ARBOVIRUS (English. arboviruses, from arthropod borne viruses the viruses transmitted by arthropods) — the ecological group of the viruses which are transmitted to vertebrata and the person by blood-sicking arthropod carriers in which organism they actively breed, but have no disease-producing effect. K A. conditionally carry group arenovirus (see) which transmissible transfer is not established.
History of studying And. began with establishment of the virus nature and transmissible transfer of yellow fever (see).
After this participation of mosquitoes in transfer of so-called three-day (mosquito) fever was revealed (see. Flebotomnaya fever ) and mosquitoes in spread of fever dengue (see) which virus etiology was confirmed much later. In the USSR the beginning of researches A. it was necessary discovery in 1937 of a virus of a tick-borne encephalitis (see viral tick-borne Encephalitises). Till 40th years And. were studied for establishment of an etiology of diseases which spread was connected with participation of carriers. Later researches of arthropod and vertebrate animals for the purpose of allocation of viruses and identification of the natural centers began to be conducted. Possibility of opening new And. is in direct dependence on efficiency of the used methods. After in 1933 for allocation of a virus St. Louis intracerebral infection of white mice was successfully used, by means of this method by 1950 it was open apprx. 40 A. The new wave of opening was connected with new methodical reception — use of newborn white mice. Thanks to it for the next 20 years more than 200 viruses were allocated. By July 1, 1972 302 A is registered. In the USSR by 1973 20 A is allocated and identified.
And. in group classify by the principle of an antigenic structure and as carriers (mosquito and tick-borne viruses). And. in universal classification of viruses are not uniform taxonomical group. Separate antigenic groups differ among themselves according to fundamental characteristics of virions and therefore hold different position. Antigenic classification And. it is constructed at association in uniform group of the viruses finding antigenic bonds in hemagglutination-inhibition reaction (RTGA) or RSK. Groups are designated by letters of the Latin alphabet (And, Century. C) or are called on the most studied virus (Bunyamvera, Bvamba, etc.).
By July 1, 1972 apprx. 80% And. were distributed on 41 antigenic groups (tab).
Number of viruses in each group from 2 to 40, are most numerous Bunyamver's supergroup, groups A and B. V of antigenic classification differs the certain hierarchy based on degree of an antigenic affinity: group, complex (subgroup), virus (serotype), options, strains. In group A, e.g., complexes are allocated: a) virus of the Venezuelan encephalomyelitis of horses, b) virus of east encephalomyelitis of horses, c) virus of the western encephalomyelitis of horses, d) Semliki's woods, e) Middelburg, e) Nduma. In group B distinguish complexes: a) virus of a tick-borne encephalitis, b) Japanese encephalitis, c) yellow fever. It is possible to give following as an example of further hierarchy of an antigenic structure: Semliki's complex consists of viruses of the wood of Semliki, Chikungunya (the O'Nyong-nyong subtype and several geographical options), Get (a subtype Sagiyam, Bebara, Ross River), Mayaro (a subtype Oona and two options).
The viruses having a similar antigenic structure have also other general characteristics: type nucleinic to - you, morphology, nucleotide structure and such physical parameters, as weight, the floating density, a sedimentation constant. These characteristics are among the main taxonomical signs (see. Viruses , classification). In process of accumulation of data separate groups A. or individual viruses from among nongrouped find the place in universal classification of viruses.
Antigenic groups A and B make respectively childbirth of Alpha virus (type-species of Alphavirus sindbis) and Flavovirus (type-species of Flavovirus febricis, i.e. a virus of yellow fever). They are a part of the Togaviridae family. Viruses of these groups represent spherical particles with a diameter on average of 45 nanometers for flavovirus and 60 nanometers for alpha viruses. Pier. weight of virions of alpha viruses 1 X 10 8 dalton, flavovirus — 46 X 10 6 dalton; floating density — 1,19 — 1,24 g/ml, a sedimentation constant — 208 — 215 S at flavovirus and 240 — 280 S at alpha viruses. Thus, alpha viruses differ from flavovirus in the big sizes and weight, higher sedimentation constant.
Virions consist of nucleoid or a nucleocapsid and the outside lipoprotein cover which is closely adjoining to it with fibers. Thickness of a cover is 2 — 7 nanometers, diameter of fibers apprx. 2 nanometers, length is 4,5 — 5 nanometers (flavovirusa) or 6,5 — 10 nanometers (alpha viruses). Diameter of nucleoid of alpha viruses 30 — 50 nanometers, flavovirus of 20 — 30 nanometers. The nucleocapsid consists of proteinaceous subunits of a ring-shaped form which number is definitely not determined. Presumable number of such subunits at alpha viruses 32 or 92 with dia. 7 nanometers.
The genome presents one-filamentous linear RNA in the form of a single whole of a piece about a pier. weighing 4 x 10 6 dalton. RNA infectious also makes 4 — 8% of weight of virion. Flavovirus as a part of virions have three structural proteins, for alpha viruses — two or three.
Pier. the weight of polypeptides of flavovirus makes: I \90009000 dalton, II (nukleoidny) — 13 500 — 16 000 dalton, III (glycoprotein) — 51 000 — 52 000 dalton. Pier. the weight of two polypeptides of alpha viruses is equal respectively 30 000 and 53 000 dalton. The heaviest protein (53 000 dalton) is localized in an outside cover and bears functions of hemagglutinin.
Alpha and flavovirusa breed in cytoplasm of cells. Maturing of virions happens on membrane structures: passing through membranes, nucleoid becomes covered by an outside cover and gemmates from it. Budding of alpha viruses happens hl. obr. through a marginal membrane, and flavovirusa generally form on internal membrane structures.
At replication in cells viruses of both groups induce formation of RNA-dependent of a RNA polymerase. In the course of reproduction of viruses several nonstructural proteins which number at alpha viruses from 3 to 15, and at flavovirus — 4 — 5 are formed.
Viruses are steady at pH = 3,0 thanks to presence of lipids as a part of virion, sensitive to effect of ether and a dezoksikholat of sodium.
Viruses of supergroup of Bunyamver make the sort Bunyamvirus with type-species of Bunyamvirus bunyamwera. Virions isometric, are covered with a cover, have the size 90 — 100 nanometers in the diameter. The external membrane consists of three layers, has the fibers bearing hemagglutinin. Thickness of a membrane is 15 — 20 nanometers, length of fibers is 5 — 6 nanometers.
The genome of a virus is presented to one-filamentous RNA, perhaps, consisting of 3 — 6 fragments. It is not possible to extract infectious RNA neither from virions, nor from the infected cells. The nucleocapsid has spiral type of symmetry and represents ribonukleoproteidny tyazh 2 — 2,5 nanometers thick, curtailed into a spiral 9 nanometers wide.
Floating density is 1,18 — 1,20 g/ml, a sedimentation constant 450 S. Breed in cytoplasm of the infected cells, ripen budding in bubbles of the device of Golgi or near it.
Because of presence of lipids as a part of virions viruses collapse under the influence of zhirorastvoritel (ether, chloroform) and a dezoksikholata of sodium.
Perhaps, on the basis of a community of morphology and the genetic characteristic antigenic groups Ukuniyemi, Tyurlok, Mapputta, Kaicuodi, Gandzham, KGL — Congo, viruses of fever Rift-Valli and a sheep disease of Nairobi will be a part of this sort.
The sort Orbivirus which is a part of the Reoviridae family (see. Viruses , classification), consists of antigenic groups of viruses of blue language of sheep, the African disease of horses, Korripart, Changinola, Kemerovo, Varry, an epizootic hemorrhagic disease of deer, Palyam, viruses of the Colorado fever and Lebombo.
Key parameters of this sort are given on the basis of studying of a virus of blue language of sheep which is type-species. Other antigenic groups of viruses are joint as a part of this sort on the basis of morphological criteria as viruses with identical morphology possess a similar genome and other fundamental molekulyarnobiologichesky properties.
Viruses have spherical shape, have no lipidic cover though are sometimes covered with a pseudomembrane of a cellular origin. Diameter of virions is 65 — 80 nanometers, weight 60 X 10 6 dalton, floating density is 1,36 — 1,38 g/ml, a sedimentation constant 550 S. The nucleocapsid is two-layer, total number of proteinaceous subunits of which it consists — 1600. The structure of an enveloping layer of a nucleocapsid is not established, the inner layer consists of 32 capsomeres. The genome is presented in the form by the two-filamentous linear RNA consisting of 10 — 12 fragments with the general a pier. weighing 12 X 10 6 dalton. RNA makes 20%, and protein of 80% of weight of virion. 7 proteins about a pier are a part of virion. weighing from 32 000 to 155 000 dalton. Virions are unstable at pH = 3,0, concerning a rezistentna to effect of ether and a dezoksikholat of sodium as they do not contain lipids.
Viruses of antigenic group of vesicular stomatitis formed a basis of creation of the sort Rhabdovirus (type-species — the Indiana virus). It is a sort, difficult on structure, where viruses of vertebrata, invertebrates and plants enter. Except mentioned, viruses Chandipur, Peary, bats Lagos, Klamat, Flanders, Mokola, Kern-Kanyon enter into this group (see. Rhabdoviruses ).
Viruses of group of vesicular stomatitis have the bullet form, are covered with an outside lipoprotein cover. The sizes — 175 X 68 nanometers, a pier. weight is equal to 1,2 — 4,0 X 10 8 dalton, floating density — 1,18 — 1,20 g/ml, a sedimentation constant — 625 S. The sizes of nucleoid — 155 X 49 nanometers. Nucleoid represents the cylinder with flat on the one hand and hemispherical on the other hand the ends. This cylinder is formed by turbinal ribonukleoproteidny thread, i.e. has spiral type of symmetry. Fibers of an external cover have length of 8 — 10 nanometers and diameter apprx. 3 nanometers.
The genome is presented by linear one-filamentous RNA in the form of whole thread about a pier. weighing 4 X 10 6 dalton. RNA makes 2%, proteins — 64%, lipids — 20% and carbohydrates — 13% of weight of virion. As a part of a virus there are 5 proteins: L-protein (pier. weight 190 000 dalton), apparently, is connected with a nucleocapsid; G-protein (75 000 dalton) represents a glycoprotein and is probably localized in fibers; N-protein, or nukleokapsidny protein (57 000 dalton), is connected with RNA and is major structural protein of a nucleocapsid; NS protein (45 000 dalton) — minor nukleokapsidny protein, perhaps, is a product of degradation of N-protein; M-protein (33 000 dalton) — membrane protein which ties a nucleocapsid to an outside cover. Each of proteins plays a part in serological tests. So, e.g., the antibodies specific to G-protein while for neutralization of a nucleocapsid (i.e. the virus deprived of an external cover) antibodies to N-and M-proteins are necessary participate in a neutralization test of an intact virus.
Virus of vesicular stomatitis and some other representatives have the hemagglutinating properties. Viruses of this group are stable at rn = 5,0 — 10,0, are sensitive to ether and a dezoksikholat of sodium.
Separate And. are interspersed in other taxonomical groups. So, Nodamur's virus belongs to the sort Enterovirus. Only DNA-containing A. the African fever of pigs is a part of the sort Iridovirus (see. Viruses , classification). The largest of an arbovirus — Kotia, has ospopodobny morphology.
Despite the distinctions noted above, And. have many similar biological properties, and for their studying use identical methodical receptions. And. pathogens for many wild and domestic animals, causing in them as clinically expressed, and an asymptomatic infection. From laboratory animals newborn white mice at their infection in a brain are the most susceptible. They are used both for allocation, and for browning of a virus in laboratory. Some And. pathogens also for adult white mice. Sensitivity, almost identical with mice, to And. it is noted at the Syrian hamsters. Incubation interval of a disease at newborn white mice on average for viruses of group A — 36 — 48 hours, groups B — 60 — 72 hours, Ukuniyemi's groups — 72 — 96, the Congo groups — 96 — 120 hour. Duration of an incubation interval at primary infection in the course of allocation of a virus is usually more long. The infection at newborn white mice proceeds as lethal encephalitis and in most cases is followed virusemia (see). At infection of monkeys the virusemia against the background of fever is one of constant signs. Majority And. breed in chicken embryos, the most effective is infection in a vitellicle. For cultivation And. use also fabric and cellular cultures including cultures of fabric from arthropods. Ability to breed in cultures of different types is individual for each virus, however are most sensitive to the majority And. the intertwined cultures of cells of the Syrian hamster (a strain of VNK-21) and a renal epithelium of green monkeys (a strain of VERO), a kidney of duck embryos, a kidney of an embryo of a pig, and from cultures of arthropods — the line of cells from mosquitoes of Aedes albopictus. In cellular cultures hron, the infection often develops. One of the general properties inherent in about 60% And., their ability to cause agglutination of erythrocytes of geese is. Source of receiving the hemagglutinating antigen is the brain, and for some And., napr, representatives of group C or a virus of the wood This faces — blood serum of newborn white mice at height of a disease. Identification of hemagglutinin requires the special processing directed to removal of the inhibitors blocking hemagglutinin. The method of sakharozo-acetonic extraction, alkaline extraction, processing is for this purpose applied by trichloro-trifluoroethane, acetone, the twin-80 and ether. At some viruses communication of hemagglutinins with inhibitors very strong and, except the specified methods, in addition apply to release from inhibitors still ultrasound and fermentation by trypsin. Hemagglutinins A. remain only in alkaline condition, but the phenomenon of hemagglutination is shown at the indicators of pH defined for each virus in an acid zone. Depending on existence of an external lipoprotein cover And. show different sensitivity to ether and a dezoksikholat of sodium. Viruses of groups A, B, Bunyamver's supergroups lose infectious properties under the influence of these substances whereas these properties of viruses of antigenic groups of Kemerovo, blue language of sheep, the African disease of horses, an epizootic hemorrhagic disease of deer, Korripart decrease slightly.
Majority And., except large rhabdoid, pass through milliporovy filters of 100 nanometers in size. As all And., except a virus of the African fever of pigs, RNA-containing are, their reproduction is not suppressed at addition on Wednesday of cultivation of inhibitors of synthesis of DNA. All listed above signs, namely: pathogenicity for mice suckers, the hemagglutinating properties, sensitivity to ether and a dezoksikholat of sodium, filterability via milliporovy filters and nonsensitivity to inhibitors of synthesis of DNA are indirect signs during the studying of new izolyant for their reference to And. Direct indicator is ability to breed and be transferred by arthropods in a natural cycle or in an experiment or establishment of antigenic bonds with already classified And.
For serological identification And. use generally hemagglutination-inhibition reaction (RTGA), the reaction of binding complement (RBC), a neutralization test which reveal both group-specific, and species-specific antibodies. Specificity of these reactions for different antigenic groups varies. So, in group B the broadest intra group bonds are revealed by RTGA, to a lesser extent RSK. The neutralization test accurately reveals specific antigens. It defines the sequence of use of different reactions in establishment at first of belonging to group, then to a complex and a serotype. For group identification use the polystandard serums received by immunization by several representatives of antigenic group. The most standard method is immunization of white mice suspension of a brain of the infected newborn white mice. In this case the accompanying antifabric antibodies are practically not formed that is observed at immunization by heterological virussoderzhashchy material. In recent years instead of serum use the astsitny liquid of mice induced by introduction of cells of sarcoma 180/TG. Thanks to lack of antifabric antibodies of advantage of the immune serums or astsitny liquids received from mice especially come to light in RSK, an immunofluorescence and precipitation.
About 80 And. are pathogenic for the person. Arboviral diseases (see) are described as large epidemic flashes and as sporadic cases. Pathogenicity of some And. it is established only at laboratory infections. The last circumstance allows to assume, as others And. are potentially pathogenic.
Ecological data speak about primacy of relationship of the most part of Ampere-second arthropod. And. can remain in various bodies and fabrics throughout all life of arthropods, without bringing at the same time, as a rule, harm. From arthropods more than 80% known are allocated And., from them 55% — from mosquitoes. Majority mosquito And. also Aedes (37%) is allocated from representatives of the sort Culex (47%). Then on number allocated And. there is childbirth of Anopheles, Mansonia, Sabethes, Psorophora, Haemagogus, Culiseta, Eretmapodites, Trichoprosopon, Wyeomyia, Limatus, Phomiomyia, Aedemyia. In development of epidemic flashes the special place belongs to a synanthropic type of Ayodes aegypti. In some cases And. are closely connected with one main carrier, in other cases a set of types of carriers is broad. A susceptibility of mosquitoes to And. is defined by a lower limit of a titre of a virus in blood of a vertebrate, at Krom 50% of carriers, and also number of the caught carriers capable to transmit a virus to a vertebrate through a sting catch. Quantitative assessment of these parameters is defined experimentally. Besides, 7% And. it is allocated from mokrets and 4% — from mosquitoes.
Ixodic and argasovy mites thanks to a long cycle a metamorphosis, abilities to transphase and transovarial transfer are not only carriers, but also a natural tank adapted to them And. From family of ixodic mites 13% are allocated And., from argasovy — 5% A. Rol of gamazovy and krasnotelkovy mites in circulation And. so far it is found out insufficiently. One of the major features And. their ability to a reproduction in arthropods at animal temperatures, rather low for viruses (an optimum is 27 °). But if temperature falls below threshold value (18 ° + 3 °), then the reproduction of a virus in a carrier stops. The size of an external incubation interval in a carrier, during to-rogo the virus which got with blood shall pass through a peritrophic membrane, be implemented into an epithelium of intestines, get through its wall into a perigastrium, reach sialadens and collect there in the quantities sufficient for infection of susceptible vertebrata at a krovososaniye, depends on the sum of effective temperatures above threshold level. If heating environments at least throughout the infecting phase a metamorphosis of a carrier do not reach necessary values, then in this area emergence of the centers is impossible And. All this first of all concerns to ecological group A., reflected by mosquitoes. At mosquitoes transovarial transfer of viruses therefore at the longevity of an imago of mosquitoes which is not exceeding in active season 1 — 1 x / 2 month the area is limited to an average monthly isotherm 18 — 20 ° for the hottest month is not established. It corresponds to a part of the temperate climatic zone, boundary with subtropics. In case of ecological group A., given by ixodic and argasovy mites, infection of a carrier can occur in a phase of a larva, the nymph and an imago. There is a transphase and transovarial transfer of a virus. The virus is capable to winter in various phases a metamorphosis of a carrier. All these circumstances define a possibility of expansion of an area of a virus to borders of an area of a carrier as throughout a long cycle a metamorphosis or several cycles the sum of effective temperatures, necessary for end of an external incubation interval, will be provided. Area of many And. it is limited to a season, in time to-rogo the main ways of circulation are interrupted. In moderate and subtropical belts it is the winter period of low temperatures. Conditions of experience And. during the winter period gradually improve in process of advance from a moderate belt to subtropics and tropics. Within a tropical belt heating environments for year-round circulation of viruses appear. Possibly, it approximately corresponds to an average monthly isotherm 16 ° for the coldest month. In process of further advance to subequatorial and equatorial belts these conditions become optimum. However in tropical and subequatorial belts circulation of a row A. can stop or be at a loss during a dry season. It results from a depression of population of main types of mosquitoes carriers. And only in the equatorial climate abiotic factors do not interfere with year-round active circulation And. Therefore number A. it is reduced in process of advance from the equator in the polar directions, and it occurs at the expense of mosquito And. In the conditions of dry zones of tropics-sub-tropics a significant role in preservation And. play argasovy mites. Hl are widespread in the temperate climatic zone and in the subarctic region. obr. And., given by ixodic mites. Vertebrata along with arthropods are the major link in a chain of circulation of A. Osobenno mass types on which the main carriers eat are important. Intensity of circulation in many respects is defined by titres of a virus in blood and duration of a virusemia (see). Value hron, and latent forms of an infection for preservation of virus population during the periods, adverse for active circulation, and for distribution is big And. on new territories, in particular, migratory birds. In circulation And. species of the vertebrata belonging to mammals, birds, amphibians and reptiles take part. The person as a tank matters only for some And. — causative agents of antroponozny arboviral infections (city form of yellow fever, Chikungunya, O'Nyong-nyong, Oropuge, dengue, mosquito fever). From wildings rodents and birds have the greatest value. From rodents it is allocated apprx. 20% And. Owing to a high susceptibility of mice suckers to And. these animals are most often used as «sentry» (indicator) for isolation And. by exhibiting in the places available to attack of carriers. With this method it is allocated 32 various And. Besides, for allocation And. use rabbits and the Syrian hamsters. Hares and wild rabbits play an important role in circulation of viruses of the California group. The preferential role of the South American rodents, especially the sorts Oryzomys and Proechimys in circulation is proved And. Bunyamver's supergroups, group of mosquito fever, Venezuelan encephalomyelitis of horses of group A. Rats of Calomys callosus are the main natural tank of a virus of Machupo — the causative agent of the Bolivian hemorrhagic fever, a Hesperomys laucha — the virus Junin causing the Argentina hemorrhagic fever. Are known And., allocated from rodents who, apparently, have no transmissible transfer and infection it is carried out in the alimentary or respiratory way (Modok, Apoi). For many And. on the African continent a tank are rats of the sort Arvicanthis. The role of rodents in circulation of viruses of a complex of a tick-borne encephalitis is big. 18 various And. it is allocated from bats, from them 9 are not isolated from other sources. The distant seasonal migrations made by nek-ry species of bats can promote transcontinental exchange of A. Nekotorye A., widespread in the equatorial and subequatorial climate, are ecologically connected with monkeys. Many viruses of group C in South America, a dzhunglevy form of yellow fever in Africa and South America, a kyasanursky forest disease in India and some other concern to them. The epidemic flash among people is often preceded by an epizooty among monkeys. The virus of an epizootic hemorrhagic disease of deer in North America is connected with wild hoofed animals. Domestic animals are an important link in development of an epizooty and epidemic flashes at the Japanese encephalitis (pigs, horses), east, western and Venezuelan encephalomyelitis (horse), Rift-Valli (sheep) and others. 53 A is isolated from birds and bird's ectoparasites., from which at least 15 cause in people and domestic animals arboviral infections. Birds in the evolutionary plan are one of the most ancient tanks A. Plotnost of populations of a number of types is very high that is one of conditions of development of an epizooty. Some bird species closely contact to an environment of the person that creates premises for development of an epizooty among domestic animals, and then and epidemic flashes. Annual migrations cause a regular intercontinental drift And., ecologically connected with birds. In this process value hron, and latent infections which in an experiment are often observed also at bats and cold-bloodeds, especially at the lowered temperatures of external environment is big. The virusemia in these cases has cyclic rhythm. Possibly, hron, the infection can serve in these cases to one of the leading mechanisms of a perezimovyvaniye of some And. in the conditions of temperate and subtropical climate. Reflected by mosquitoes
an arbovirus of groups A, B (vast majority), Anofeles And yes B is among, Bakau, Bunyamver's supergroup, vesicular stomatitis, Korriparta, Mapputta, Mossuril, Nyando, Palyam, Timbo, Tyurlok, Yubinangi. Mosquitoes transfer also not less than 12 nongrouped arbovirus from which Rift-Valli (East Africa) matters in pathology of the person and domestic animals. Mites transmit some viruses from group B: a complex of the tick-borne encephalitis including viruses of a tick-borne encephalitis, the Omsk hemorrhagic fever, the Scottish encephalomyelitis of sheep, a kyasanureky forest disease, Langat, Negisi, Povassan and a number of the viruses entering into other antigenic groups. Mokretsa are carriers of viruses of blue language of sheep, the African disease of horses and some other. Mosquitoes preferential participate in circulation of viruses of groups of mosquito fever and Changinola.
Bibliography: Arbovirus, the general and private questions, under the editorship of S. Ya. Gaydamovich, M., 1971; Zhdanov V. M. and the Guide and - m about in and the p C. Ya. Virusologiya, page 292, M., 1966; Laboratory diagnosis of viral and rickettsial diseases, under the editorship of E. Lennet and N. Schmidt, the lane with English, page 185, M., 1974; Levkovich E. H., Karpovich L. G. and 3 and with at x both N and G. D. Genetika and evolution of an arbovirus, 19 71, bibliogr.; Moshkina. Century and Lebedev A. D. An arbovirus of the world and their geographical distribution, in book: Medical geography, under the editorship of A. D. Lebedev, t. 6, page 9, M., 1974, bibliogr.; Semenov B. F. and L in about in D. K. Distribution and role in infectious pathology of the person of the arbovirus which is given by mites (excepting members of group B), Medical parazitol., t. 40, No. 3, page 259, 1971, bibliogr.; Catalogue of arthropod-borne viruses of the world, J. trop. Med. Hyg., v. 19, No. 6, Suppl., p. 1082, 1970; Catalogue of arthropod-borne and selected vertebrate viruses of the world, ibid., v. 20, jsft 6, pt 2, Suppl., p. 1018, 1970; Viral and rickettsial infections of man, ed. by F. L. Horsfall a. I. Tamm, Philadelphia, 1965.
S. Ya. Gaydamovich. D. K. Lvov.