key: cord-0772602-5mtd8ftz authors: McCutcheon, A. R.; Roberts, T. E.; Gibbons, E.; Ellis, S. M.; Babiuk, L. A.; Hancock, R.E.W.; Towers, G.H.N. title: Antiviral screening of British Columbian medicinal plants date: 1995-12-01 journal: Journal of Ethnopharmacology DOI: 10.1016/0378-8741(95)90037-3 sha: b5159e520976d43aab858a8c019a48bc9434e5b8 doc_id: 772602 cord_uid: 5mtd8ftz Abstract One hundred methanolic plant extracts were screened for antiviral activity against seven viruses. Twelve extracts were found to have antiviral activity at the non-cytotoxic concentrations tested. The extracts of Rosa nutkana and Amelanchier alnifolia, both members of the Rosaceae, were very active against an enteric coronavirus. A root extract of another member of the Rosaceae, Potentilla arguta, completely inhibited respiratory syncytial virus. A Sambucus racemosa branch tip extract was also very active against respiratory syncytial virus while the inner bark extract of Oplopanax horridus partially inhibited this virus. An extract of Ipomopsis aggregata demonstrated very good activity against parainfluenza virus type 3. A Lomatium dissectum root extract completely inhibited the cytopathic effects of rotavirus. In addition to these, extracts prepared from the following plants exhibited antiviral activity against herpesvirus type 1: Cardamine angulata, Conocephalum conicum, Lysichiton americanum, Polypodium glycyrrhiza and Verbascum thapsus. The search for selective antiviral agents, principally focused on an,&human immunodeficiency virus (HIV) agents, has been vigorous in recent * Corresponding author. years (De Clercq, 1988) but progress in the development of useful new antivirals has been painstakingly slow (Galasso, 1988) . Meanwhile, frequencies of viral resistance to the relatively few anti-viral drugs currently used are increasing (De Clercq, 1993) and the problem of viral latency, the greatest obstacle to treatment of some viral infec-0378-8741/95/%09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0378-8741(95) tions, remains unsolved. The increasingly urgent need to find effective therapeutics justifies not only an accelerated search for new agents but also a broader scope to such research. Ethnopharmacological screenings provide scientists with an alternative avenue to discovery from the current mainstream approach of attempting to design narrow spectrum drugs for specific molecular targets. The ethnopharmacological approach has equal potential for identifying new antiviral compounds, yet relatively few antiviral screenings of plant ethnomedicines have been conducted to date. 'In view of the significant proportion of plant extracts that have yielded positive results in these screenings, it seems reasonable to conclude that there are probably numerous types of antiviral compounds in these materials. Further characterization of the active ingredients of some of these plants should reveal some useful compounds' (Hudson, 1990) . It seems prudent, if not imperative, that researchers continue to investigate these sources before the knowledge or the plants themselves are lost. In this report, the results of an antiviral screening of 100 methanolic plant extracts against seven viruses, as well as background ethnobotanical and pharmacological activity data for those species which exhibited antiviral activity are presented. This research was conducted as part of an ongoing project investigating the pharmacological activities of medicinal plants used by the British Columbian native peoples (McCutcheon et al., 1993; McCutcheon et al., 1994) . A sampling of the British Columbian ethnobotanical literature (Turner, 1975 (Turner, , 1978 Turner et al., 1980 was surveyed to compile a representative list of those plants used medicinally by the native peoples of this area. The list was used in the field as a selection guide for the plant species and type of material to be collected. The collecting was carried out from May-July 1991, in five areas of the province: the Wyndel region in the Kootenay mountains, the Princeton-Penticton region in the interior, the Queen Charlotte Islands, the Fraser River canyon and the Lower Mainland. From the several hundred plant species on the ethnobotanical list, 100 specimens were collected. In order to ensure accurate botanical identilications, only plants which were in flower were collected, introducing a seasonal bias into the selection. A voucher specimen was made for each collection and these vouchers have been filed in the University of British Columbia Herbarium. The plant material was air dried and ground in a Wiley grinder with a 2 mm diameter mesh. Twenty grams of the ground material were extracted with (3 x 100 ml) methanol over 24 h. The crude methanolic extract was filtered through cheesecloth and cotton wool, and then through a Biichner funnel with a No. 4 paper filter. The filtrate was evaporated to dryness on a rotary evaporator and reconstituted with 10 ml of methanol. The effect of the methanolic plant extracts on the replication of seven selected viruses representing a spectrum of viral families was assayed. The viruses selected were: bovine coronavirus (BCV, Coronaviridae), bovine herpesvirus type 1 (BHVl, Herpesviridae), bovine parainfluenza virus type 3 (BP13, Paramyxoviridae), bovine rotavirus (BRV, Reoviridae), bovine respiratory syncytial virus (BRSV, Paramyxoviriaize), vaccinia virus (Poxviridae) and vesicular stomatitis virus (VSV, Rhabdoviridae). Viruses were propagated in established cell lines which were maintained in vitro as monolayer cultures using Eagle's minimal essential medium (MEM) supplemented with fetal bovine serum (10% v/v) and gentamicin (10 pg/ml). The cells were incubated at 37°C in a humidified environment containing 5% CO,. BCV, BHVl and VSV were grown in Madin-Darby bovine kidney (MDBK) cells; BRV and vaccinia virus in African green monkey kidney (MA104) cells; BRSV in Georgia bovine kidney (GBK) cells; and BP13 in African green monkey kidney (Vero) cells. The abilities of dilute plant extracts to inhibit virus-specific cytopathic effects were used as a measure of antiviral activities. Near-confluent 0.3 cm2 cell monolayers in 96-well plates (Flow Laboratories) were rinsed with serum-free MEM then each was treated with 0.2 ml of a plant extract diluted in serum-free MEM. The extracts were tested at dilutions raqging from 1 x 10-l through 1 x lo-'. Antiviral ( 10-3 to eliminate any microscopically observable toxic effects. Two samples (Ql and 42) demonstrated residual toxicity at that level, hence they were scored after application at a dilution of 2.5 x 10e5. After 12 h of treatment at 37"C, the medium was removed and the cultures were infected with stock preparations containing approximately 100 plaque-forming units (PFU) of the respective infectious virus in 0.1 ml of MEM. Mock-infected controls received sterile cell-culture medium. After a 1 h absorption perio'd, the inoculum was removed, the cells were washed twice with MEM then overlaid with 0.2 ml of fresh diluted plant extract. Plates were incubated at 37°C for 2-7 days, depending upon the virus-cell combination used. Cytopathic effects were scored after microscopic observation. Each treatment (+I-plant extract, +/-virus) was performed in triplicate and the entire regimen was repeated at least once for each extract tested. One hundred crude methanolic extracts of plants, 96 of which were used medicinally by British Columbian native peoples were screened for antiviral activity against seven viruses. The botanical names of the plants tested are listed alphabetically by genus in Table 1 . Twelve plant extracts each demonstrated some antiviral activity against one virus. Scores of the degrees of inhibition of virus-induced Icytopathic effects caused by treatment with these extracts are presented in Table 2 . Results for vaccinia virus and VSV are not shown as none of the plant extracts were observed to inhibit the cellular cytopathology induced by these virus.es at the extract dilutions used. One of the inherent drawbacks of in vitro antiviral testing is the environmental sensitivity of animal cells in culture. Preparations which exert antiviral effects in vivo may not be detected in in vitro assays because of the extremely low concentrations of extract tolerated by cells in the artificial system. Even with this limitation, 12 of the 100 methanolic plant extracts screened exhibited some antiviral activity. Six of these active extracts completely inhibited virus induced cytopathic effects at the non-cytotoxic concentrations tested. As has been found in previous antiviral screenings (see Hudson, 1990 for ovlerview), none of the extracts exhibited broad spect.rum activity. Each active extract was effective against only one of the seven viruses screened. Three of the most active extracts in this study were members of the same plant family. The branch extracts made from Rosa nutkana and Amelanchier alnifolia, both members of the Rosaceae, completely inhibited the cytopathic effects of an enteric coronavirus. A root extract of another member of the Rosaceae, Potentilla arguta, completely inhibited respiratory syncytial virus. Coronavirus and respiratory syncytial virus are similar in that they are both single-stranded RNA viruses which Infect mucosal surfaces. A branch tip extract of Sambucus racemosa (Caprifoliaceae) also completely inhibited the cytopathic effects of respiratory syncytial virus while an inner bark extract of Oplopanax horridus (Araliaceae) exhibited partial inhibition. A root extract of Ipomopsis aggregafa (Polemoniaceae) completely inhibited cytopathology induced by parainfluenza virus type 3, another single-stranded RNA virus which causes respiratory disease. None of the extracts was effective against the fourth single-stranded RNA virus used in the screening, vesicular stomatitis virus. Rotavirus is a double-stranded RNA virus that causes gastroenteritis, one of the major infectious diseases in the world today, as judged by mortality statistics (Vesikari, 1988) . The only extract which exhibited activity against this serious pathogen was a Lomatium dissecturn (Umbelliferae) root extract whidh completely inhibited the cytopathic effects. Two double-stranded DNA viruses were used in this screening, herpesvirus type 1 and vaccinia virus. Herpesviruses cause respiratory, genital, conjunctival or encephalitic infections which become latent in the trigeminal ganglion. There is also a growing body of evidence that Kaposi's sarcoma is caused by a newly discovered type of herpesvirus (Chang et al., 1994; Cohen, 1995; Chang, 1995) . Five of the plant extracts were found to partially inhibit the cytopathic effects of herpesvirus: Cardamine angulata (Cruciferae) roots, Conocephalum conicum (Conocephalaceae) thallus, Lysichiton americanum (Araceae) roots, Polypodium glycyrrhiza (Polypodiaceae) rhizomes and Verbascum thapsus (Scrophulaiiaceae) leaves. None of the extracts exhibited activity against vaccinia virus at the non-cytotoxic concentrations tested. Given the pressing need for new antiviral agents and the inherent limitations of in vitro antiviral testing for such agents, the results of this screening were promising. It is possible that the elucidation of the active constituents in these plants may provide useful leads in the development of antiviral therapeutics. It is interesting to note that 10 of these 12 active plant species were traditionally used to treat what are now known as viral ailments. Eight of the active plants were used to treat the specific diseases caused by the virus that they exhibited activity against. The traditional medicinal uses, reported chemical constituents and pharmacological activities of the 12 plants which exhibited antiviral activity in this study are summarized below. Decoctions of the aerial parts of A. alnifolia were traditionally used to treat respiratory ailments such as colds and coughs, as well as diarrhea, influenza and smallpox. Serviceberry decoctions were also used as a general tonic, reportedly good for any type of sickness (Turner, 1'980; 1990) . The cyanogenic glycoside, prunasin, is a constituent of A. alnifolia (Majak et al., 1978) . Majak et al. (1981) reported that the vegetative parts of this plant were potentially hazardous to ruminants. In spite of this toxic constituent, extracts of this plant were found to exhibit little antibacterial activity (McCutcheon et al., 1993) , no antifungal activity (McCutcheon et al., 1994) and no in vitro cytotoxicity at a dilution of 1 x 10m4. Work is in progress to isolate and identify the antiviral compound present in this plant. Caruizmiw angdata Hook. (Cruciferae) Common name: Bitter Cress. Root extract showed activity against bovine herpesvirus type 1. The Haida people used the roots of C. angulata mixed with 'water lily medicine' (Nuphar lufea ssp. polysepala) in a poultice for sores (Turner, personal communication) . In eastern North America, the Iroquois and the Cherokee peoples were reported to have also used Car&mine sp. to treat colds, sore throats, fever and heart problems (Moerman, 1986) . This plant extract only exhibited very slight antibacterial and antifungal activity in previous screenings, although N. lutea ssp. polysepala, the plant that it was compounded with, had very strong antimicrobial activity (McCutcheon et al., 1993; . No other reports of pharmacological activity nor reports on the chemical constituents of this plant were found in the literature. (L.) Dum. (Conocephalaceae) (Hepaticae) Common name: Liverwort. Thallus extract showed activity against bovine herpesvirus type 1. The Haida people of the Queen Charlotte Islands used this plant as a cold medicine (Turner, personal communication) . On Vancouver Island, the Coast Salish used it to treat cankers and sores (Turner and Bell, 1973) while the Dididaht employed it as an eye medicine (Turner in Pojar and MacKinnon, 1994) . This plant extract also exhibited some antifungal activity, particularly against Aspergillus fiigatus (McCutcheon et al., 1994) . The flavonoid, monoterpene, sesquiterpene, lipid and alkane chemistry of this species has been investigated quite extensively with over 100 chemical constituent reports in the literature. Zpomopsis aggregata (Pursh) Grant var. aggregata (Polemoniaceae) (Can&a aggregafa Pursh, Cilia aggregata (Pursh) Spreng.) Common names: Scarlet Gilia, Sky-rocket. Root extract demonstrated activity against bovine parainfluenza virus type 3. A root infusion of Ipomopsis aggregata was drunk for high fever by the Okanagan-Colville people. An infusion of the aerial parts of A. ag-gregata was used to 'clean out your system' and as a laxative (Turner, 1980) . Moerman (1986) cited reports of the Navaho, Paiute, and Shoshone also using Z. aggregata for colds, stomach disease, gonorrhea, syphilis, rheumatism, cathartic, emetic, physic and blood tonic. It was previously reported that methanolic extracts of this plant showed good antibacterial and antifungal activity (McCutcheon et al., 1993; . The compounds responsible for these activities have now been identified and tests are underway to determine if these compounds also have antiviral activity. Six tumor inhibiting compounds (three cucurbitacins, ipomopsin, hydroquinone and schottenol glucoside) have also been isolated from 1. aggregata (A.risawa et al., 1985) . As one of the common names of this plant implies, the major medicinal use of this plant was in the treatment of pulmonary complaints such as colds and coughs, p:neumonia, tuberculosis, lung hemorrhage and asthma. The root was also used in a poultice for sores, cuts, boils, bruises, sprains and broken bones. The powdered root was sprinkled on bums, boils, wounds and fresh sores to make them heal. The roots were also used in a steam-bath for rheumatism, sprains, pains of any sort and even pneumonia (Turner, 1980; 1990) . Moerman (1986) cited reports that the Blackfeet, Kawaiisu, Paiute, Shoshone and Washoe peoples also used this plant medicinally. Potential antiviral applications. included: colds, coughs, sore throat, pneumonia, bronchitis, smallpox and influenza. L. dissectum was previously reported to have antimicrobial activity (Cardellina and Vanwagenen, 1985; McCutcheon et al., 1993) . The active compounds were identified as a pair of unstable, homologous 2-alkenyl-3-hydroxy-penta-2,4-dien-4-olides (Vanwegene.n and Cardellina, 1986 ). It is not yet known whether these compounds are also responsible for the antiviral activity exhibited by this plant. The Haida used young roots from this plant to treat fever and skin disease (Turner, personal communication) . The southern Kwakiutl compounded the roots in a medicine for general weakness or undefined sickness (Turner and Bell, 1973) . The Bella Coola and Gitksan peoples used the root in the treatment of stomach troubles, influenza, rheumatism, blood poisoning and boils (Smith, 1928) . The Shuswap used a cold infusion of the roots for any sickness (Palmer, 1975) . Gunther (1973) reported that the Klallam, Kwakiutl, Makah, Quileute, Quinault, Skokomish and Swinomish peoples in neighbouring Washington state also used L. americanum roots for carbuncles, body parts sore with scrofula, for general weakness or undefined sickness, as a tonic, blood purifier and physic. Inner bark extract showed activity against respiratory syncytial virus. 0. horridus was a very important spiritual and physical medicine among the aboriginal peoples of western North America. The extensive traditional uses of Devil's Club were reviewed by Turner (1982) . The inner bark was used fresh, dried or ashed in a wide variety of preparations. For external applications, it was applied directly, as a salve or as a poultice for burns, swellings, sores, boils, swollen glands and sore areas. Poultices were also applied to wounds to prevent blood poisoning and to broken bones, toothaches and abrasions to relieve the pain and swelling. Devil's Club was used in steam-baths for arthritis, rheumatism, body pain, lameness, stomach trouble and as a skin tonic (Turner, 1982) . For internal use, the inner bark was either chew-ed and the juice swallowed or a decoction or infusion was drunk. One of the primary medicinal uses was in the treatment of respiratory ailments such as colds, coughs, chest pains, blood spitting and tuberculosis. Devils's Club was also used internally to treat a variety of diseases including: influenza, measles, diabetes, cancer, arthritis and rheumatism. It was often taken as a tonic or blood purifier for any general sickness and especially those with fever, weight loss and general debility. In higher doses, it was used as a purgative, emetic and cathartic (Turner, 1982 ). An inner bark infusion was also drunk for stomach troubles such as stomach pain, ulcers, gallstones, indigestion and constipation. It was commonly used to relieve internal pains caused by ailments as varied as broken bones to black eyes. Women drank a decoction to help expel the afterbirth, to start menstruation post partum and to treat uterine cancers (Turner, 1982) . See also Smith (1928) , Steedman (1929) , Perry (1952) , Gunther (1973) , Turner and Bell (1973) , Moerman (1986) , Gottesfeld and Anderson (1988) and Turner and Hebda (1990) . The methanolic extract of 0. horridus also exhibited good antibacterial and antifungal activity in previous screenings (McCutcheon et al., 1993; . The essential oil of the related species Oplopanax elatus has also been reported to have antifungal activity (Mi et al., 1987) . Chemical isolation work is in progress to identify the pharmacologically active constituents of this plant. Polypodium glycyrrhizu D.C. Eaton (Polypodiaceae) (P. vulgare L., P. fakatum Kellogg) Common names: Licorice Fern, Licorice Root. Rhizome extract exhibited activity against bovine herpesvirus type 1. This plant extract also exhibited antibacterial and antifungal activity (McCutcheon et al., 1993; . Girre et al. (1987) reported that P. vulgare had antiviral activity against a herpesvirus. They found that the active principles were catechin tannins. Husson et al. (1986) also reported that the ferns P. aureum and P. vulgare had antiviral properties. The Haida people made a poultice from the rhizomes to treat colds and coughs (Turner, per-sonal communication) . They were compounded in a decoction for stomach pain (Smith, 1928) . The rhizomes were chewed and the juice swallowed for sore throats (Turner, 1973) . The Thompson people also chewed the rhizomes or made an infusion from them to treat sore throats, colds and sore gums . The Oweekeno, Hanaksiala, Haisla and Kitasoo peoples also used the rhizomes for colds, coughs and sore throats (Compton, 1993) . The Kwakiutl people used the rhizome juice to stop vomiting blood and a decoction of the rhizome was taken for diarrhea (Turner and Bell, 1973) . The Cowlitz took an infusion of the crushed stems for measles (Gunther, 1973) . Gunther (1973) also reported that the Green River, Khallam, Makah and Quinalt peoples used the rhizome as a cough medicine. et al., 1993; 1994) . Polyphenols from the related species P. erecta were reported to exhibit antimicrobial, antiinflammatory and capillary resistance effects (Selenina et al., 1973) . The Okanagan-Colville people steeped the roots to make an infusion drunk by women after childbirth (Turner, 1980) . The Chippewa used the root to stop the bleeding after bloodletting and to treat convulsions, people who seemed to have too much blood (headache?), cuts and dysentery (Densmore, 1928) . Other medicinal uses of Potentilla sp. were fever, sore throat, diarrhea, inflammation and as a tonic (Moerman, 1986 (Smith, 1928) . The Thompson people drank a rosehip infusion for sore or itchy throat, cough and especially coughs in babies. For diarrhea, vomiting, or women's illnesses, the branches were compounded with Prunus virginiana and Cornus sericea. The roots of Rosa species were boiled to make a decoction taken for syphilis and by women after childbirth (Turner, 1!)90). A bark decoction was used as an eyewash for cataracts or to enhance eyesight (Turner in Pojar and MacKinnon, 1994) . The Skagit people used a root decoction for sore throats and as an eye wash. Decoctions were also administered to ease labor pains and used as a wash to strengthen babies. The Quileuete and Quinault peoples used the ashes in a poultice for swellings and syphilitic sores (Gunther, 1973) . The Okanagan-Colville people chewed the leaves and then applied them directly to bee stings to reduce swelling and pain (Turner, 1980) . This plant extract exhibited some antibacterial activity, particularly against Escherichia coli but showed only slight antifungal activity (McCutcheon et al., 1993; . Work is now in progress to isolate and identify the antiviral constituents. The most common medicinal use of the root or bark of this shrub was as an emetic or purgative (Moerman, 1986; Turner, 1973 Turner, , 1980 Turner and Bell, 1973) . The Haida, Saanich and Cowichan Coast Salish peoples used the bark in a female medicine (Turner and Hebda, 1990; Turner, personal communication) . Among the Gitksan, weakness, general illness and inability to eat were presenting symptoms indicating the use of a S. racemosa root bark decoction (Gottesfeld and Anderson, 1988) . Gottesfeld and Anderson (1988) reported that patients were treated with this decoction during the deadly 1918 influenza epidemic. The Thompson people compounded the roots to make an infusion drunk for liver disease . Perry (1952) reported that the Thompson and Okanagan peoples used this plant for rheumatism, erysipelas and toothache. Other traditional uses cited in Moerman (1986) included: measles, poultice to reduce swellings, footbath for aching legs and feet, lotion on open cuts or sores, wash on area infected with blood poisoning, applied to breast after childbirth to start milk flow, steam-bath to relax a woman's body after childbirth and as a physic. This plant extract exhibited very slight antibacterial and antifungal activity in previous screenings (McCutcheon et al., 1993; . Chemical isolation and identification of the antiviral constituents is in progress. Verbuscum tAapsus L. (Scrophulariaceae) Common names: Common Mullein, Flannel Plant. Leaf extract demonstrated activity against bovine herpesvirus type 1. This extract showed only slight antibacterial and antifungal activity (McCutcheon et al., 1993; . Mullein was used extensively as a medicinal by aboriginal peoples throughout North America. Moerman (1986) contains sixty-nine citations for medicinal uses of V. thapsus, compiled from the ethnobotanical literature on eighteen different tribes. One of the most common uses cited was in the treatment of pulmonary complaints such as cold, cough, consumption and asthma. Other reported uses which imply possible antiviral activity include mumps, swollen glands, croup and fever. Among the British Columbian peoples, the Thompson drank a leaf tea for colds and coughs. Leaf juice was rubbed on warts to remove them . The Okanagan-Colville people drank a deco&ion of the leaves for consumption (Turner, 1980) . Plant anticancer agents VI. Schottenoi Glucoside from Baccharis cordifolia and Ipomopsis aggregata Antifungal agents from Lomatium dissectum. Abstracts of the International Research Congress on Natural Products Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma AIDS mood upbeat -for a change Upper North Wakashan and Southern Tsirnshian Ethnobotany Recent advances in the search for selective antiviral agents Antiviral agents: characteristic activity spectrum depending on the molecular target with which they interact Uses of plants by the Chippewa Indians Promises to keep: clinical use of antiviral drugs On the antiherpetic activity of extracts from marine and land plants and the standardization of the study of antiviral properties Ethnobotany of Western Washington Antiviral Compounds from Plants Research into antiviral properties of a few natural extracts The cyanide potential of Saskatoon Serviceberry Amelanchier alnifolia and Chokecherry Prunus virginiana Prunasin, the cyanogenic glycoside in Amelanchier alnifolia Antibiotic screening of medicinal plants of the British Columbian native peoples Antifungal screening of medicinal plants of the British Columbian native peoples Studies on chemical constituents and antifungal activites of essential oil from Oplopanax elatus Medicinal Plants of Native America. Research Reports in Ethnobotany, Contribution 2. University of Michigan Museum of Anthropology Shuswap Indian Ethnobotany. Dept. of Anthropology Ethnobotany of the Indians of the interior of British Columbia Plants of Coastal British Columbia Polyphenols of Potentilla erecta and their biological activity Materia medica of the Bella Coola and neighboring tribes of British Columbia The ethnobotany of the Thompson Indians of British Columbia The ethnobotany of the Bella Coola Indians of British Columbia Food Plants of the British Columbian Indians Coastal Peoples. British Columbia Provincial Museum Handbook No. 34. Royal British Columbian Provincial Museum Food Plants of the British Cohunbian Indians: Interior Peoples. British Columbia Provincial Museum Handbook No. 36. Royal British Columbian Provincial Museum Traditional use of Devil's Club (Oplopanax horridus, Araliaceae) by native peoples in western North America Ethnobotany of the Southern Kwakiutl Indians of British Columbia Contemporary use of bark for medicine by two Salishan native elders of southeast Vancouver Island Ethnobotany of the Okanagan-Colville Indians of British Columbia and Washington. British Columbia Provincial Museum No. 21 Thompson Ethnobotany: Knowledge and Uses of Plants by the Thompson Indians. British Columbia Provincial Museum Memoir No. 25. British Columbia Provincial Museum Native American food and medicinal plants 7. Antimicrobial tetronic acids Rotavirus: new vaccine and vaccination Our thanks to Dr. N.J. Turner for lending her expertise and encouragement throughout this project. Financial support for this research was provided by the Canadian Bacterial Diseases Network and by the Natural Sciences and Engineering Research Council of Canada.