Biomedical subjects
Publications and source records attributed to J M Mezencio.
Amplification by RT-PCR of the RNA present in foot-and-mouth disease virus particles is inhibited by substances present in the sera of several species. This inhibition appears to be caused by a direct interaction of the substances with the RNA and not the enzymes used for its amplification.
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We had shown in preliminary studies with a small number of animals that antibodies against 2C could be detected in cattle and pigs which had been infected with FMDV but not in animals which had been vaccinated against the disease. To determine whether this test was generally applicable, sera from several hundred animals which had been vaccinated with different products in many countries have been tested in an ELISA using baculovirus expressed 2C. Our results show that only 1-2% of the sera gave a positive reaction by this method. In contrast, 100% of sera from convalescent animals gave a positive reaction. To be useful in differentiating between convalescent and vaccinated animals it is necessary to know how long these antibodies can be detected by our ELISA. We have determined the levels of antibodies against 2C and also other virus-specific proteins which are present in cattle and pigs following infection with FMDV. Our results show that levels of anti-3ABC antibodies could be detected by ELISA with baculovirus-expressed protein up to one year after infection. In contrast, the levels of anti-2C antibodies fell more rapidly than those against 3ABC indicating that the latter protein may be preferable for detecting convalescent animals. Nevertheless, we envisage that the final test format should include several virus-specific proteins to determine accurately the immune status of an animal.
The replication of Mayaro virus in BHK-21 cells was studied by electron microscopy. The infected cells show an intense vacuolization and proliferation of membranous structures. At 5 h post-infection, precursor virus particles were seen in the cytoplasm of infected cells. Later, mature virus particles were found outside the cells and budding from the plasma membrane. Enveloped virus particles were also observed inside the vesicles and budding across their membrane. The release of virus particles into the extracellular space by exocytosis was also observed. In a later stage of the infection, inclusion bodies were sometimes present in the cytoplasm of infected cells. We conclude that in BHK-21 cells, budding from the plasma membrane is the main process of Mayaro virus maturation, and in this kind of cell replication differs significantly from that observed in Aedes albopictus cells.
The replication of Mayaro virus in Aedes albopictus cells, was studied by electron microscopy at various times post-infection. In infected cells we observed the presence of cytoplasmic vesicles containing viral nucleocapsids and mature virus particles but at no time did we detect virus budding into such vacuoles. Budding of virus through plasma membrane was rarely observed. Our results are discussed considering the possibility of the release of virus particles to the extracellular space by exocytosis.
The multiplication of Mayaro virus in Aedes albopictus cells was drastically inhibited after incubation at 37 degrees C. The effect of short-term exposure of infected cells to high temperatures (heat shock) produced a preferential translation of the heat shock messengers when compared to the viral mRNAs. When cells were shifted back to 28 degrees C (the optimum growth temperature for Aedes albopictus cells), preferential translation of viral mRNA occurred. Although the infected cells were programmed for preferential translation of viral messengers, the thermal treatment was able to shift the translational machinery towards synthesis of heat shock proteins.
The sensitivity of several group C arboviruses (Bunyaviridae) to human amnion interferon was compared to that of vesicular stomatitis virus (VSV) and Sindbis virus. In CPE inhibition assays. Apeu virus was the most sensitive of the group C arboviruses tested; it was significantly more inhibited than VSV, but was in the same range as Sindbis virus. In plaque reduction assays, the increasing order of sensitivity was Apeu, Marituba, VSV and Sindbis viruses. Single-cycle yields of VSV and Apeu virus were reduced to the same extent with 1-10 interferon units; with 230 units, VSV growth was inhibited to a much greater extent (100-fold) than Apeu virus.
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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.