Multiplication in and transmission by Aedes aegypti of vesicular stomatitis virus

JOURNAL

OF INVERTEBRATE

PATHOLOGY

Multiplication

G. H. Dcpartamento

l&406-428

(1968)

in and Transmission of Vesicular Stomatitis 0. M.

BERGOLD,

de Virohgia, Institute Apartado 1827, Received

by Aedes aegypti Virus 1

SU~~REZ,

Venezoluno Caracas,

November

AND

K.

MUNZ

de lnvestigaciones Venezuela 23,

Cientificas,

1967

The multiplication of vesicular stomatitis virus (VSV) in Aedes aegypti mosquitoes is about 4.5 logs 7 days after intrathoracic injection of 25 PFU of the “Indiana” strain and about 3.5 logs after injection of 2.5 PFU of the “New Jersey” strain. Higher doses cause no increase of multiplication. The multiplication in eight different organs follows a similar pattern. The concentration is highest 2-3 days after injection and shows a lower peak after 6-8 days before it drops. The specific virus concentration per microgram of wet organ is highest in the thoracic and abdominal ganglion, and after 20 days in the salivary glands. “Indiana” but not “New Jersey” could easily be expelled from the lumen of the intestine by slight pressure resulting in titers up to 3 logs PFU/mosquito. Therefore expelling of virus may take place during oviposition. Electron microscopic investigations of the insect’s tissues reveal VSV particles in the salivary glands, intestine, thoracic and abdominal ganglion, and midgut, but not in the cerebrum and both diverticuli in spite of high virus titers present. The particles occur nearly always in groups and within cisterns and tubuli of the endoplasmic reticulum. They are located in all three acini towards the periphery of the salivary glands or close to the internal duct. In the intestine they are found close to the wall neighboring the coelom. Transmission to baby mice by mosquitoes after intrathoracic injection takes place very regularly commencing from the 2nd4th day until at least 20 days. Transmission efficiency is independent of the titers in the mosquitoes within a range of 2.18-5.48 logs for “Indiana” and within 1.16-2.89 logs for “New Jersey.” Indiana virus was transmitted by 45% of the mosquito lots ( 5 mosquitoes/lot) to about 11% of baby mice. New Jersey virus was transmitted by 20% of the mosquito lots to only 5.2% of baby mice. After 15 days incubation Indiana virus was found in 31% of the 45% transmitting mosquito lots with titers between 1.8 and 3.7 logs PFU/5 mosquitoes; New Jersey virus was detected in 8.3% of the 20% transmitting mosquito lots with titers of 0.5 and 0.7 logs PFU/5 mosquitoes.

Mussgay and Suarez (1962) found that vesicular stomatitis virus (VSV), “Indiana” strain, multiplies in and is transmitted by the yellow-fever mosquito, Aedes aegypti, after intrathoracic injection. Ingestion of virus in aqueous sugar solution also induces multiplication of VSV, and mosquitoes so infected can transmit the virus to baby mice. 1 This investigation Health Service Grant

was supported A105247-01.

by

It was of interest to look for the site of virus multiplication by titration and electron microscopic investigation, to titer the virus amount, and to check survival of the virus in A. uegypti. Furthermore, the multiplication and transmission of VSV after feeding on infected baby mice was investigated. MATERIALS

The VSV “Indiana” strains

Public

were

and kindly 406

AND

METHODS

Jersey” in 1958, supplied by Dr. C. Palacios of isolated

and “New

in

Venezuela

VESICIJLAR

STOMATITIS

VIRUS

the Ministerio

de Agricultura y Cria. The Indiana strain was passaged about 20 times and the New Jersey strain about 15 times in baby mice. Brain suspensions (10%) in bovalbumin (0.75%) borate buffer, pH 8, was used in all experiments. The Aedes aegypti strain dates back to a capture of mosquitoes in Los Teques, Estado Miranda, Venezuela, in 1960, and has been reared for about 70 generations in the laboratory since then. The intrathoracic injections of 0.25 ~1 were performed with a microsyringe (Bergold, 1941) with the glass capillary drawn out to about 50 rnp at the tip. Mosquitoes 3 days old, without having fed, were used in all experiments. They were held by suction onto a fine metallic grid soldered over the flattened tip of a No. 13 injection needle. A cylinder 6 mm in diam pushed over the tip enlarged the supporting area. After injection the mosquitoes were fed with 10% sugar water. Dissection of the mosquitoes was carried out in a drop of sterile distilled water under a dissecting binocular microscope. Insects or their

Injected log,” PFU

1

OF VSV INDIANA AND NEW 7 DAYS AFTER INTRATHORACIC Virus 1 hr

407

MOSQUITOES

organs were triturated in a 1 or 5 ml TenBroeck glass tissue grinder with 0.5 ml of bovalbumin (0.75%) borate buffer, pH 8. Such suspensions were centrifuged for 15 min at 3000 r-pm and the supernatants stored at -70°C until titration. Often whole insects were stored at -70°C until trituration. Virus titration was carried out in baby hamster kidney cells (BHK 21) (Macpherson and Stoker, 1962) using 30-ml disposable Falcon plastic flasks. The 3-day-old cell sheets were washed with PBS (phosphate-buffered saline) and 0.5 ml of the test suspensions added. After an incubation period of 2 hr at 37°C the suspension was sucked off and the cell sheet overlaid with 4 ml of any overlay or a special overlay (Bergold and Mazzali, 1968). Plaques [PFU (plaque-forming units ) /ml] were counted 20 to 24 hr later with or without staining with neutral red. All operations with live VSV were carried out in reduced light to minimize light inactivation. A Mettler S6 Microbalance was used for weighing the mosquito organs.

TABLE MULTIPLICATION

IN

concentration

JERSEY STnIuN\'s INJECTION log, ,, PFU/mosquito

after infection Expts.

7 days

1

2

3

4.4 3.4 2.4 1.4

0 0 0 0

3.48 2.25 0 0

1.82 0.6 0 0

3.4 2.4 1.4 0.4

0 0 0 0

2.23 1.53 0 0

Indiana

IN Aedes

aegypti

n after Expts.

infection

1

2

3

4.45 4.45 4.5 4.62

4.65 4.81 4.76 4.84

3.71 3.59 3.69 3.49

3.95 4.15 3.65 3.6

2.3 1.3 0 1.3

strain

New Jersey strain

a Based

on

average

of five

mosquitoes.

2.06 0.3 0 0

3.62 3.25 4.04 3.25

2.98

3.87

3.60 4.18

0

0 0

2.14 3.46

1

0

0

0

lhrb

4.45 4.61

4.72

5.02

3.37 3.56

3

levels in italics.

3.89 4.54

4.70

4.52

2.92 3.70

2

IN

4.08 4.08

4.85

4.99

2.96 3.08

4

3.65 4.38

4.75

5.06

2.76 2.58

5

Virus concentration

OF VSV (INDIANA STRAIN) 1-18 DAYS AFTER INTRATHORACIC

a Based on average values of 5; maximum b 1 hr as control.

Abdomen Expt. 1 Expt. 2

Thorax Expt. 1 Expt. 2

Head Expt. 1 Expt. 2

Days after injection:

MULTIPLICATION

2

6

4.07 3.69

3.62

4.98

1.61 2.58

log,,

AND ABDOMEN

3.62 3.40

3.59

4.49

2.54 1.08

7

1.4)

OF Aedes

0.0 3.71

3.83

0.0

0.0 0.32

8

2.87 2.75

2.80

3.21

1.20 1.34

10

of 1 mosquito a

25 PFU (log,,

PFU/part

HEAD,THORAX, INJECTION OF

TABLE

2.03 2.43

2.52

2.96

1.08 1.45

12

aegypti

2.58 1.34

0.0

2.75

0.0 0.0

14

1.80 -

-

2.00

0.70 -

16

1.93 -

-

2.54

0.70 -

18

3N

5

2 i? w

iiH

R

VESICULAR

STOMATITIS

VIRUS

IN

MOsQuI’rOsS

409

410

BERGOLD, sukR~2

AND MUNZ

PFU/ml (4.5 log) with the Indiana strain For electron microscopic investigations, and about 3.5 with the New Jersey strain, the dissected organs were fixed in 2% osmium tetroxide and after dehydration, whether 4.4 or 1.4 logs of Indiana virus or embedded in butyl-methyl methacrylate 3.4 or 0.4 of New Jersey virus were injected. (7:3). Sections were cut with a FernandezTherefore in all further experiments about I.4 logs of Indiana virus and 0.4 logs of Moran, type microtome (FernandezMoran, 1956) using a diamond knife. A New Jersey virus were injected. Siemens Elmiskop I was used for the elecThe multiplication of WV in head, tron microscope studies. thorax, and abdomen of Aedes aeggpti 1 to I8 days after intrathoracic injection is RESULTS shown in Table f! (Indiana) and Table 3 (New Jersey). On the basis of the zero Multiplication of VSV in Mosquitoes after value at 1 hr multiplication is very fast and Zntrathoracic Injection reaches a maximum value in the head Table 1 indicates that recovery of virus (about 3.5 logs Indiana and up to 1.5 logs after 1 hr is irregular, but up to IA0 of the in New Jersey) within 2-3 days. Similarly, injected virus amount can be found. However, the multiplication appears to be in- maximum values up to 5 logs with Indiana and 3.5 logs with New Jersey are acdependent of the injected concentration and cumulated in the thorax (with salivary can be titered reproducibly. The concentration is just as high, i.e., about lo&.” glands) and somewhat lower values in the

a....-.-..o v o-----+ -----4

FIG. 1. intrathoracic

The concentration of VSV injection of 25 PFU.

Indiana

So!l”ary gland Thor gongllon Cerebrum Dar dlvertlculum

in cig ht different

organs

of Aedes

aegypti

l-20

days

VESICULAR

STOMATITIS

VIRUS

IN

411

MOSQUITOES

---.o Veniral dlverl!culum c----o lntcstlne a.. .. . . .a ,,,,jqu,

12345678

FIG.

after

2. The intrathoracic

concentration injection

of WV of 2.5 PFU.

10

New

Jersey

in eight

abdomen in the same time. After 4 to 6 days virus concentration drops, but there are still about 3 logs after 18 days in the thorax and abdomen of Indiana, and about 1 log after 7 to 9 days of New Jersey strain (neglecting the probably erroneous high values of the 8th day). Virus multiplication in cerebrum, salivary gland, dorsal diverticulum, thoracic gan-

INDIANA MAXIMAL

CONCENTHATION OUTPUT, RELATION

8 DAYS, TO Vmus

wet weight (a)

Organ Brain Salivary gland Dorsal diverticulmn Thoracic ganglion Ventral diverticulum Abdominal ganglion Midgut Hindgut ‘I Average 0 Estimated

of 10 organs. value.

( logI,, AND

(’

16 14 5 2.7 11 3.8 b 227 97

of organs 3.72 4.32 3.50 4.67 4.01 4.81 4.50 4.45

orgnns

of Act/es

cleg@i

l-12

days

4

PFU)

IN &CANS

20 DAYS

CONCENTRATION

Max.

different

p I

glion, ventral diverticulum, abdominal ganglion, midgut, and hindgut l-20 days after intrathoracic injection, are outlined in Fig. 1 for the Indiana strain and in Fig. 2 for the Kcw Jersey strain. Multiplication of VSV Indiana reaches about 100 times higher levels in all eight organs than New Jersey. The peak of Indiana (Fig. 1) in all organs is followed at Z-3 days by a drop

TABLE VW

12 Days

AFTEH

OF L~oSQUITOXs INTHATHOHACIC

PEH pg

____~~output

2.52 3.17 3.80 4.24 2.97 4.23 2.14 2.46

WET

After

vsv

per Pug

OF

AT

TI~IE

INJECTION

o? IN

O~~axs 8 days

After

20

days

at d>lYS

of organs

per m

of organs

per

2 2 3 3 3 2 2 4

3.01 3.41 2.0” 3.38

1.81 5.26 1.32 2.95

1.45 2.29 0.24 2.13

0.95 1.14 0.032 0.70

2.11

1.07

0.00

0.00

2.33 9.72 3.49

1.75 0.36 1.50

1.01

0.043 0.079 0.51

1.15 -

/I!2

diverticulum

I

I

I -

1

MOX.3 8 90 --

Days PA.

Ventral diverticulum I

DOYS

P.’

Abdominal ganglion

,

0

I

2

log,,

3

4

5

PFU

FIG. 3. Comparison of VSV Indiana concentrations maximum levels ( 2, 3, or 4 days) and 8 and 20 days indicate concentrations in the total organs and empty

4

N

FIG. 4. Tracing the salivary gland particles; N, nuclei,

in after bars

6 0

TOTAL ORGAN PERpg wet weight

eight different intrathoracic concentrations

organs of Aedes aegypti at injection of 25 PFU; black bars per microgram of wet weight.

f

of a mosaic of 24 electron of Aedes negypti; numbers ID, internal duct. 2,500

micrographs of l-84 indicate X. 412

a cross section through sites of accumulations

the three of VSV

a&i of Indiana

VESICULAR

STOMATITIS

and a lower peak again after 6-8 days. The virus concentration drops in all organs but remains relatively high in the salivary glands, thoracic ganglion, and intestine even after 20 days. VSV New Jersey also shows the first peak after 2-3 days but the virus levels are low and drop fast. No virus could be found in any organ after 12 days in mosquitoes infected with New Jersey virus. The specific virus concentration is quite different in the eight organs. This is clearly shown in Fig. 3 and Table 4, in which the virus concentration per wet weight is compared with the concentration of the total organ at peak levels after 8 and 20 days, The VSV concentration per microgram at peak level is highest in the thoracic and abdominal ganglions (4.2 logs). After 8 days the thoracic ganglion is still leading with about 3 logs/Ec.g. However, after 20 days the specific concentration is highest in the salivary glands. Table 5 shows that in 12 out of 20 mosquitoes, VSV Indiana could be found (up to 3 logs) in the lumen of the gut 3 days after intrathoracic injection when the virus concentration in the intestine was quite uniformly high, that is about 4.5 logs. However, no New Jersey virus could be detected in the gut lumen even when the highest concentration in the intestine was about 2.3 logs. Electron

Microscopic

Investigations

The electron microscopic demonstration of VW in the organs of infected mosquitoes was much more difficult than expected in spite of the relatively high virus titers, VSV Indiana particles were found in salivary glands (Figs. P21), intestinal cells (Figs. 2231) , abdominal ganglia ( Figs. 32-35), thoracic ganglia (Figs. 36-39), and midgut cells (Figs. 40 and 41), but not for certain in the cerebrum, and the cells of either diverticuli.

VIRUS

IN

MOSQUITOES

413

In salivary glands, the particles are readily seen in all three acini (Fig. 4) where they are mostly located near the periphery and less frequently in the neighborhood of the duct. They occur almost exclusiveIy in groups and within limiting membraneous structures. These are most likely cisterns or tubuli of the endoplasmic reticulum. As an exception, rodshaped particles, apparently identical with VW were observed at two occasions (Fig. 20) in the nucleus of a cell of the salivary gland. The dimensions of the particles are within the range of those characteristic for VSV Indiana. They resemble rods, mainly hemispherical at both ends, and spheres, surrounded by a membrane and having a more-or-less dense core. Frequently differentiations are seen within the VSV particles (Figs. 10-15, 17, 21, and 36), which could be interpreted as developmental stages. Once a somewhat crystalline arrangement of particles was observed (Fig. 18). Occasionally much smaller spherical particles with a diameter of about 25mp without membranes are seen in the salivary glands (Fig. 18). In some infected cells of the salivary gIands there appear to be signs of pathological destruction such as changes in the endoplasmic reticulum (Figs. 16 and 19). The appearance of the virus particles within the intestinal epithelium (Figs. 2231) , abdominal ganglia ( Figs. 32-35), thoracic ganglia (Figs. 36-39)) and midgut epithelium (Figs. 4041), is much the same as described above for the salivary gland. In the intestine the VSV particles are found close to the outside wall towards the coelom (Fig. 22). Once VSV particles were seen embedded in a dense inclusion (Fig. 26). Occasionally one can see spherical particles with varying diameters of about 30-117 rnp. They appear to have some granular structure internally. Some of them can be mistaken for VSV particles if they have a similar diameter.

a Positive

lumen: epithelium:

Intestinal

of total.

0 2.13

0 0.00

2.08 3.95

0 0.00

0.00 4.48

0 0.00

0.87 3.04

0 1.87

2.52 4.70

5

0 1.00

0.95 4.67

6

0 1.30

1.18 4.51

7

0 2.34

0.00 4.51

8

0 1.78

0.40 4.03

9

New

0 2.19

Jersey

3.00 4.84

Indiana

10

1.00 4.08

11

0.00 4.95

12

13

0.00 4.24

OF 20 Aedes OF NEW JERSEY,

5

0.08 4.93

14

aegypti TITERS

0.08 4.30

15

0.00 4.61

16

.~

0.00 4.31

17

0.08 4.59

18

3 DAYS AFTER INTHATHORACIC IN ( loglo) PFU/MOSQUITO

0.00 4.65

19

0.00 4.52

20

INJECTION

12/20 20/20

P/Tn

OF

“!

8

0 2.04

0 1.30

0 0.00

0 1.00

0 0.00

0 2.10

0 2.22

0 2.16

0 1.78

0 2.27

o/20 15/20

;

;

t5

2.20 4.00

4

TABLE EPITHELIUM (log,” 0.4)

1umen : epithelium:

3

OF INTESTINE AND INTESTINAL. 1.4) OF INDIANA AND 2.5 PFU

2

2

IN LUMEN 25 PFU (log,,

Intestinal

1

CONCENTRATION

Mosquito:

VW

VESICULAR

FIGS. 5-10. Enlnr~ed sites where virus particles

Transmission

STOMATITIS

portions from were found.

of VSV to B&J

the mosaic 50,000 X.

Mice

Transmission of VSV Indiana and New Jersey to baby mice by Aecles acpypti infected by intrathoracic injection can easily be demonstrated. The results are summarized in Table 6, and indicate that transmission commences 3 days after injection and continues for at least 20 days. Transmission of lNew Jersey virus is noted on the 2nd day. Transmission efficiency is apparently independent of the titer within the range of 2.18-5.48 logs for the Indiana strain and within 1.16-2.89 Iogs for the New Jersey strain. In three cases transmission occurred without detection of New Jersey virus in the mosquitoes.

VIRUS

IN

MOSQUITOES

415

of Fig. 4; the numbers correspond to the numbered Figs. 5, 6, and IO, 50,000 >(; Figs. 7-9, 44,600 X.

The results of experiments on transmission of VSV Indiana to baby mice by feeding mosquitoes after ingestion on viremic mice are summarized in Table 7. In the first group of 16 lots of mosquitoes (5 mosquitoes/lot per mouse) transmission of Indiana virus occurred after 15 days incubation of 3 to 16 lots of mosquitoes which fed (5 mosquitoes/lot per mouse) on baby mice 24 hr after ip infection with 2000 PFU. This dose caused death of all mice in 72-96 hr, although no virus could be detected in the blood at 24 hr. In the group of 16 Iots of mosquitoes ingesting on 36-hr viremic mice 4 lots transmitted on the 3rd and 10th day to 4 of 64 baby mice. In the last group

416

BERGOLD,

FIGS.

SUhEZ

5-10.

AND

( Continued).

MUNZ

VESICULAR

STOMATITIS

FIGS.

5-10.

VIRUS

IN

( Continued).

MOSQUITOES

417

FIGS. 11-21. VSV

Indiana

virus

Electron micrographs particles within

of tubuli

sections through the salivary glands showing and cisterns of the endoplasmic reticulum.

accumulations 11-15, 43,500

of X.

VESICULAR

STOhIATITIS

-.”

.

VIRUS

-

IN

MOSQUITOES

410

.

17, 19, and 21 sllo\v particles with various structural differentinFIGS. 11-21 (contkc~ct~) . Figures 17 and 19. 50,000 ;(I 21, 100,000 X. Fig,rure 18 depicts some virus partions of developmental stages. ticles in a “crystalline” orcler; arrows indicate sm;~ll particles of unknown nature. 50,000 X. Figure 20 shows unusual localization of virus pnrticlcs \vithiu the nucleus close to the membrane. .50,000 X.

FIG. numbers FIGS. bered FIG. 25,000

22.

Mosaic of six electron micrographs of a section through the intestine of 1-14 indicate sites of accumulations of VW Indiana particles. 2,400 X. 23-25. Enlarged portions from the mosaic of Fig. 22; the numbers correspond sites where virus particles were found. 43,500 X. 26. Dense inclusion within a cell of the intestine, showing mitochondria and x. 420

Aedes

uegypti;

to the virus

num-

particles.

BERGOLD,

27. Enslarged portions where virus particles were

FIG.

site

from the found.

mosaic

SU6REZ

of Fig.

AND

22.

491

MUNZ

23,600

X.

The

numbers

correspond

to the

FIGS. 28-31. Electron micrographs of intestine cells with nature. Fig. 28, 22,500 X; Figs. 29-31, 45,500 X. FIGS. 32-35. Electron micrographs of a section through Arrow in Fig. 32 indicates the site of virus particles shown 33-35, 45,500 x.

granulated

30-117

an abdominal enlarged in Fig.

mp particles ganglion 33; Fig.

of unknown

of Aedes 32, 4,500

aegypli. X; Figs.

VESICULAR

STOMATITIS

VIRUS

IX

MOSQUITOES

423

424

BERGOLD,

SUAREZ

AND

MUNZ

VESICULAR

STOMATITIS

VIRUS

IN

MOSQUITOES

425

426

BERGOLD,

SdREZ

AND

MUKZ

VESICULAR

STOMATITIS

of mosquitoes ingesting on 4%hr viremic mice, 15 of the 16 lots transmitted on the 3rd, Cith, lOth, and 15th day to 15 of 64 mice. Thus 22 of a total of 192 mice (11%) were infected by Zi! or 45% of the 48 mosquito lots used in this experiment. The virus titers in the viremic mice at 36 and 48 122 ranged from 0.7 to 3.0 log PFU/pl of blood. In.the three groups of mosquito, virus was found after 15 days incubation in 1 of 16, 3 of 16, and 11 of 16 lots of mosquitoes or a total of 15 of 48 or about 31%. The titers ranged between 1.8 and 3.7 log PFU/5 mosquitoes. Therefore 7 of the 48 lots of mosquitoes transmitted Indiana virus although the virus could not be detected in them. The results of experiments on transmission of VSV New Jersey are summarized in Table 8. Transmission commenced after 3 days incubation in mosquitoes which fed on 36-hr viremic mice, but efficiency was lower. Only 5 of 24 mosquito lots or about 20% transmitted New Jersey virus to 5 or 5% of the 96 mice used. Virus could be foulid in the blood of 6 of 24 mice only by ic injection in baby mice but not by titration in BHK 21 cells. Virus was found in only 2 or 8.3% of the 34 mosquito lots. The titers were very low, being of 0.5 and 0.7 log PFU/lot. However, 3 of 24 lots transmitted New Jersey virus although the virus could not be detected in them. I)ISCUSSION

The results of this study confirm in general the classical investigation of La Alotte (1960) of the multiplication of Japanese B encephalitis virus in organs of mosquitoes, It is, however, remarkable that such different viruses as Japanese B and VSV multiply in a similar manner. The lack of dependence between the injected concentration of virus and the amount of virus found after 7 days in mosquitoes (Table 1) is of interest. It suggests that there are only

VlRUS

IN

MOSQUITOES

427

a certain number of susceptible cells in a mosquito, in which the virus multiplies during the life span of a mosquito. Similarly it can be learned from Table 7 that a mosquito ingesting 1 121of viremic blood, or about 10 PFU, becomes infected, and trammits to baby mice. Initially only about two cells mk2y be infected, assuming equal sensitivity between BHK 21 cells and mosquitoes. According to Devine et al. (1965), a mosquito deposits about 5 118 of saliva in a mouse while taking a blood meal. Therefore, one can calculate from the data of Table 4 that almost one-third of the weight of the salivary gland or about 500 and 70 PFU respectively are deposited during the blood meal S days or 20 days after infection. The results shown in Tables 6 and 7 leave no doubt that Aenes aegypti may act as a vector for VSV. However, the efficiency of transmission of the New Jersey strain of virus is so low that A. aegypti cannot play an important role as a vector in nature. It is of interest that VSV Indiana but not I%ew Jersey is present in the gut lumen in such a high concentration and that it can be expelled so easily (Table 5). One could imagine that in this way, virus is released during oviposition or rectal discharge into a water hole. The hatching larvae may then take up the virus, since Collins (1964) has demonstrated that larvae of C&x quinquefascintm can thus become infected with St. Louis encephalitis virus and that the resulting adults transmit it to l-day-old chicks. Similarly Chamberlain et al. (1963) found that up to 92% of egg rafts laid by experimentally infected G&X quinquefmciutus contained St. Louis encephalitis virus. Therefore, the possible rectal discharge of Indiana virus may be of importance for the transmission from one generation of mosquitoes to the next. Concerning the electron microscopic demonstrations of VSV in various organs 110 explanation can be given as to why the virus particles were not found in the cere-

428

BEBGOLD,

SUhEZ

brum and the diverticuli in spite of the high virus titers in these structures. NO conclusive statements can be made as to the nature of the spherical forms of the virus particles found frequently in all organs, particularly in the salivary glands. Some of these spheres show various internal structural differentiations (Figs, 10-15, 17, 21, and 36). We believe, in agreement with Mussgay and Weibel (1963), that these spherical forms are developmental stages of vsv. The occurrence of virus in high concentration at the periphery and to a lesser extent near the internal ducts (Fig. 4) or towards the gut lumen (Fig. 22) is striking. It may mean that the virus particles multiply mainly at the periphery of the salivary gland and the intestinal epithelium, and that the particles collect around the duct and gut lumen. The lower concentration of virus particles at the central part of the salivary gland and the intestinal epithelium may be caused by periodical expulsion during feeding and rectal discharges. The unusual presence of VSV particles within the nucleus close to the membrane (Fig. 20) is surprising and cannot be explained except by pinocytosis. The dense inclusion shown in Fig. 26 contains, apart from mitochondria, VSV particles but we are unable to explain its meaning. We have not attempted to determine the significance of the 25-mu particles without a membrane (Fig. 18), and of the large particles (30-117 mu; Figs. 28-31). The largest particles (Fig. 29) have a granular structure and are not confined to the endoplasmic reticulum. It is of interest that there are hardly any bullet-shaped VSV particles in any organ. Almost all particles are hemispherical at both ends. This is contrary to findings of other investigations in this field, e.g., that by Howatson and Whitmore (1962).

AND

MUNZ ACKNOWLEDGEMENTS

The and

technical assistance Mrs. C. de Decena

of Mrs. is greatly

M. de Pingarrcin appreciated.

REFERENCES BERGOLD, und 61,

G. H. 1941. Mikroburette 158-162.

Eine Mikroinjektionsspritze bis zu 0.1 cmm. Bid.

Zentr.

BERGOLD, G. H., AND MAZZALI, R. 1968. Plaque formation of Arboviruses. J. Gen. Virology, 2, 273-284. CHAMBERLAIN, R. W., SUDIA, W. D., AND GOGEL, R. H. 1964. Studies on transovarial transmission of St. Louis encephalitis virus by Culex quinquefasciatus Say. Am. J. Hyg., 80, 254-265. COLLINS, W. E. 1963. Transmission of encephalitis virus by larval-infected yuinquefasciatus mosquitoes. Ann. Sac. Am., 56, 237-239. DEVINE, T. L., VENARD, 1965. Measurement aegypti (L.) feeding Physiol., 11, 347-3ii3. FEHNANDEZ-MOHAN,

H.

St.

Louis Culex Entomol.

L.

E., AND MYSEH, W. C. of salivation by Aedes on a living host. 1. Insect. 1956.

Application

of

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