Development of an algorithm for production of inactivated arbovirus antigens in cell culture.

Arboviruses are medically important pathogens that cause human disease ranging from a mild fever to encephalitis. Laboratory diagnosis is essential to differentiate arbovirus infections from other pathogens with similar clinical manifestations. The Arboviral Diseases Branch (ADB) reference laboratory at the CDC Division of Vector-Borne Diseases (DVBD) produces reference antigens used in serological assays such as the virus-specific immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Antigen production in cell culture has largely replaced the use of suckling mice; however, the methods are not directly transferable. The development of a cell culture antigen production algorithm for nine arboviruses from the three main arbovirus families, Flaviviridae, Togaviridae, and Bunyaviridae, is described here. Virus cell culture growth and harvest conditions were optimized, inactivation methods were evaluated, and concentration procedures were compared for each virus. Antigen performance was evaluated by the MAC-ELISA at each step of the procedure. The antigen production algorithm is a framework for standardization of methodology and quality control; however, a single antigen production protocol was not applicable to all arboviruses and needed to be optimized for each virus.

Viral arthritides.

Viral infections may manifest as acute or chronic arthritis. Joint involvement arises from either direct infection of the joint, through an immunological response directed towards the virus or autoimmunity. Epidemiological clues to the diagnosis include geographic location and exposure to vector-borne, blood-borne or sexually transmitted viruses. Although not always possible, it is important to diagnose the pathogenic virus, usually by serology, nucleic acid tests or rarely, viral culture. In general, viral arthritides are self-limiting and treatment is targeted at symptomatic relief. This article focuses on the causes, clinical features, diagnosis and treatment of viral arthritides.

Retinoblastoma protein purification and transduction of retina and retinoblastoma cells using improved alphavirus vectors.

PURPOSE: To develop and use improved Semliki Forest vectors (SFVs) for functional and structural analyses of the retinoblastoma protein (RB) in developing retina and retinoblastoma cells. METHODS: Virus was harvested from cells transfected with replicon and helper plasmids. Combinations of producer and target cells were tested for optimal virus production and protein expression. The replicon was improved by adding an expanded multiple cloning site, translational enhancer, and FLAG and HIS10 epitope and affinity tags. Affinity chromatography was used to purify beta-galactosidase or RB. RB function was assessed through interaction with E2F1. The efficacy of SFV as a retinal delivery system was tested on mouse explants and cultured human retinoblastoma cells. RESULTS: The optimal producer and target cell lines were an HEK-293 derivative (Phoenix Eco) and BHK-21, respectively. Stable expression of structural proteins in the BHK-21 helper line simplified virus production and amplified virus yield 100-fold. The translational enhancer improved expression three- to eightfold. Full-length, functional RB was produced without the truncated products characteristic of bacterially produced RB and was purified using the affinity tags. SFVs efficiently transduced mouse retinal explants and multiple hard-to-transduce retinoblastoma tumor lines. CONCLUSIONS: This study describes a simple, rapid, SFV vector system to produce recombinant proteins, such as RB, in mammalian cells. These SFV vectors represent an efficient approach to purification of proteins and protein complexes and transduction of retinal or retinoblastoma cells for the purpose of in vivo analysis of protein functions.

Epidemic O'Nyong-Nyong fever in southcentral Uganda, 1996-1997: entomologic studies in Bbaale village, Rakai District.

Entomologic studies were conducted between January 27 and February 2, 1997, in Bbaale village in southcentral Uganda during an o'nyong-nyong (ONN) virus epidemic, which began in mid 1996 and continued into 1997. The objectives were to confirm the role of anophelines in ONN virus transmission and to examine other mosquito species as epidemic vectors of ONN virus. Of 10,050 mosquitoes collected using light traps and pyrethrum knockdown sprays, Anopheles (Cellia) funestus Giles was presumed to be the principal vector because it was the most abundant mosquito species from which a strain of ONN virus was isolated. This virus was isolated for the first time from a culicine species, Mansonia (Mansonioides) uniformis Theobald. Bwamba virus and Nyando virus were also isolated from An. funestus.

Alteraciones feto-placentarias inducidas en ratas por la cepa TC-83 del virus de la encefalitis equina venezolana / Fetal-placental alteration induced in rat by venezuelan equine encephalitis virus strain TC-83

El estudio macroscópico y microscópico de los fetos de ratas Sprague-Dawley inoculadas con el virus de la EEV, cepa Guajira y cepa atenuada TC-83 demostró, durante la primera semana de gestación, muerte y reabsorción de todos los fetos con cepa Guajira y una disminución considerable en el número de crías, cuando se inocularon con la cepa TC-83. El estudio histológico del sistema nervioso central de las crías nacidas vivas, no demostró lesiones. Ratas inoculadas al 4to y 7mo día de gestación con TC-83 y sacrificadas el día de la gestación mostraron lesiones placentarias, principalmente, en los vasos miometriales en los fetos de aspecto viable, así como fuerte necrosis de otros fetos. Se compara la patogenia de la infección con el virus de la EEV a las lesiones inducidas en humanos por el virus de la rubéola y con estudios previos sobre el efecto intrauterino de otros Togavirus, y se destaca la necesidad de examinar cuidadosamente a las mujeres embarazadas y a sus hijos en las áreas de riesgo epidémico así como proscribir el uso de la vacuna TC-83 durante la gestación

Efecto teratogénico del virus de la encefalitis equina venezolana: una revisión del problema / The teratogenic effect of venezuelan equine encephalitis virus: a review of the problem

Se describen las características clínicas de la encefalitis equina venezolana (EEV) y la capacidad teratogénica de diversos Togavirus. Se destaca la similitud entre las alteraciones intrauterinas inducidas por el virus de la EEV y el de la rubeola. Se señalan las observaciones descritas en 1967 por Wenger sobre necrosis cerebral masiva en fetos de mujeres que presumiblemente habían padecido de encefalitis y se describe el desarrollo de un modelo experimental en ratas a finales de la década de 1970-1980. La patogenia de la infección intrauterina parece relacionarse con alteraciones vasculares placentarias y del sistema nervioso central en ratas sobrevivientes a la infección viral; se destaca la similitud entre estos hallazgos y lesiones vasculares cerebrales descritas en niños con síndrome de rubeola congénita. Se hace énfasis en la necesidad de estudios multidisciplinarios en las áreas endémicas de EEV como una vía para detectar secuelas del efecto del virus in utero. Finalmente se proponen algunos modelos experimentales en animales para esclarecer diversos aspectos sobre el efecto intrauterino provocado por el virus de la EEV

Resistance to influenza virus and vesicular stomatitis virus conferred by expression of human MxA protein.

MxA and MxB are interferon-induced proteins of human cells and are related to the murine protein Mx1, which confers selective resistance to influenza virus. In contrast to the nuclear murine protein Mx1, MxA and MxB are located in the cytoplasm, and their role in the interferon-induced antiviral state was unknown. In this report we show that transfected cell lines expressing MxA acquired a high degree of resistance to influenza A virus. Surprisingly, MxA also conferred resistance to vesicular stomatitis virus. Expression of MxA in transfected 3T3 cells had no effect on the multiplication of two picornaviruses, a togavirus, or herpes simplex virus type 1. Treatment of MxA-expressing cells with antibodies to mouse alpha-beta interferon did not abolish the resistance phenotype. The conclusion that resistance to influenza virus and vesicular stomatitis virus was due to the specific action of MxA is further supported by the observation that transfected 3T3 cell lines expressing the related MxB failed to acquire virus resistance.

Uso de células de Aedes albopictus C6/36 na propagaçäo e classificaçäo de arbovirus das famílias Togaviridae, Flaviviridae, Bunyaviridae e Rhabdoviridae / The use of Aedes albopictus C6/36 cells in the propagation and classification of arbovirus of the Togaviridae, Flaviviridae, Bunyaviridae and Rhabdoviridae families

Colônias de células de mosquito Aedes albopicus C6/36 foram infectadas com 23 arbovirus, sendo 19 destes existentes no Brasil, pertencentes às famílias Togavitidae, Flaviviridae, Bunyaviridae e Rhabdoviridae. A Replicaçäo viral foi detectada por imunofluorescência indireta com todos os vírus estudados enquanto que o efeito citopático foi observado durante a infecçäo por alguns deste. No teste de imunofluorescência indireta utilizou-se fluidos ascíticos imunes de camundongos, especificos para os vírus estudados. A replicaçäo viral caracterizada por grande produçäo de antígeno recomenda a utilizaçäo de células C6/36 na propagaçäo e em tentativas de isolamento desses arbovírus. A técnica de imunofluorescência ofereceu subsídios na classificaçäo e identificaçäo de vírus que replicam nestas células

[The use of Aedes albopictus C6/36 cells in the propagation and classification of arbovirus of the Togaviridae, Flaviviridae, Bunyaviridae and Rhabdoviridae families]. / Uso de células de Aedes albopictus C6/36 na propagação e classificação de arbovírus das famílias Togaviridae, Flaviviridae, Bunyaviridae e Rhabdoviridae.

C6/36 Aedes albopictus cells were infected with Brazilian arbovirus from the families Togaviridae, Flaviviridae, Bunyaviridae and Rhabdoviridae. Replication was obtained with all the studied viruses and cytopathic effect was observed with some. Viral antigen was assayed in C6/36 cell cultures for antigen was assayed in C6/36 cells by an indirect immunofluorescence test using specific mouse immune ascitic fluid. Antigen production was detected in C6/36 cells infected with all the studied viruses. The author recommends the inoculation of C6/36 cell cultures for isolation of virus from the four studied families. The immunofluorescence technique is an important tool for classification and identification of virus growing in C6/36 cells.

Incomplete replication of human parainfluenza virus type 2 in mouse L929 cells.

Human parainfluenza virus type 2 (HPIV-2) was tested for its ability to replicate in murine L929 cells. L929 cells were non-permissive for replication of HPIV-2. Interferon produced endogenously played no role in its incomplete replication. The mechanism by which growth of HPIV-2 was suppressed in L929 cells was studied. Synthesis of virus-specific polypeptides, particularly glycoprotein(s), was suppressed in HPIV-2-infected L929 cells. The HN mRNA could scarcely be detected in virus-infected L929 cells.

The effect of gold sodium thiomalate in adult Swiss/A2G mice infected with togaviruses and flaviviruses.

Treatment of adult mice with gold sodium thiomalate made the normally non-lethal Semliki Forest virus and Sindbis virus infections lethal and increased the virulence of Langat and West Nile viruses. These changes were associated with an enhanced virus invasion of the brain. Depression of the humoral antibody response was not observed.

[Chiroptera viruses transmitted or not transmitted by arthropods]. / Virus des chiroptères transmis ou non par arthropodes.

The authors make a review of literature on viruses isolated from chiroptera's tissues. They study especially two of these viruses: a true arbovirus, the Japanese B encephalitis virus and a virus of the same genus but not arthropod-borne, the Rio Bravo virus. They consider successively for each virus: isolations from bats, serosurveys in bat and in man, and experimental studies with these viruses, allowing to estimate the role of chiroptera in the diffusion and the maintenance of some arboviruses. They also report some personal results of serosurveys in bats from Tunisia and Spain.

Elimination of persistent simian hemorrhagic fever (SHF) virus infection in patas monkeys.

Devastating epizootics of simian hemorrhagic fever (SHF) have been iatrogenically initiated in captive colonies of macaque monkeys by strains of SHF virus emanating from asymptomatic persistently infected patas monkeys. We have found that persistently infected patas monkeys can be cleared of their infection by superinfection with strains of SHF virus which cause acute infections in this species. All 20 persistently infected animals subjected to this procedure have been cleared of their infection within 3 months. These animals were shown to be virus free by the most sensitive in vitro and in vivo tests currently available and periodic tests of serum from these animals over several years have shown them to remain virus free. Superinfection has in some cases caused some adverse clinical symptoms (anorexia, lethargy, facial edema, dehydration, and mild subcutaneous hemorrhages), but with supportive care, no fatal infections have occurred. Thus, superinfection with acute strains of SHF virus is a highly effective method of eliminating persistent infection in patas monkeys.

Differences among isolates of simian hemorrhagic fever (SHF) virus.

Simian hemorrhagic fever (SHF) virus is a member of the Togaviridae family which currently is unclassified to genus. We have studied the relatedness of four different SHF virus isolates obtained from infected macaque or patas monkeys. Differences were found among isolates in type and severity of disease produced in patas monkeys, cell sensitivity to infection, viral antigens, and levels of specific antibody induced in patas monkeys. Based on these criteria, the four isolates have been grouped in two categories: those producing acute infections in patas monkeys (LVR, P-180) and those producing persistent infections (P-248, P-741). The P-180 isolate induced the most severe disease in experimentally infected patas monkeys, but only occasionally were their infections fatal. Persistently infected patas monkeys were viremic over a period of years, but showed no signs or symptoms of infection. All four isolates were found to be antigenically related by use of enzyme-linked immunosorbent assay (ELISA); the P-248 isolate showing the weakest antigenic relationship. However, none of the four isolates induced cross-neutralizing antibodies in infected patas monkeys. High titers of specific IgG antibody (up to 31,250 as determined by ELISA) were induced in acutely infected patas monkeys (LVR, P-180), but antibody was barely detectable (less than or equal to 50) in persistently infected patas monkeys (P-248, P-741). LVR lytically infected USU-104 cells, patas monkey peritoneal macrophages (PMAC), and rhesus monkey PMAC. The P-180 isolate lytically infected both patas monkey PMAC and rhesus monkey PMAC, but not USU-104 cells. The isolates producing persistent infections (P-248, P-741) lytically infected only rhesus monkey PMAC. These results show that marked differences exist among isolates of SHF virus from naturally infected animals. These differences should be useful in categorizing new isolates.

Electron microscopic observation of a newly isolated flavivirus-like virus from field-caught mosquitoes.

Of many unidentified virus strains which were isolated from field-caught mosquitoes by using C6/36 cells (a virus-sensitive clone of Aedes albopictus cells), three strains which formed small size plaques (SP virus) in C6/36 cells were investigated by electron microscopy. Although the SP virus strains did not react with antisera against known arboviruses in serological tests, they closely resembled flaviviruses in morphology. However, when they were compared to Japanese encephalitis (JE) virus, several differences in morphogenesis were observed. Proliferating membranous structures and electron-dense amorphous areas involving precursors of the virus were observed only in cells infected with the SP virus strains. Enlarged areas of endoplasmic reticulum containing mature virions were often observed adjacent to these structures. Since the SP virus strains were isolated from wild mosquitoes and multiplied only in mosquito cells, it seems appropriate to classify them as insect viruses which resemble togaviruses morphologically.

[Propagation of Pixuna virus in rat embryo cultures]. / Propagación del virus Pixuna en cultivos de embrión de ratón.

The behaviour of cell cultures from 11 to 18 days-old Swiss albino mouse embryo infected with Pixuna virus was studied. Eleven day-old embryo cultures showed to be a permissive system supporting a productive and cytocidal infection. In contrast, the 13 to 18 day-old embryo cultures were also permissive and productive but no evidence of cytopathic effect was observed. D-glucosamine did not affect the infectivity titer of the Pixuna virus when 11 day-old embryo cultures were used. In these cultures the eclipse phase was observed between 15 and 90 min post-absorption (Figure 1).

The effects of interferon and double-stranded RNA upon the virus-host interaction: studies with togavirus strains in mice.

Partially purified fibroblast interferon and double-stranded RNA (dsRNA) of fungal origin were administered as single graded doses to A2G and Balb/c mice shortly before intraperitoneal infection by specified virulent or avirulent strains of representative togaviruses (Semliki Forest virus, Venezuelan equine encephalomyelitis virus and yellow fever virus). Changes of efficiency of infection arising from interference at the level of clearance or replication and changes of the expression of virulence or protective immunity, were compared for different initial doses of interferon or dsRNA in relation to different levels of infection by defined virus strains. Interferon and dsRNA, although acting through quantitatively different mechanisms, both reduced effective dose of virus and influenced only the extent of primary replication and host stimulation. Neither agent changed in any way the outcome of infection in terms of expression of virulence (regulatory immunity) or protective immunity. These results are discussed in terms of the control of virus infections at stages before or after immune stimulation can be effective.

Amino acid requirements for the growth of Aedes albopictus clone C6/36 cells and for the production of dengue and Chikungunya viruses in the infected cells.

Amino acid requirements for the growth of Aedes albopictus, clone C6/36, cells and for the production of dengue (DEN) and Chikungunya (CHIK) viruses were examined by growing the cells or the viruses in media which were deprived of one of the 20 amino acids. Cell growth was markedly inhibited when cystine was omitted from the medium, and to a lesser extent by arginine deprivation. On the other hand, omission of alanine, asparagine, aspartic acid, and glutamic acid at the same time did not affect cell growth. Marked accumulation of alanine was observed in the medium when the cells were grown for 8 days in complete medium, with concomitant depletion of aspartic acid and glutamic acid. The production of CHIK virus was inhibited markedly by omission of cystine from the medium after virus infection, while the production of DEN viruses was more affected by glycine deprivation, although cystine deprivation also inhibited virus production to a lesser extent. On the other hand, production of CHIK and DEN viruses was not affected when alanine, asparagine, aspartic acid, and glutamic acid were omitted from the medium at the same time.