key: cord-0004855-uw2iwcij
authors: Rossi, C. R.; Kiesel, G. K.
title: Bovine respiratory syncytial virus infection of bovine embryonic lung cultures: Enhancement of infectivity with diethylaminoethyl-dextran and virus-infected cells
date: 1978
journal: Arch Virol
DOI: 10.1007/bf01317851
sha: 143454c41d8e309d5680819eaa3e5b0e06cad022
doc_id: 4855
cord_uid: uw2iwcij

The effects of incorporating diethylaminoethyl-dextran (DEAE-D) in the inoculum with bovine respiratory syncytial virus (BRSV) on the infectivity of BRSV was evaluated. A concentration of 40 µg DEAE-D/ml provided maximal enhancement of infection as determined by the time of onset of cytopathic effect (CPE), the percentage of cells infected by the inoculum, and the amount of virus produced. When DEAE-D was used in the inoculum, the CPE appeared a day earlier, the percentage of cells infected by the inoculum, as determined by the fluorescent antibody test, was increased 11 times, and the viral titer was increased 2 times as compared to results obtained without DEAE-D. Bovine respiratory syncytial virus-infected cultures contained much cell-associated virus which could be liberated by sonication to increase the titer of virus stocks. The use of BRSV-infected cells rather than supernates from BRSV-infected cells increased the rate at which a cytopathic effect developed, although it did not substantially increase the titer of virus which was harvested. The use of DEAE-D in the inoculum and the passage of BRSV-infected cells instead of viral suspensions was found to be the quickest and most effective method of consistently obtaining BRSV with a titer of about 10(5.5) TCID(50)/ml.

Bovine respiratory syncytial virus (BRSV) is closely related to respiratory syncytial virus (RSV) (1) , and although the two viruses cross-react considerably in serum neutralizing tests, they are antigenieally distinct (13, 21) . gespiratory syncytial virus is considered to be the most, important virus in initiating severe respiratory disease in infants (8) . Its counterpart in the bovine, BRSV, has been

Bovine embryonic lung (BEL) cultures were initiated from the lungs of embryos obtained at a nearby abattoir and were used between the 3rd and 8th passages. Second passage BEL cultures were stored in liquid nitrogen. Cultures were passaged a month in serum free of bovine viral diarrhea (BVD) virus and examined by the fluorescent antibody (FA) technique to be certain they were free of BVD virus and, therefore, satisfactory for use. Medium consisted of minimal essential medium of Eagle (MEM) supplemented witlh different concentrations of fetal bovine serum (FBS) for different conditions and containing penicillin, streptomycin, and neomycin at concentrations of 200 units, 200 ~g, and 100 y~g per ml, respectively. Medium for microtiter titrations was supplemented with organic buffers as described (18) .

Bovine respiratory syncytiM virus (BRSV) originally isolated by S~i I~ et al. and kindly provided by Dr. t%. M. Phillips was purified by terminal dilution for use in these experiments.

Stock BRSV was prepared on BEL cultures. Calves were inoculated intratracheMly and intranasally with t07.4 median tissue culture infective doses (TCIDs0) BI%SV. Three months later 5 intravenous injections containing 106.9 TCIDs0 each of BRSV were given at three-week intervals. Two weeks after the last injection the cMves were bled and the immunoglobulin G (IgG) fraction obtained and conjugated with fluorescein isothiocyanate (FITC) at a concentration of 25 ~g FITC/mg IgG (20) .

Viral suspensions and cells which were titrated were diluted with pipetes in tubes with microtiter medium as diluent. Eight replicates of two-fold dilutions were placed in microtiter plates with microtiter pipets which delivered 0.025 ml per drop. A drop of medium and a drop of BEL cells at a concentration of 300,000 cells per ml were added. Cultures -were incubated at 37 ° C as previously described (18) , and the titer of the virus was calculated by the method of REED and MVE~C~ (14) .

Diethylaminoethyl-dextran (DEAE-D) was prepared as a stock solution in saline at a concentration of 4000 ~g/rnl, filtered, and diluted in appropriate medium before use. I t was tested for its toxicity for B E L cells at different concentrations, different volumes of inocula, with different media used subsequent to treatment, with DEAE-D, and with cells in different stages of growth.

Three methods were used to evaluate the effectiveness of D E A E -D added to the inoculum in enhancing the infectivity of BRSV for B E L cells. These included i) the percentage of cells infected with BRSV as determined by the F A technique, ii) the extent of cytopathie effect (CPE) produced, and iii) the amount of virus produced.

I n the fluorescent antibody technique, B E L coverslip cultures were inoculated with BRSV and different concentrations of DEAE-D. Twenty-four hours later, a time at which fluorescence was maximal and no fluorescence due to a second cycle of infection was present, the cultures were fixed, stained, and examined for fluorescence along with uninoeulated cultures and cultures inoculated with heat-inactivated BRSV. Cells infected with BRSV were identified by their typical fluorescence (20) . Evans blue at a final concentration of 0.04 per cent was added to the FITC-conjugate to eliminate background fluorescence.

Bovine embryonic lung cultures in 25 cme flasks were inoculated with BRSV conraining 40 ~xg D E A E -D / m l and allowed to absorb at 37 ° C for two hours. Cultures were washed four times with Hanks' balanced salt solution (HBSS) to remove unattached virus and replaced with M E N containing 5 per cent FBS. At appropriate times the supernate was removed and centrifuged to remove unattached ceils and a sample of supernate was used for titration. Attached ceils were removed with tr?xosin-versene and dooled with the cells from the supernate, resuspended in medium equal to the volume of supernate, and a sample was used for titration. To compare titers of infectious virus obtained from the i) supernate, ii) cells, and iii) a mixture of supernate and sonicated cells, the procedure was varied so that after suspending the cells, they were centrifuged, suspended in a small volume of supernate, sonicated, and resuspended in the entire volume of supernate for titration. Titrations of infected cells and viral suspensions were carried out by microtitration. Results showed t h a t growing cultures t h a t did not reeeive serum after t r e a t m e n t with D E A E -D were much more susceptible to the toxic effects t h a n cultures which received serum. F u r t h e r m o r e , cultures which received serum often recovered from the toxic effect of D E A E -D whereas those which did not receive serum did not recover. Cultures which exhibited toxicity and subsequently recovered due to the presence of serum usually had fewer cells t h a n cultures which exhibited no initial toxicity. W h e n serum was not used, concentrations of D E A E -D as low as 30 ~g/ml produced some cytotoxieity. W h e n serum was used, concentrations of 60 to 120 p,g D E A E -D / m l were usually satisfactory, whereas higher concentrations were toxic. Confluent monolayers of B E L cells in 25 eme flasks were also tested for susceptibility to D E A E -D . R e p e a t e d trials showed t h a t inocula of 0.2 ml were not toxic up to concentrations of 2000 ag/ml when 5 per cent or more serum was added subsequent to t r e a t m e n t , whereas cultures inoculated with 1.0 ml or more sometimes exhibited a slight degree of toxicity at concentrations of 1000 and 2000 pog D E A E -D / m l ; in some experiments no t o x i c i t y at these concentrations was evident. Table 1 . Infection was enhanced as much as 10,9 times at a concentration of 40 [zg D E A E -D per mt. Considerable enhancement was also present at concentrations of 20 and 80 tzg D E A E -D / m l . Using young, r a p i d l y growing cells, higher concentrations of D E A E -D were toxic. (Table 3) .

On the second, third, and fourth d a y after inoculation of confluent B E L culture flasks with BRSV, supernate and cells were t i t r a t e d separately. The distribution of infectious B R S V between supernate and cells is shown in Table 4 . During the early stages of infection when there was a 1 and 2 + CPE, the m a j o r i t y of B R S V was found associated with cells, whereas when a 3 + CPE was present, even t h o u g h most of the cells were still a t t a c h e d to the glass, the m a j o r i t y of B R S V was found in the supernate. (Table 5 ). 

T h e c a p a c i t y to t r a n s Viral suspensions with a titer from 105 to 105.s produced a 3 + CPE on the third day, whereas the cells from these suspensions produced a 3 + CPE on the second day. Titers of virus which have been obtained by using BRSV-infeeted cells have been consistently as high, or slightly higher, than maximal titers in which viral suspensions have been used as the inoculum.

The toxicity of DEAE-D for B E L cultures primarily depended upon the condition of the cells. Rapidly growing cells were more susceptible than resting, confluent, cultures to the toxic effects of DEAE-D. Concentrations above 60 or 80 ~g/ml were usually toxic to rapidly growing B E L cells and prevented the use of higher concentrations in other experiments, but concentrations up to 1000 and 2000 ~g/ml were often tolerated by resting cultures. The effect of different concentrations of DEAE-D on the infectivity of BRSV for B E L cells was evaluated by several criteria: number of cells infected by the inoculum, degree of CPE, and amount of infectious virus produced. Because of the ease of counting cells in semiconfluent cultures compared to the difficulty of counting ceils in a confluent culture, semi-coD_fluent cultures were used in FA tests to determine the number of cells infected with BRSV. Under these conditions, enhancement was maximal with 40 [zg DEAE-D/ml and infection was enhanced almost 11 times as compared with B E L cultures in which DEAE-D was not added to the inoculum ( Table 1 ).

The CPE produced by BRSV occurred a day earlier when DEAE-D was present in the inoculum than when it was absent. This effect occurred over a large range of concentrations of BRSV (Tables 2 and 3 ). The principal object of determining the capacity of DEAE-D to enhance dilute solutions of BRSV was to evaluate the feasibility of using DEAE-D in isolating BRSV from the respiratory tract of cattle. In both naturally and experimentally infected cattle, the isolation of BRSV has posed problems in that a CPE has not been identified until after subculture, and/or because of the short period of time over which the virus was recoverable (6, 1t, 13, 21) . Difficulties have been reported in the isolation of RSV from humans (4), which to some extent have been due to the loss of infectivity which occurs on freezing and thawing of the samples. However, even when nasal washings and nasal swabs are placed directly on susceptible cultures, BRSV has been difficult to isolate (Rossi and Kiesel, unpublished observations) . Using confluent cultures, DEAE-D enhanced infection with BRSV to the extent that the amount of virus harvested was about twice the amount harvested when DEAE-D was not used ( Table 3 ). The reason for the greater amount of virus harvested with DEAE-D in the inocutum ~han when it was not present probably reflects the greater infectivity of the inoculum. Since virus is produced more quickly with higher than with lower concentrations of BRSV, the sooner the virus can be harvested, the greater is the probability that the virus will not be inactivated.

The use of polyions, especially DEAE-D, to enhance infection has been investigated with a variety of viruses, including RSV (10, 12, 16, 22) . Although the mechanism of action of DEAE-D in enhancing BRSV infection was not investigated, work with laryngotracheitis virus (LTV), an avian herpesvirus, is probably applicable in explaining enhancement of other viruses with DEAE-D. Studies with LTV have conclusively shown that enhancement of infection with DEAE-D is due to increasing the attachment of virus to cells by the action of DEAE-D on cells, and that DEAE-D has no effect on virus particles themselves (16, 17) . Furthermore, work with LTV has shown that there is not a subpopulation of virus particles which lacks the capacity to attach to cells, but that the entire population is deficient in its ability to attach to cells. This by no means rules this out as a possibility for BRSV. The efficacy of using DEAE-D in the inoculum of :BI%SV in order to experimentally infect cattle is open to question, since DEAE-D probably has little affinity for the virus and might be diluted to insignificant concentrations in the respiratory tract of cattle. One should note, however, that in mice its use with eucephalomyocarditis virus has been shown to have a considerable enhancing effect (3).

Since BI~SV is an RNA virus which completes its development at the cell membrane (1), disruption of Bl~SV-infected cells cannot liberate infectious intracellular virus but can only remove virus adherent to the call membrane. Sonication of BRSV.infected cells was found to be an effective method for liberating BRSV from infected cells in order to obtain higher titered virus stocks ( Table 5 ). The use of virus-infected cells was found to be more efficient in infecting cells than viral suspensions. Viral suspensions are limited in their infectivity by the capacity of one virus particle to infect only one cell, and by the fact that the percentage of virus absorbed from a large inoculum is much less than that absorbed from a small inoeulum (2) . As a consequence of the latter hmitation, increasing the volume of inoculum is not especially effective in increasing infection. However, virusinfected cells all settle to the monolayer and are probably capable of infecting more than one celI.

Since virus stocks of BRSV lose considerable virus because of freezing and due to storage, we have found the following method to be the most suitable to get stock virus preparations to maximal titers of around 105.9 TCIDs0/ml. Virus taken from the freezer is mixed with DEAE-D to a final concentration of 40 ~g/ml. When a 3~-CPE has been reached, cells from the supernate and those attached to the glass are used to inoculate a culture of BEL cells which cover u/4 the surface of the flask. When the CPE again reaches a 3 ~-CPE, the procedure is repeated until a sufficient number of flasks have been inoculated. Passage of cells is made at a 1 : 1 ratio to begin with and at a 1 : 2 ratio thereafter. When a sufficient number of flasks have been inoculated and the CPE reaches 3 + on the second to third day, the supernate is harvested and the cells sonieated in a small volume of supernate. This type preparation has been found to give the most consistently high titers of 105.3 to 105.9 TCID~0, although it is considerably lower than titers of 107.9 which we have obtained with RSV on ttEp-2 cells. However, the amount of BRSV produced can be estimated to be at least ll-fold greater than that determined in microtiter assays, or in any assay in which DEAE-D is not used in the inoculum. Since DEAE-D enhances RSV-infection, titers of RSV can also be considered to be low estimates of the infectivity which might be obtained under different conditions. It is possible that further studies using Bl%SV-infected cells as inoeula on BEL cells will reveal methods of obtaining higher titers of BRSV than have been achieved to date.

W h e n using B R S V in serum neutralization tests, one should be aware of the large a m o u n t of uninfective a n d inactivated virus in the suspensions a n d be cognizant of the reduction this introduces in the sensitivity of the test to detect antibody. Because of this, one can predict t h a t the prevalence of BRSV infection of cattle is greater t h a n t h a t based upon results of serum neutralization tests.

Supported by the Alabama Agricultural Experiment Station, Auburn University, Auburn, AL and General Cooperative Agreement 12-14-3001-520 with the U.S. Department of Agriculture, Agricultural Research Service, U.S.A.

The authors thank Barbara Cuenco for excellent technical assistance.

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