[Virology of the family Togaviridae].

Many pathogens important for medicine, veterinary medicine or public health belong to the genera alphavirus and rubivirus within the family Togaviridae. 29 species of alphaviruses have been reported, and most of them are arboviruses. Chikungnya virus re-emerged in Kenya in 2004 and the epidemics spread to the Indian Ocean islands and many countries in South Asia, South-East Asia and Europe. On the other hand, rubella virus, a sole member of the genus rubivirus, is the causative agent of rubella and congenital rubella syndrome (CRS). Because human is only a natural host of the virus and effective live attenuated vaccines are available, immunization activities are strengthened globally to eliminate rubella and CRS, together with measles.

Encephalitic Arboviruses of Africa: Emergence, Clinical Presentation and Neuropathogenesis.

Many mosquito-borne viruses (arboviruses) are endemic in Africa, contributing to systemic and neurological infections in various geographical locations on the continent. While most arboviral infections do not lead to neuroinvasive diseases of the central nervous system, neurologic diseases caused by arboviruses include flaccid paralysis, meningitis, encephalitis, myelitis, encephalomyelitis, neuritis, and post-infectious autoimmune or memory disorders. Here we review endemic members of the Flaviviridae and Togaviridae families that cause neurologic infections, their neuropathogenesis and host neuroimmunological responses in Africa. We also discuss the potential for neuroimmune responses to aide in the development of new diagnostics and therapeutics, and current knowledge gaps to be addressed by arbovirus research.

X-ray inactivation of RNA viruses without loss of biological characteristics.

In the event of an unpredictable viral outbreak requiring high/maximum biosafety containment facilities (i.e. BSL3 and BSL4), X-ray irradiation has the potential to relieve pressures on conventional diagnostic bottlenecks and expediate work at lower containment. Guided by Monte Carlo modelling and in vitro 1-log10 decimal-reduction value (D-value) predictions, the X-ray photon energies required for the effective inactivation of zoonotic viruses belonging to the medically important families of Flaviviridae, Nairoviridae, Phenuiviridae and Togaviridae are demonstrated. Specifically, it is shown that an optimized irradiation approach is attractive for use in a multitude of downstream detection and functional assays, as it preserves key biochemical and immunological properties. This study provides evidence that X-ray irradiation can support emergency preparedness, outbreak response and front-line diagnostics in a safe, reproducible and scalable manner pertinent to operations that are otherwise restricted to higher containment BSL3 or BSL4 laboratories.

Wolbachia significantly impacts the vector competence of Aedes aegypti for Mayaro virus.

Wolbachia, an intracellular endosymbiont present in up to 70% of all insect species, has been suggested as a sustainable strategy for the control of arboviruses such as Dengue, Zika and Chikungunya. As Mayaro virus outbreaks have also been reported in Latin American countries, the objective of this study was to evaluate the vector competence of Brazilian field-collected Ae. aegypti and the impact of Wolbachia (wMel strain) upon this virus. Our in vitro studies with Aag2 cells showed that Mayaro virus can rapidly multiply, whereas in wMel-infected Aag2 cells, viral growth was significantly impaired. In addition, C6/36 cells seem to have alterations when infected by Mayaro virus. In vivo experiments showed that field-collected Ae. aegypti mosquitoes are highly permissive to Mayaro virus infection, and high viral prevalence was observed in the saliva. On the other hand, Wolbachia-harboring mosquitoes showed significantly impaired capability to transmit Mayaro virus. Our results suggest that the use of Wolbachia-harboring mosquitoes may represent an effective mechanism for the reduction of Mayaro virus transmission throughout Latin America.

Diverse Viruses Require the Calcium Transporter SPCA1 for Maturation and Spread.

Respiratory and arthropod-borne viral infections are a global threat due to the lack of effective antivirals and vaccines. A potential strategy is to target host proteins required for viruses but non-essential for the host. To identify such proteins, we performed a genome-wide knockout screen in human haploid cells and identified the calcium pump SPCA1. SPCA1 is required by viruses from the Paramyxoviridae, Flaviviridae, and Togaviridae families, including measles, dengue, West Nile, Zika, and chikungunya viruses. Calcium transport activity is required for SPCA1 to promote virus spread. SPCA1 regulates proteases within the trans-Golgi network that require calcium for their activity and are critical for virus glycoprotein maturation. Consistent with these findings, viral glycoproteins fail to mature in SPCA1-deficient cells preventing viral spread, which is evident even in cells with partial loss of SPCA1. Thus, SPCA1 is an attractive antiviral host target for a broad spectrum of established and emerging viral infections.

Insect-Specific Viruses: A Historical Overview and Recent Developments.

Arthropod-borne viruses (arboviruses) have in recent years become a tremendous global health concern resulting in substantial human morbidity and mortality. With the widespread utilization of molecular technologies such as next-generation sequencing and the advancement of bioinformatics tools, a new age of viral discovery has commenced. Many of the novel agents being discovered in recent years have been isolated from mosquitoes and exhibit a highly restricted host range. Strikingly, these insect-specific viruses have been found to be members of viral families traditionally associated with human arboviral pathogens, including but not limited to the families Flaviviridae, Togaviridae, Reoviridae, and Bunyaviridae. These agents therefore present novel opportunities in the fields of viral evolution and viral/vector interaction and have tremendous potential as agents for biocontrol of vectors and or viruses of medical importance.

Interferon and antibody titrations using haemagglutinating Togaviridae and trypsinized human erythrocytes.

Several Togaviridae of the alphavirus and flavivirus genera agglutinate trypsinized human group O erythrocytes (THOE) (Shortridge and Hu, 1976). Haemagglutinin titers of Semliki Forest virus (SFV) and Japanese encephalitis virus (JEV) measured with THOE were equivalent to, if not higher than, those obtained with Embden gander erythrocytes, even with unextracted haemagglutinin. Results obtained with THOE in JEV haemagglutination-inhibition tests on sera taken from a previously infected individual over a 20-yr period were similar to those measured during the initial JEV infection. The inhibition of SFV haemagglutinin production as measured with THOE was a very sensitive bioassay for chicken interferon: interferon titers were 6- to 10-fold higher than those obtained with the vesicular stomatitis virus plaque-reduction method. The generally greater availability of human erythrocytes (including those stabilized with glutaraldehyde), the simplicity of the trypsin treatment, and the possibility of using unextracted haemagglutinin recommend this technique for use with haemagglutinating Togaviridae.

The use of scanning electron microscopy in the study of haemagglutination induced by various arboviruses.

Scanning electron microscopy was used to study haemagglutination by 10 arboviruses belonging to the Togaviridae and Bunyaviridae families. The pictures obtained did not allow to clarify the precise mechanism of haemagglutination by these viruses. The data suggest that no fundamental differences exist in the mechanism of haemagglutination between these two arbovirus families.

Inhibition of Mayaro virus replication by cerulenin in Aedes albopictus cells.

The antibiotic cerulenin, an inhibitor of lipid synthesis, was shown to suppress Mayaro virus replication in Aedes albopictus cells at non-cytotoxic doses. Cerulenin blocked the incorporation of [3H]glycerol into lipids when present at any time post infection (p.i.). Cerulenin added at the beginning of infection inhibited the synthesis of virus proteins. However, when this antibiotic was added at later stages of infection, it had only a mild effect on the virus protein synthesis. The possibility that cerulenin acts by blocking an initial step in the Mayaro virus replication after virus entry and before late viral translation is discussed.

[Fractions from mouse brain which non-specifically inhibit the hemagglutination of various togaviruses]. / Fracciones de cerebro de ratón que inhiben inespecíficamente la hemaglutinación de algunos togavirus.

Brain fractions isolated from Swiss albino mice were studied from the physical, chemical and biological viewpoints. The isoionic point gave a value of 4.16 +/- 0.17 indicating the presence of acid groups. UV absorption experiments showed an opening of the structure when the fractions were heated. The presence of neuraminic acid group was demonstrated using o-phenanthroline and, when heated, the structure exposed the hidden neuraminic acid. The biological property of inhibiting viral hemagglutination increased when the fractions were heated. EEE virus was used as hemagglutinating antigen. The treatment with alpha-chymotrypsin destroyed the inhibiting viral hemagglutination property in 120 seconds. Polyacrylamide gel electrophoresis showed that the fractions are proteic in nature. Glyco- and lipoproteins were only present in the fraction with the inhibitory property. Only neutral lipids were demonstrated.

[Mechanisms of the stimulating action of the Rauscher leukemia virus on togavirus reproduction in the bodies of BALB/c strain mice]. / Izuchenie mekhanizmov stimuliruiushchego deistviia virusa leikoza Raushera na reproduktsiiu togavirusov v organizme myshei linii BALB/c.

Reciprocal stimulation or inhibition are observed in double infection of mice with Rauscher leukemia virus (RLV) and togaviruses Sindbis or West Nile (WN). Stimulation of togavirus infections is manifested by the enhancement of the visceral phase of pathogenesis without subsequent activation of togarivus reproduction in the CNS. This effect is accompanied by enhanced togavirus replication in splenocytes, a decrease in the number of antibody-producing cells in the spleen, a decrease of antihemagglutinins titer in the blood without any significant change in the virus-neutralizing antibody and interferon titers. RLV-induced immunosuppression is termporary and of varying intensity with regard to individual parameters of immune response and to different variants of WN virus (highly virulent, poorly virulent). It is assumed that the differentiated and temporary nature of the immunosupressive effect of RLV is conducive to selective stimulation of the visceral phase of togavirus reproduction followed by inhibition of the neural phase under the influence of restored immune mechanisms and interferon. Because of the defects of humoral and cellular immunity, however, no complete elimination of togavirus occurs and conditions for its long-term persistence are created.

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.

Mayaro virus proteins.

Mayaro virus was grown in BHK-21 cells and purified by centrifugation in a potassium-tartrate gradient (5-50%). The electron microscopy analyses of the purified virus showed an homogeneous population of enveloped particles with 69 +/- 2.3 nm in diameter. Three structural virus proteins were identified and designated p1, p2 and p3. Their average molecular weight were p1, 54 KDa; p2, 50 KDa and p3, 34 KDa. In Mayaro virus infected Aedes albopictus cells and in BHK-21 infected cells we detected six viral proteins, in which three of them are the structural virus proteins and the other three were products from processing of precursors of viral proteins, whose molecular weights are 62 KDa, 64 KDa and 110 KDa. The 34 KDa protein was the first viral protein synthesized at 5 hours post-infection in both cell lines studied.

Virus inoculation in mice bearing Ehrlich ascitic tumors: antigen production and tumor regression.

Ehrlich ascitic carcinoma, as developed in albino mice, has been used as a source of hemagglutinating and complement-fixing antigens, and it proved to be suitable for one type of antigen, or both, for at least 12 viruses of 16 tested. Hemagglutinins were obtained with members of arbovirus groups A, B, and C; complement-fixing antigens were obtained for at least one member of each antigenic group tested. Ehrlich ascitic tumor was compared with sarcoma 180 as a source of antigens; although sarcoma 180 showed many advantages over Ehrlich tumor, the latter gave, in general, better results for complement-fixing antigens. Oncolytic effect with complete recovery of the mice was observed in some instances. The highest recovery rate resulted with Congo and UNA viruses (40%), and the second highest rate resulted with dengue 2, St. Louis, Hazara, and Uukuniemi viruses (20%). The best survival was observed, in decreasing order, with Congo, St. Louis, dengue 2, Tacaribe, Sindbis, Junin, and Amapari viruses.

Simian haemorrhagic fever (SHF): new virus isolate from a chronically infected patas monkey.

A new strain of simian haemorrhagic fever (SHF) virus was isolated from chronically infected patas monkey no. 248 (P-248) in USU-104 cells. The P-248 isolate had the same size, morphology and cytoplasmic site of replication as the prototype LVR strain. However, the P-248 isolate caused a persistent infection without noticeable cytopathology in USU-104 cells rather than the strongly lytic infection produced by prototype LVR virus. The capacity of P-248 virus to produce a persistent, non-lytic infection of USU-104 cells was a very stable characteristic of the isolate. Extensive serial passage of this isolate through USU-104 cells (over 50 passages) and rhesus monkeys (six passages) failed to unmask virus with lytic properties for USU-104 cells. Culture medium from persistently infected cultures assayed in rhesus monkey peritoneal mononuclear phagocytes, where measurable cytopathology occurs, was found to contain about 10(5) to 10(6) TCID50/ml of cell-free P-248 virus. Immunolabelling techniques showed only a low percentage of infected cells in persistently infected cultures. The mechanism of persistence of the P-248 isolate in USU-104 cells has not been determined but evidence suggests it does not involve interferon or defective interfering particles.

Replication of non-respiratory viruses in tracheal organ cultures.

Employing Hoorn's technique, tracheal explant cultures were set from adult hamsters, rabbits and human foetuses. To determine the susceptibility of these cultures they were infected with nine different mainly non-respiratory viruses. Assay of virus was carried out in susceptible cell lines. The results of these studies indicated that herpes simplex type I (HSV-1) and vaccinia viruses multiplied in these cultures and caused ciliostasis. Herpes simplex type 2 (HSV-2) although multiplying in all, produced ciliostasis only in human foetal tracheal cultures. However, Chandipura (CHP), West Nile (WN), sandfly fever (SF-N) and polio-1 viruses multiplied without ciliostasis. These cultures did not support multiplication of Japanese encephalitis (JE) and Chikungunya (CHIK) viruses. The studies indicated that mammalian tracheal cultures support replication of the non-respiratory viruses. The continued and undiminished multiplication of viruses over long periods without ciliostasis suggests a role for the trachea in the transmission of viral infections by aerosol.