Archives of Virology

Archives of Virology 78, 309--314 (I983)

© by Springer-Verlag 1983

The Replication of Roeio Virus in Brain Tissue of Suckling Mice. Study by Electron Microscopy Brief Report By H. TANAKA 1, D. ][~. WEIGL 1, and O. DE SOUZA LOPES2 Seq~o de Microseopia EletrSnica do Instituto Adolfo Lutz, 2 Seato de Virus Transmitidos por Artrop6dos do Instituto Adolfo Lutz, Sao Paulo, Brasil With 4 Figures Accepted July 26, 1983 Sunmmry By electron microscopy studies, Rocio virus particles were about 43 nm and spherically shaped. They were found within the cisternae of the endoplasmic retieulum and Gotgi complex of infected neurons. No precursor particles were detected nor virus budding was evident. Beginning in April 1975, several outbreaks of h u m a n encephalitis occurred in coastal areas of the State of S~,o Paulo, Brasil. The etiologic agent was demonstrated to be a new flavivirus, subsequently named l~ocio virus (3). The biological characteristics of this virus have been described as well as its epidemiological (4), clinical (10) and human pathological characteristics (8). This report deals with ultrastructural aspects of Rocio virus replication in experimentally infected suckling mice. We have compared our observations with those describing other flavivirus which are the causative agents of h u m a n encephalitides. Rocio virus (strain S P H 34675) was received from the "Seato de Virus Transmitidos por Artrdpodos" of the Instituto Adotfo Lutz, S~o Paulo, Brasil, in its sixth suckling mouse brain passage. Two-day-old Swiss mice from a colony maintained b y the Instituto were inoculated intraeerebrally (i.c.) with virus suspension at 10 -1, diluted in 0.15 M phosphate buffered sahne with 0.75 per cent bovine albumin. Brain tissue was harvested at 10, 16, 25, 40, 48, 59 and 73 hours after inoculation. These tissues were processed and included by classic techniques of electron microscopy. Uttrathin sections were stained with t~eynoIds lead stain (7) and examined at 60 kV in a Philips EM-200 and EM-400 electron microscope. I n these sections, l~ocio virus particles were always found in the lumen of membra-

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nous cytoplasmic organelles, principally within Golgi membranes and endoplasmie retieulum, intraeytoplasmie vesicles and the nuclear envelope. These particles were found in rows within the organelles, however individually scattered particles were also common. In infected cells there was a hypertrophy of endoplasmic reticulum and Golgi complex. No particles were seen within the cytoplasmic matrix. The diameter of t~oeio virus particles was found to be around 43 nm in thin section studies. I n order to measure the virus particles, we compared it with band repeating intervals of collagen fibril that was known to be of 64 nm in negative staining.

Fig. 1. Cytoplasm of mouse brain cell infected with Roeio virus, 73 hours post inoculation. Virus particles are present within eisternae of the endoplasmie retieulum. M mitoehondrium 16,500 × ; N mteleous, Insert 90,000 × I~ocio virus particles are spherical (Fig. l); they have an electron dense core surrounded by a well delimited homogeneous transparent layer followed by a slight membrane. No virus partieles could be detected in brain tissues collected before 40 hours after inoculation. From 40 hours onward the number of virus particles in infected cells increased rapidly, reaching its maximum when animals were sick and moribund, at 73 hours. However, no Roeio virus particles were found in the cytoplasmic matrix, no precursor particles could be detected, and there was no evidence of virus budding. Nuclei and mitoehondria of infected cells appeared normal until very late in infection and there was not morphological evidence of nuclear participation ill the virus replication. Within virus infected

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Fig. 2. C y t o p l a s m infected, 73 hours post inoculation. L u c e n t vesicles (LV), tadpole shaped structures (TSS) and virus particles (V) are present within eisternae of t h e endoplasmie retieulum. 70,000 ×

Fig. 3. U n i n f e c t e d brain cell of suckling mice. M m i t o c h o n d r i u m t 7,500 × ; N nucleous 21 a Arch. Virol. 78[3--4

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cells there were abnormal structures (Fig. 2) which were associated with virus replication since they were never found in brain cells of uninoculated mice (Fig. 3). These were lucent vesicles, tadpole shaped structures, and inclusion bodies. The lucent vesicles had an almost spherical shape, were always associated with endoplasmic reticulum membranes and were first seen in those samples collected after 40 hours of infection (Fig. 2). Tadpole shaped structures were found within vacuoles, and never free in the cytoplasmic matrix (Fig. 2). Inclusion bodies consisting of electron dense masses were found in the cytoplasm of infected cells, and different from all other cytoplasmic organelles (Fig. 4).

Fig. 4. Inclusion body within the cytoplasm of a mouse brain cell, 73 hours post inoculation. 46,000 × Several morphologie and morphogenetic studies have been done on flaviviruses which are the causes of sporadic and epidemic human encephalitis. All of these flaviviruses have been found to be spherical in shape, with a size ranging from 30 to 60 nm, and have an electron dense inner core when stained ~dth uranium or lead citrate (1, 2, 5, 6). Similar morphologic details were found in Rocio virus (9). I t was difficult to establish the real parameters of the virus replication, and some changes associated with infection, such as hypertrophy of cytoplasmic organelles, increase of perinuclear space and the appearance of cytoplasmic vacuoles, could not be ordered into a precise chronology. We suppose t h a t the sequential events were obscured because the disease caused by Rocio virus in suckling mice developed very quickly, and the animals did not survive after 75 hours.

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The a p p e a r a n c e of precursor signs of celt infection a n d t h e i n v o l v e m e n t of Golgi c o m p l e x a n d e n d o p l a s m i e r e t i e u l u m in l~oeio virus infection were similax to t h a t o b s e r v e d b y F I L s m n (2), OYASAOI (6), M v ~ Y (5) a n d CALBERG-BACQ (1) in t h e i r studies on M u r r a y Valley, J a p a n e s e E n c e p h a l i t i s , S a i n t Louis E n c e p h a l i t i s , a n d others flaviviruses. C y t o p l a s m i c m a t r i x d i d n o t seem to be a p l a c e for virus m a t u r a t i o n or a c c u m u l a t i o n . M i t o e h o n d r i a were p r e s e r v e d l a t e in infection. A l t h o u g h no v i r u s particles were visualized inside t h e nuclei of infected cells, nuclear p a r t i c i p a t i o n in virus replieat:ion could n o t be e x c l u d e d b y the m e t h o d s used. Concerning t h e a n o m a l o u s lucent vesicles o b s e r v e d (Fig. 2), it seemed t h a t t h e y m u s t be associated w i t h virus replication. According t o Mv~PgY (5), t h e y are a l w a y s f o u n d a s s o e i a t e d with e n d o p l a s m i c r e t i e u l u m m e m b r a n e s a n d o t h e r c y t o p l a s m i c m e m b r a n o u s organelles. T a d p o l e s h a p e d structures, which functions are unknown, a p p e a r e d more f r e q u e n t l y in t h e final p h a s e of t h e virus infection (Fig. 2) a n d seemed to be identifical to those f o u n d in S a i n t Louis E n c e p h a l i t i s virus infection in sucMing mice (5). Inclusions bodies (Fig. 4) were also f o u n d in final stages of infection, suggesting t h a t t h e y could be f o r m e d b y a n a c c u m u l a t i o n of electron dense m a t e r i a l p r o d u c e d b y cellular d e g e n e r a t i o n a n d m e t a b o l i c changes. F r o m our observations, Rocio virus a p p e a r s to be a t y p i c a l f l a v i v i r u s ; it has morphologic a n d m o r p h o g e n e t i c characteristics in b r a i n s of suckling mice which are v e r y similar to o t h e r a g e n t s of this v i r u s genus.

Acknowled~lments The authors gratefully acknowledge the revision of Dr. Frederick A. Murphy and the technical assistance of Dr. Terezinha Lisieux M. Coimbra and Dulee Maria de SoL1za.

Grant This work was supported in part b y Conseiho Naeional de Desenvolvimento Cient.iffco e Tecndlogieo (CNPq).

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7. REYNOLDS, E. S. : The use of lead-citrate at high pH as an electron opaque stain in electron microscopy. J. Cell. Biol. 17, 208--212 (1963). 8. I~OSEIY[BEI~G, S. : Neuropathology of S~o Paulo, South Coast Epidemic Encephalitis (Rocio Flavivirus). J. Neurol. Sci. 45, i.--12 (1980). 9. TANAKA, H. : Observa95es preliminares sobre a replicag~o do virus Roeio (Togavfrus, Flavivlrus) em c@rebro de camundongos rec4m-nascidos. Rev. Inst. Med. Trop. S. Paulo 21, 228--230 (1979). 10. TI~IBA, A. C., MIZIARA, A. M., LO~EN¢O, I%. : Encefalite humana prim£ria, epid@mica, por arbovirus, observada no litoral sul do Estado de Sao Paulo. Rev. Ass. Med. Bras. 22, 415--420 (1976). Authors' address: Seg£o de Microseopia Eletr6nica, Instituto Adolfo Lutz, Caixa Postal 7027, 01000-S~o Paulo, Brasil. Received March

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