APPLIED MICROBIOLOGY, Jan. 1975, p. 81-89 Vol. 29, No. 1Copyright i 1975 American Society for Microbiology Printed in U.S.A.

Plaque Production by Arboviruses in Singh'sAedes albopictus Cells

C. E. YUNKER* AND J. CORYNational Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratory,

Hamilton, Montana 59840

Received for publication 27 September 1974

We report plaquing tests of 124 virus strains, mostly arboviruses of 21serological groups, in Singh's line of Aedes albopictus cells. Thirty of theseplaqued; all were arboviruses of six groups and were known or presumed to bemosquito borne. Failing to plaque were 86 strains of arboviruses, mostlytick borne, two strains of insect pathogens, and six animal viruses not classifiedas arboviruses. Among mosquito-borne agents, plaquing ability appeared relatedto serological classification. California group and most A-group viruses failed toplaque, but nearly all members of B and Bunyamwera groups readily plaqued.Within serological group B, 14 of 16 mosquito-borne agents plaqued, but none of13 tick-borne or vector-unassociated viruses did so. Some implications of theseresults for recognition and classification of arboviruses are discussed.

In 1969 Suitor (20) reported that Japanese Bencephalitis (JBE) virus, a member of Casals'serological group B known to cause cytopatho-genic effect (CPE) in Singh's line of Aedesalbopictus cells, was also capable of producingplaques in monolayers of these cells. However,the broad and practical implications of thisdemonstration for virus-vector studies re-mained questionable, possibly because CPE ischaracteristically inapparent in virus-infectedarthropod cells in culture (3) and the strain ofvirus Suitor used was a unique variant thatgrows well at the lower temperatures usuallyrequired to incubate insect cells. Later, usingvirus strains not adapted to grow at low temper-atures, we confirmed Suitor's observation ofplaque production by JBE virus in A. albopic-tus cells and, further, reported similar resultswith. five related Flaviviruses, West Nile, yellowfever, and dengue viruses 1, 2, and 4, and theunrelated rhabdovirus, Indiana vesicular sto-matitis (7). In the present study we reportresults of plaquing attempts with 124 viruses,mostly arthropod borne, in Singh's A. albopic-tus cells.

MATERIALS AND METHODSCell line and culture conditions. The A. albopic-

tus cell line of Singh (15), received by us in its 49thpassage from Sonja Buckley, Yale Arbovirus ResearchUnit, New Haven, Conn., was used in its 87th to 135thpassages. Since receipt at this laboratory, these cellshave been grown as monolayers in a lactalbuminhydrolysate growth medium in Hanks basic salt

solution (Grand Island Biological Co.) to which wasadded the following (per liter): 100 ml of fetal calfserum inactivated at 56 C for 1 h, 1 g of bovine plasmaalbumin (fraction V) (Armour Co.), 105 U of penicillinG (sodium salt), 100 mg of streptomycin sulfate, and40 mg of neomycin sulfate. The pH was adjusted to6.8 if necessary, and the medium was sterilized bypressure filtration. Stock cultures were grown asmonolayers in 12 ml of medium in 250-ml plasticflasks (Falcon Plastics) in ambient air at 27 C andwere subcultivated at intervals of 7 to 10 days.Normal cell stocks were routinely monitored for the

presence of adventitious or latent animal viruses, withnegative results, by screening in suckling mice andVero cell cultures. In addition, neither known virusnor virus-like particles could be detected in thisparticular strain of Singh's A. albopictus cells byelectron microscopy thin-section technique (H. Hi-rumi, personal communication). Mycoplasma couldnot be demonstrated by testing of selected samplesof cells (both plaquing virus infected and uninfected)by conventional methods and also by a commercialtesting laboratory. Species identity of the A. albo-pictus cell line was confirmed by the Institute forMedical Research, Camden, N.J.

Preparation of monolayers for virus inoculation.Growth medium was decanted from confluent youngmonolayers of cells grown in four 250-ml flasks andreplaced with -fresh growth medium. Cells were dis-sociated with the aid of a 10-ml plastic pipette byvigorously drawing and expelling medium over themonolayers. This was facilitated by using a pipette onwhich the tip had been bent in a flame. Cell suspen-sions were pooled in a 100-ml Erlenmeyer flask andstirred by a magnetic stirrer adjusted to medium-slowspeed. Cell density was adjusted to 2 x 10' to 3 x106/ml with growth medium, and 30-ml Falcon plastic

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flasks were each seeded with 5 ml of suspension.Cultures were incubated at 27 C for 2 to 7 days.

Virus inoculation and assay. Virus stocks, storedfrozen usually as 10% mouse brain suspensions (vol/vol) in 7.5% bovine plasma albumin or 50% normalrabbit serum in 0.15 M phosphate-buffered saline,were mostly from the Rocky Moutain Laboratorycollection. These were thawed and prepared as serial10-fold dilutions in growth medium. Medium wasdecanted from cell monolayers, 0.2 ml of each virusdilution was introduced into a flask, caps were re-placed, and the inoculum was distributed over themonolayer by tilting the flask. Cultures were thenincubated at room temperature for 1 h on a rockingplatform. Primary overlay medium consisted of 3parts of growth medium mixed at 44 C with 1 part ofmolten 2% agarose (Seakem) in Hanks solution. Thiswas brought to 41 to 42 C and introduced in 4-mlamounts into each flask by means of a needlelessCornwall syringe. Caps were replaced, the flasks werereturned to a horizontal position, and the mediumwas allowed to solidify for at least 15 min. Cultureswere incubated at 35 or 37 C for 5 to 7 days,whereupon 2 ml of secondary overlay medium wasintroduced into each. This medium was identical toprimary overlay medium except that it contained1:5,000 neutral red dye. Plaques were counted after 1to 5 days of further incubation in the dark at 35 to 37C, and titers were expressed as plaque-forming unitsper milliliter. Plaquing in A. albopictus cells wasconfirmed by replicate tests. Identities of certainplaquing viruses were established by plaque-reduc-tion neutralization tests in A. albopictus or Africangreen monkey kidney (Vero strain CCL-81) cells (9).In addition, comparative titers for some virus stockswere obtained through parallel inoculation of Verocells. All virus stocks that failed to plaque in A.albopictus cells, except insect viruses, were demon-strated to possess levels of virus adequate to infectVero cells or newborn mice.

RESULTSThirty of 124 virus strains tested produced

plaques in A. albopictus monolayers. Thosepositive were members of the serological groupsA, B, C, Bunyamwera, Kemerovo, and vesicularstomatitis (Table 1). Of the remaining 94 vi-ruses failing to induce plaques in these cells, 39were members of the first five groups men-tioned, 47 were arboviruses of 16 miscellaneousserological groups or were not group associated,six were vertebrate viruses not arthropod borne,and two were mosquito pathogens (Table 2).Two lots of normal mouse brain, each testedtwice as serial 10-fold dilutions, also failed tocause plaques or CPE in A. albopictus cells.Group A arboviruses. Of nine viruses tested,

only one, Chikungunya, formed plaques in A.albopictus monolayers (Fig. 1). Two or moreattempts to induce plaques in these cells witheach of the remaining eight failed; these viruseswere Eastern equine encephalomyelitis, Mid-

delburg, O'nyong nyong, Semliki Forest, Sind-bis, Una, Uruma, and Western equine enceph-alomyelitis.Group B arboviruses. Fourteen of 28 viruses

tested produced plaques: dengues 1, 2, 3, and 4,Ilheus, Israel turkey meningoencephalitis, JBE(two strains), Kunjin, Murray Valley encephali-tis, Ntaya, St. Louis encephalitis, Uganda S,West Nile (three strains), and yellow fever (Fig.2). Viruses of this group failing to plaque on twoor more attempts were Apoi, Bussuquara, Ka-dam, Langat, Louping Ill, Modoc, Negishi,Omsk hemorrhagic fever, Powassan, Rio Bravo,Royal Farm, tick-borne encephalitis, Tuleniy,and Zika. Two of these, Bussuquara and Zika,unlike the remaining 12, consistently caused awell-defined CPE in agar-overlaid cells.Group C viruses. Two of four viruses of this

group, Marituba and Murutucu (Fig. 1), causedplaques; negative after two attempts were Apeuand Itaqui viruses.Bunyamwera group viruses. Seven viruses

of this group caused plaques: Bunyamwera,Cache Valley, Chittoor, Germiston, Guaroa,Ilesha, and Wyeomyia (Fig. 1). One virus, Kairi,failed to plaque, although it was tested threetimes. With four members of this group, opti-mal development of plaques required prolongedincubation periods of up to 12 days postinocula-tion. Here it was necessary to reduce incubationtemperatures to 35 C to prevent deterioration ofcells.Kemerovo group viruses. Of nine members

of this group tested only Tribe6 virus formedplaques. Others tested, each two or more times,were Chenuda (two strains), Great Island, Hua-cho, Kemerovo, Lipovnik, Sixgun City, WadMedani, and Yaquina Head (three strains)viruses.

Vesicular stomatitis group viruses. BothIndiana and New Jersey serotypes of vesicularstomatitis virus plaqued in A. albopictus cells.No other rhabdoviruses were tested.

Plaque-reduction neutralization tests per-formed in A. albopictus or Vero cells confirmedthe identity of selected plaquing viruses fromeach serological group. These were: Chikun-gunya, dengue 1, 2, and 4, Israel turkey menin-goencephalitis, JBE, West Nile, yellow fever,Murutucu, Chittoor, Tribec, and Indiana vesic-ular stomatitis.

Titers (dex plaque-forming units/ml) in A.albopictus cells were obtained for all virusesexcept one (Table 1). The exception was Gua-roa, in which plaques were extremely large anddiffuse. For comparison, titers in Vero cells weredetermined for 15 viruses (Table 1). These werecomparable in both Vero and A. albopictus cells

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TABLE 1. Plaque formation in A. albopictus cells by 27 arboviruses of six serological groups

_|Virus st cks |Plaques in A. albopictusVirusstocks ~~cells

Virusname | Strain Serological Vector5 T Vero cell TiterVirusname designation group" Passagetiter (log1. (log0 Avhistory plaque- formu diam Read|hisoryc forming ming (mm) on day

units/ml) ml)unlts

ChikungunyaODengue ldDengue 2dDengue 3Dengue 4dIlheusIsrael turkeymeningo-encephalitisd

JBEJBEdKunjinMurray Valley enceph-

alitisNtayaSt. Louis encephalitisUganda SWest Nile"West NileWest NileYellow feverdMaritubaMurutucudBunyamweraCache ValleyChittooreGermistonGuaroaIleshaWyeomyiaTribe&dVesicular stomatitis

IndianadNew Jersey

23161HawaiiNGBH-87H-24126None

K-29NakayamaMRM164286

B25833798-55B309577259-60Ar95-60ArlO8-6017DBeAnl5BeAn974255106V633AMM2222AR105031498KO/2B26 380VR 468

438Hazelhurst

ABBBBBB

BBBB

BBBBBBBCCBUNBUNBUNBUNBUNBUNBUNKEM

VSVS

MMMMMMU

MMMM

MMMMUUMMMMMMMMMMT

MU

m174m116m45m23m9m28m33

m12e41m6m4m13

m24m34m22m27m6m7x

m19m13m38m7ml8m12m2m4m207ml8

elm5e2Oml

8.4

7.1

6.65.59.6

7.4

9.0

4.3

9.0

8.310.0

8.2

10.2

8.59.4

8.68.38.66.07.45.0

10.3

7.610.08.09.4

6.08.04.09.09.09.06.07.78.79.36.49.67.6_e

8.79.78.2

8.09.4

4.03.53.53.53.05.05.0

4.52.52.55.0

3.52.01.52.02.02.03.05.05.04.02.57.0

10.010.02.52.52.0

7.014.0

6776776

6779

99766697686

111212666

56

a BUN, Bunyamwera group; KEM, Kemerovo group; VS, vesicular stomatitis group.° Vector known or presumed to be: mosquito or other biting diptera (M), tick (T), or undetermined (U). West

Nile strains Ar95-60 and 108-60 were isolated from ticks.c Passage level of stock in mouse brain (m) or embryonated chicken egg (e), or of complex, high passage

history (x).d Identity confirmed by plaque-reduction neutralization test.e Plaques were large, diffuse, and not countable.

for Chikungunya, dengue 4, Ilheus, Israel turkeymeningoencephalitis, JBE, Uganda S, WestNile, Murutucu, Bunyamwera, and Indiana andNew Jersey vesicular stomatitis viruses. Dengue2 and Chittoor viruses gave significantly highertiters in the mosquito cells than in Vero cells,whereas Kunjin and Tribec virus titers weresignificantly lower in the former cells.Of 146 plaquing attempts with viruses even-

tually determined to cause plaques in A. al-

bopictus cell monolayers, 25 (17.1%) were nega-tive. Among these, the rate of success wasmarkedly higher with group B agents (87%)than with those of other groups (79%). Somefactors involved in these rates are discussedbelow.Miscellaneous groups and ungrouped vi-

ruses. Negative plaquing results were obtainedwith 30 arboviruses representing 16 serologicalgroups and with 10 ungrouped arboviruses

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TABLE 2. Viruses failing to cause plaques in A. albopictus cells

Virus namea Strain designation 1 Serological| Vectorc hPssage No.mofEEE 85 A M m9 4Middleburg Ar749 A M m12 2O'nyong nyong MP30 A M m12 3Semliki Foreste (ATC) A M m8 6Sindbis Ar339 A M mlO 4Una BT1495-3 A M m9 2Uruma Mayaro A U m9 3WEE 999-51 A M cdm6 3Apoi Apoi B U m6 2Bussuquarae BeAn4O73 B M m13 4Kadam MP6640 B T m4 2Langat TP21 B T m9 2Louping III 334-64 B T ml 4Modoc M544 B m5 2Negishi B U mU 2Omsk Guriev B T ?+ml 2Powassan M794 B T m6 1Powassan M791A B T m12 1Rio Bravo M64 B m9 2Rio Bravo Burns bat B ml5 2Royal Farm EgArt371 B T m8 2CETBE Hypr B T ?m2 2Tuleniy 3-Arch B T m3 2Zikae B24982 B M m147 4Apeu BeAn848 C M m5 2Itaqui BeAn12752 C M m5 2Anopheles A 166 ANA M ?+m2 1Bakau M2325 BAK M m13 3Bluetongue Cal BT8 BLU M e90m54 2Kairi TRVL8900 BUN M m15 3CE BFS283 CAL M -ml5 2La Crosse PR105826A CAL U m9 3Melao TRVL9357 CAL M m2 2Snowshoe hare MC3150 CAL U m16 2Tahyna 92 CAL M m21 2Trivittatus 993 CAL M ml5 2Trivittatus 7941 CAL M m3 1Hazara JC280 CON T m9 1Dugbe Ar1792 GAN T m16 1Ganjam G619 GAN T m8 3Farallon Ar846 HUG T m5 1Hughes Original HUG T ml1 1Punta Salinas Ar888 HUG T m3 1Raza 5/18/64 HUG T m9 2Sapphire II 14 HUG T m3 1Chenuda Ar1170 KEM T ?+m4 1Chenuda Ar1152 KEM T m19 1Great Island Main 45 KEM T m3 1Huacho 883 KEM T m6 3Kemerovo R10 KEM T m7 4Lipovnik 91 KEM T ccl+m3 3Mono Lake Ar861 KEM T m4 1Sixgun City 52451 KEM T v4m2 1Wad Medani Ar492 KEM T m5 2Yaquina Head 62 KEM T m5 1Yaquina Head 15 KEM T m4v9 3Yaquina Head 90 KEM T v13 1Lanjan TP94 LJN T m14 1Silverwater 131 LJN T m8 1

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TABLE 2-Continued

85

Virus namea Stra1 n designation Serological Vectore Passage No. ofgroup, historyd attempts

MidwayNyamaniniPalyamSandfly feverJohnston AtollQuaranfilBandiaQalyubTacaribeTurlockUukuniemiUukuniemiGrand ArbaudJosLone StarMatucareMutSakhalinSakhalinSapphire ISawgrassThogotoUpoloWanowrieAnopheles BCTFCTFEMCHVH(II)Mosquito iridescentMosquito iridescentPoliovirus murisMyxomaPseudorabiesVaccinia

Green KureAr1304G5287NaplesLBJ39579Arl113IP/A611Ar370TRVL11573MP847-32S2356298-38Argas27Iban17854TMA138121343EgAn4996Liev-71c56300-86IxH5aPR96406IIAC5581G700178FlorioSS18EMC75c(wild)RTGD7MosesAujeszkyLilly

NYMNYMPALPHLQRFQRFQYBQYBTACTURUUKUUKUUKUNGUNGUNGUNGUNGUNGUNGUNGUNGUNGUNGUNGUNGUNGn.a.n.a.n.a.n.a.n.a.n.a.n.a.n.a.

TTMMTTTT

MTTTTTTTTTTTTTTMTT

t

I!!

ml3vlm16m2m55-m7?+m4m7m3m6elm8mlOm3v6m6m5m4m12m9m6m7m8m9m9m8m7?+m2x+66mm7m4vlm2larlarm52vlmlr?r43ml?+vl

11111111211111111331111125522522222

aCE, California encephalitis; CETBE, Central European tick-borne encephalitis; CTF, Colorado tick fever;EEE, Eastern equine encephalomyelitis; EMC, encephalomyocarditis; HVH(II), Herpesvirus hominis (type II);WEE, Western equine encephalomyelitis.

b ANA, Anopheles A; BAK, Bakau; BLU, Bluetongue, BUN, Bunyamwera; CAL, California; CON, Congo;GAN, Ganjam; HUG, Hughes; KEM, Kemerovo; LJN, Lanjan; NYM, Nyamanini; PAL, Palyam, PHL,Phlebotomus; QRF, Quaranfil; QYB, Qalyub; TAC, Tacaribe; TUR, Turlock; UUK, Uukuniemi; UNG,ungrouped; n.a., not applicable.

Known or presumed to be: borne by mosquito or other biting diptera (M), tick borne (T), borne byundetermined vector (U), not vector borne (!).

dPassage level of stock in wet chick (c), chicken embryo cells (cc), embryonated chicken egg (e), groundmosquito larvae (lar), mouse brain (m), rabbit tissue (r), Vero (African green monkey kidney) cells (v), or ofcomplex, high passage (x) or unknown history (?).

eWell-defined CPE produced by this virus.

(Table 2). In addition, six viruses that are notarthropod borne, as well as two insect viruses,failed to induce plaques in A. albopictus cells(Table 2).

DISCUSSIONSince Suitor's original (20) demonstration of

virus plaquing in insect cells, only two pub-lished reports describing use of this phenome-

non have appeared (7, 10). We believe that therarity of these reports reflects the technicaldifficulties involved in preparing and maintain-ing satisfactory monolayers of insect cells.These cells, in comparison with many of verte-brate origin, grow more slowly and are lesstolerant of manipulative and qualitative varia-bles. Monolayers must be confluent and firmlyattached to the vessels. In our experience,

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FIG. 1. Plaque production in Singh's Aedes albopictus cells by some arboviruses of various serologicalgroups. (1) Uninfected culture with overlay, day 6; (2) Chikungunya virus, day 6; (3) Murutucu virus, day 6; (4)Bunyamwera virus, day 6; (5) Ilesha virus, day 6; (6) Chittoor virus, day 11; (7) Wyeomyia virus, day 6; (8)Tribee virus, day 6. Scale, 0.7 x.

inadequate confluence and poor attachment ofcells are most often attributable to qualitativedifferences among lots of fetal calf serum. Suchvariations can generally be overcome by pro-longed heat inactivation of the serum (1 h at 56C). In addition, at various times we have beenable to ascribe plaquing difficulties to advancedage of stock or assay cultures, excessive temper-atures (>42 C) of primary overlay, and toxicityof some lots of agarose. Incubation tempera-tures of infected cells may also affect the

development of plaques with certain agents.Many arboviruses, especially those of group B,plaqued best when overlaid cultures were incu-bated at 37 C (in fact, this temperature wasmandatory for plaquing dengue 4 virus), butBunyamwera group agents and Tribec virusyielded best plaques when cultures were incu-bated at 35 C. Also, as in vertebrate cells, highmultiplicities of virus have often preventedplaque formation in A. albopictus cells. Thus,in screening for a plaquing virus, care must be

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FIG. 2. Plaque production in Singh's Aedes albopictus cells by some group B arboviruses. (1) Uninfectedculture with overlay, day 7; (2) dengue 2 virus, day 8; (3) dengue 3 virus, day 8; (4) dengue 4 virus, day 8; (5)Ilheus virus, day 7; (6) Israel turkey meningoencephalitis virus, day 6; (7) Kunjin virus, day 7; (8) MurrayValley encephalitis virus, day 9. Scale, 0.7 x.

taken to ensure that some cultures are exposedto dilute inocula.Our results show that the plaquing technique

in A. albopictus cells is applicable to a variety ofarboviruses, nearly all of which are mosquitoborne. Among these mosquito-borne agents,plaquing ability in these cells appears to berelated to the serological group to which thevirus belongs. California group viruses and mostmembers of group A tested failed to plaque, but

nearly all B group and Bunyamwera groupviruses (the latter are related to those of theCalifornia group) readily did so. The possibilitythat such contrasting patterns may be used asclassifying characteristics is noted.Within serological group B the ability of a

virus to plaque correlates well with its vectorrelationship. Except for two, all mosquito-borneor suspected mosquito-borne group B agentsplaqued in the mosquito cells. The exceptions,

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Bussuquara and Zika, did, in fact, produce awell-defined CPE in the cell monolayer afteragar overlay, indicating that a plaquing poten-tial may exist. Conversely, no tick-borne groupB virus could be made to plaque despite re-peated attempts. Also failing to plaque weregroup B viruses (Modoc and two strains of RioBravo) for which arthropod transmission isquestioned on the basis of evidence supportingdirect transmission from infected to uninfectedhosts (1, 8, 11).These results parallel those from studies of

virus growth in fluid overlay cultures of Singh'sA. albopictus cell lines, wherein group B viruseswould propagate in the mosquito cells only ifmosquito borne (3-5, 16, 17, 21). Obviously,plaquing ability may be used to infer vectorrelationships or corroborate suspected ones forgroup B isolates having undetermined modes oftransmission. For example, Israel turkey menin-goencephalitis virus, as yet unassociated withany arthropod vector and experimentally onlypoorly transmissible by Aedes aegypti (13),behaves as do mosquito-borne group B virusesin A. albopictus agar overlay cultures. Simi-larly, the two West Nile virus strains isolated bySchmidt and Said (14) from field-collected ticks(Ar95-60 and ArlO8-60) are indistinguishable inthis plaquing system from a strain isolated fromthe classical vector, mosquitoes (Table 1). Thistends to support an alternative hypothesis pro-posed by these authors that "the actual sourceof the virus was blood from infected birds, andnot from the tissues of ticks which were main-taining the virus biologically."

Additional group B viruses whose vector asso-ciations (or lack thereof) are suggested in partby results of this study are Apoi and Negishi.The former, known only from rodents in Japan,failed to multiply in Culex tritaeniorhynchusmosquitoes that had fed on virus, and for thisreason its status as an arbovirus has beenquestioned (19). Negishi virus, recovered onlyfrom two persons dying of encephalitis andantigenically a member of the Russian spring-summer encephalitis complex of group B ar-boviruses, also lacks demonstrated vector asso-ciations. Failure of these two agents to plaque inA. albopictus cells provides in vitro evidencethat they are not mosquito borne.Of 53 tick-borne agents tested, only one,

Tribec, caused plaques in the Aedes cells. Thisvirus, along with Kemerovo, Lipovnik, andColorado tick fever viruses, are placed in the"relatively solvent-resistant arbovirus" taxo-nomic group recently designated as Orbiviruses(2). Unlike mosquito-borne arboviruses of

groups A and B, a number of tick-borne Or-biviruses, including the above-mentioned four,are capable of in vitro growth in arthropod cellsthat are unrelated to natural vectors of thearboviruses (4, 5, 22). For this reason we maderepeated attempts to plaque Colorado tick fe-ver, Kemerovo, and Lipovnik viruses, as well ascertain well-known arboviruses (Semliki Forest,Sindbis, and California encephalitis) found tomultiply in the A. albopictus cell line (3, 12, 16).Uniformly negative results may indicate thatvirus replication, in these instances, is limitedto a small proportion of the mosquito cells.A potential application of the A. albopictus

plaquing system is suggested by these results.The sensitivity and utility of Singh's A. albopic-tus cells for the isolation of various dengue virustypes in epidemic situations was demonstratedby Singh and Paul (18) and Chappell et al. (6).However, identification of the isolates byplaque-reduction neutralization test requirestheir passage into LLC-MK2 cells (6). We showthat the four major types of dengue viruses willproduce clear plaques in the mosquito cells.Thus, if wild strains of dengue viruses willplaque as readily as the strains tested here,these cells may be used as a single system forthe rapid isolation and identification of theseviruses in routine screening or large-scale opera-tions.We conclude that the plaquing technique

offers a means for direct assay of growth of anumber of arboviruses in mosquito cells, that itis especially applicable to the study of virusesfor which CPE in these cells has not beendemonstrated, and that, with regard to thewell-known (lipid-solvent susceptible) ar-boviruses as opposed to the Orbiviruses, itprovides a useful device for determination ofvirus-vector relationships or a potential aid toarbovirus classification.

ACKNOWLEDGMENTSWe thank Harry Meibos, Rocky Mountain Laboratory, for

capable technical assistance. Certain viruses tested in thisstudy were supplied through the courtesy of Jordi Casals andhis colleagues, Yale Arbovirus Research Unit, Yale UniversitySchool of Medicine, New Haven, Conn., for whose continuedcooperation we are grateful. John D. Paschke, Department ofEntomology, Purdue University, West Lafayette, Ind., andH. C. Chapman, U.S.D.A., Entomology Research Division,Lake Charles, La., kindly provided stocks of mosquito irides-cent virus. We are indebted to Hiro Hirumi, Boyce ThompsonInstitute for Plant Research, Yonkers, N.Y., for electronmicroscope examination of the cells used in this study.

ADDENDUM IN PROOFAdditional tests of Lipovnik virus have shown that

it is capable of producing plaques, inconsistently, inA. albopictus cells.

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LITERATURE CITED

1. Baer, G. M., and D. F. Woodall. 1966. Bat salivary glandvirus carrier state in a naturally infected Mexicanfreetail bat. Amer. J. Trop. Med. 15:769-771.

2. Borden, E. C., R. E. Shope, and F. A. Murphy. 1971.Physicochemical and morphological relationships ofsome arthropod-borne viruses to bluetongue virus-anew taxonomic group. Physicochemical and serologicalstudies. J. Gen. Virol. 13:261-271.

3. Buckley, S. M. 1969. Susceptibility of the Aedes albopic-tus and A. aegypti cell lines to infection with ar-

boviruses (33940). Proc. Soc. Exp. Biol. Med.131:625-630.

4. Buckley, S. M. 1971. In vitro propagation of tick-borneviruses other than group B arboviruses, p. 43-52. In MGresikova (ed.), Proceedings of the International Sym-posium on Tick-Borne Arboviruses (Excluding GroupB). Publishing House of the Slovak Academy ofSciences, Bratislava.

5. Buckley, S. M. 1972. Propagation of 3 relatively solvent-resistant arboviruses in Singh's Aedes albopictus andA. aegypti cell lines. J. Med. Entomol. 9:168-170.

6. Chappell, W. A., C. H. Calisher, R. F. Toole, K. C.Maness, D. R. Sasso, and B. E. Henderson. 1971.Comparison of three methods used to isolate denguevirus type 2. Appl. Microbiol. 22:1100-1103.

7. Cory, J., and C. E. Yunker. 1972. Arbovirus plaques inmosquito cell monolayers. Acta Virol. 16:90.

8. Davis, J. W., and J. L. Hardy. 1973. In vitro studies withModoc virus in Vero cells: plaque assay and kinetics ofgrowth, neutralization, and thermal inactivation.Appl. Microbiol. 26:344-348.

9. Early, E., P. H. Peralta, and K. M. Johnson. 1967. Aplaque neutralization method for arboviruses. Proc.Soc. Exp. Biol. Med. 125:741-747.

10. Hink, W. F., and P. V. Vail. 1973. A plaque assay fortitration of alfalfa looper nuclear polyhedrosis virus in a

cabbage looper (TN-368) cell line. J. Invert. Pathol.22:168-174.

11. Johnson, H. N. 1967. Ecological implications of antigeni-cally related mammalian viruses for which arthropodvectors are unknown and avian associated soft tickviruses. J. Med. Sci. Biol. 20:160-166.

12. Lyons, M. J., and J. Heyduk. 1973. Aspects of thedevelopmental morphology of California encephalitisvirus in cultured vertebrate and arthropod cells and inmouse brain. Virology 54:37-52.

13. Nir, Y. 1972. Some characteristics of Israel turkey virus.Arch. Gesamte Virusforsch. 36:105-114.

14. Schmidt, J. R., and M. I. Said. 1964. Isolation of WestNile virus from the African bird argasid, Argas reflexushermanni, in Egypt. J. Med. Entomol 1:83-86.

15. Singh, K. R. P. 1967. Cell cultures derived from larvae ofAedes albopictus (Skuse) and Aedes aegypti (L.). Curr.Sci. 36:506-508.

16. Singh, K. R. P., and S. D. Paul. 1968. Multiplication ofarboviruses in cell lines from Aedes albopictus andAedes aegypti. Curr. Sci. 37:65-67.

17. Singh, K. R. P., and S. D. Paul. 1968. Susceptibility ofAedes albopictus and Aedes aegypti cell lines toinfection by arbo and other viruses. Indian J. Med. Res.56:815-820.

18. Singh, K. R. P., and S. D. Paul. 1969. Isolation of dengueviruses in Aedes albopictus cell cultures. Bull. W.H.O.40:982-983.

19. Subcommittee on Information Exchange of the AmericanCommittee on Arthropod-Borne Viruses. 1971. Catalogof arthropod-borne and selected vertebrate viruses ofthe world. Amer. J. Trop. Med. (Suppl.) 20:1018-1050.

20. Suitor, E. C. 1969. Plaque formation by an arbovirus in a

mosquito cell line. J. Gen. Virol. 5:545-546.21. Yunker, C. E., and J. Cory. 1968. Growth of some group B

arboviruses in two established insect cell lines, p.

79-82. In Proc. 23rd Annual International NorthwestConference on Diseases in Nature Communicable toMan. Rocky Mountain Laboratory, Hamilton, Mont.

22. Yunker, C. E., and J. Cory. 1969. Colorado tick fevervirus: growth in a mosquito cell line. J. Virol.3:631-632.

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