Alphavirus that causes epidemic polyarthritis in humans, costing the Australian health system at least US$10 million annually. Recent progress in RRV vaccine development requires accurate assessment of RRV genetic diversity and evolution, particularly as they may affect the utility of future vaccination. In this study, we provide novel RRV genome sequences and investigate the evolutionary dynamics of RRV from time-structured E2 gene datasets. Our analysis indicates that, although RRV evolves at a similar rate to other alphaviruses (mean evolutionary rate of approx. 8x10(-4) nucleotide substitutions per site year(-1)), the relative genetic diversity of RRV has been continuously low through time, possibly as a result of purifying selection imposed by replication in a wide range of natural host and vector species. Together, these findings suggest that vaccination against RRV is unlikely to result in the rapid antigenic evolution that could compromise the future efficacy of current RRV vaccines.

Ross River virus evolution : implications for vaccine development

Ross River virus is a mosquito-borne alphavirus that causes approximately 5000 cases of epidemic polyarthritis in Australia each year and has direct medical-associated costs of approximately US$15 million annually. While mosquito control programs are able, at best, to contain rather than prevent this disease, natural infection with Ross River virus confers lifelong protection against subsequent clinical infection. A killed-virus vaccine has been developed, which is in Phase III clinical trials. Analyses of intra-host genetic diversity and of long-term evolutionary changes in Ross River virus populations suggest that antigenic variation is unlikely to pose a threat to the efficacy of this vaccine.

Outbreak detection algorithms for seasonal disease data : a case study using Ross River virus disease

Background Detection of outbreaks is an important part of disease surveillance. Although many algorithms have been designed for detecting outbreaks, few have been specifically assessed against diseases that have distinct seasonal incidence patterns, such as those caused by vector-borne pathogens. Methods We applied five previously reported outbreak detection algorithms to Ross River virus (RRV) disease data (1991-2007) for the four local government areas (LGAs) of Brisbane, Emerald, Redland and Townsville in Queensland, Australia. The methods used were the Early Aberration Reporting System (EARS) C1, C2 and C3 methods, negative binomial cusum (NBC), historical limits method (HLM), Poisson outbreak detection (POD) method and the purely temporal SaTScan analysis. Seasonally-adjusted variants of the NBC and SaTScan methods were developed. Some of the algorithms were applied using a range of parameter values, resulting in 17 variants of the five algorithms. Results The 9,188 RRV disease notifications that occurred in the four selected regions over the study period showed marked seasonality, which adversely affected the performance of some of the outbreak detection algorithms. Most of the methods examined were able to detect the same major events. The exception was the seasonally-adjusted NBC methods that detected an excess of short signals. The NBC, POD and temporal SaTScan algorithms were the only methods that consistently had high true positive rates and low false positive and false negative rates across the four study areas. The timeliness of outbreak signals generated by each method was also compared but there was no consistency across outbreaks and LGAs. Conclusions This study has highlighted several issues associated with applying outbreak detection algorithms to seasonal disease data. In lieu of a true gold standard, a quantitative comparison is difficult and caution should be taken when interpreting the true positives, false positives, sensitivity and specificity.

Characterization of epitopes for virus-neutralizing monoclonal antibodies to Ross River virus E2 using phage-displayed random peptide libraries

Ross River virus (RRV) is the predominant cause of epidemic polyarthritis in Australia, yet the antigenic determinants are not well defined. We aimed to characterize epitope(s) on RRV-E2 for a panel of monoclonal antibodies (MAbs) that recognize overlapping conformational epitopes on the E2 envelope protein of RRV and that neutralize virus infection of cells in vitro. Phage-displayed random peptide libraries were probed with the MAbs T1E7, NB3C4, and T10C9 using solution-phase and solid-phase biopanning methods. The peptides VSIFPPA and KTAISPT were selected 15 and 6 times, respectively, by all three of the MAbs using solution-phase biopanning. The peptide LRLPPAP was selected 8 times by NB3C4 using solid-phase biopanning; this peptide shares a trio of amino acids with the peptide VSIFPPA. Phage that expressed the peptides VSIFPPA and LRLPPAP were reactive with T1E7 and/or NB3C4, and phage that expressed the peptides VSIFPPA, LRLPPAP, and KTAISPT partially inhibited the reactivity of T1E7 with RRV. The selected peptides resemble regions of RRV-E2 adjacent to sites mutated in neutralization escape variants of RRV derived by culture in the presence of these MAbs (E2 210-219 and 238-245) and an additional region of E2 172-182. Together these sites represent a conformational epitope of E2 that is informative of cellular contact sites on RRV.

Sindbis virus and Pogosta disease in Finland

Sindbis virus (SINV) (genus Alphavirus, family Togaviridae) is an enveloped virus with a genome of single-stranded, positive-polarity RNA of 11.7 kilobases. SINV is widespread in Eurasia, Africa, and Australia, but clinical infection only occurs in a few geographically restricted areas, mainly in Northern Europe. In Europe, antibodies to SINV were detected from patients with fever, rash, and arthritis for the first time in the early 1980s in Finland. It became evident that the causative agent of this syndrome, named Pogosta disease, was closely related to SINV. The disease is also found in Sweden (Ockelbo disease) and in Russia (Karelian fever). Since 1974, for unknown reason, the disease has occurred as large outbreaks every seven years in Finland. This study is to a large degree based on the material collected during the 2002 Pogosta disease outbreak in Finland. We first developed SINV IgM and IgG enzyme immunoassays (EIA), based on highly purified SINV, to be used in serodiagnostics. The EIAs correlated well with the hemagglutination inhibition (HI) test, and all individuals showed neutralizing antibodies. The sensitivities of the IgM and IgG EIAs were 97.6% and 100%, and specificities 95.2% and 97.6%, respectively. E1 and E2 envelope glycoproteins of SINV were shown to be recognized by IgM and IgG in the immunoblot early in infection. We isolated SINV from five patients with acute Pogosta disease; one virus strain was recovered from whole blood, and four other strains from skin lesions. The etiology of Pogosta disease was confirmed by these first Finnish SINV strains, also representing the first human SINV isolates from Europe. Phylogenetic analysis indicated that the Finnish SINV strains clustered with the strains previously isolated from mosquitoes in Sweden and Russia, and seemed to have a common ancestor with South-African strains. Northern European SINV strains could be maintained locally in disease-endemic regions, but the phylogenetic analysis also suggests that redistribution of SINV tends to occur in a longitudinal direction, possibly with migratory birds. We searched for SINV antibodies in resident grouse (N=621), whose population crashes have previously coincided with human SINV outbreaks, and in migratory birds (N=836). SINV HI antibodies were found for the first time in birds during their spring migration to Northern Europe, from three individuals: red-backed shrike, robin, and song thrush. Of the grouse, 27.4% were seropositive in 2003, one year after a human outbreak, but only 1.4% of the grouse were seropositive in 2004. Thus, grouse might contribute to the human epidemiology of SINV. A total of 86 patients with verified SINV infection were recruited to the study in 2002. SINV RNA detection or virus isolation from blood and/or skin lesions was successful in eight patients. IgM antibodies became detectable within the first eight days of illness, and IgG within 11 days. The acute phase of Pogosta disease was characterized by arthritis, itching rash, fatigue, mild fever, headache, and muscle pain. Half of the patients reported in self-administered questionnaires joint symptoms to last > 12 months. Physical examination in 49 of these patients three years after infection revealed persistent joint manifestations. Arthritis (swelling and tenderness in physical examination) was diagnosed in 4.1% (2/49) of the patients. Tenderness in palpation or in movement of a joint was found in 14.3% of the patients in the rheumatologic examination, and additional 10.2% complained persisting arthralgia at the interview. Thus, 24.5% of the patients had joint manifestations attributable to the infection three years earlier. A positive IgM antibody response persisted in 3/49 of the patients; both two patients with arthritis were in this group. Persistent symptoms of SINV infection might have considerable public health implications in areas with high seroprevalence. The age-standardized seroprevalence of SINV (1999-2003, N=2529) in the human population in Finland was 5.2%. The seroprevalence was high in North Karelia, Kainuu, and Central Ostrobothnia. The incidence was highest in North Karelia. Seroprevalence in men (6.0%) was significantly higher than in women (4.1%), however, the average annualized incidence in the non-epidemic years was higher in women than in men, possibly indicating that infected men are more frequently asymptomatic. The seroprevalence increased with age, reaching 15.4% in persons aged 60-69 years. The incidence was highest in persons aged 50-59 years.