Физико-химические свойства и антигенная структура вируса геморрагической болезни кроликов

7. Выводы.

1. Вирус геморрагической болезни кроликов представляет собой безоболо-чечный вирус диаметром 36 нм с плавучей плотностью вирионов 1,367 г/см3 в хлориде цезия, константой седиметации 162 Б, массой 13 Мда, не содержащий в своем составе липидов и углеводов.

2. Вирус геморрагической болезни кроликов устойчив к действию липидора-створителей, рН в диапазоне 3−9, замораживанию-оттаиванию, повышенным температурам: при 65 °C — сохраняет инфекционную и гемагглютини-рующую активность в течение 3 часов, при 37 °C — не менее 2 недель. При 20 °C — его гемагглютинирующая и инфекционная активность сохраняется неизменной в течение не менее, чем 4 недель, температурах ниже 0 °C — не менее, чем 6 месяцев.

3. Вирус чувствителен к действию известных дезинфектантов (щелочь, хлорамин) и новому дезинфектанту Теотропину, который может быть использован для дезинфекции продуктов кролиководства и помещений в присутствии животных.

4. Вирус геморрагической болезни кроликов при нейтральных, слабощелочных и слабокислых значениях рН агглютинирует эритроциты человека вне зависимости от группы крови, а также эритроциты барана, курицы, утки и, в меньшей степени — эритроциты КРС, свиньи и собаки. Гемагглютинирующая активность не связана непосредственно с инфекционной — образцы вируса, утратившие при хранении гемагглютинирующую активность, могут сохранить инфекционность.

5. Гемагглютинином вируса геморрагической болезни кроликов является капеидный белок УрбО. Гемагглютинин устойчив к прогреванию (утрачивает активность полностью только при инкубации в течение 3 часов при 65°С) и протеолитическим ферментам (активность не изменяется при инкубации с трипсином в течение 18 часов при его концентрациях до 4000 Ед./мл при 37 °C.).

6. Взаимодействие вируса ГБК с эритроцитами человека группы О характеризуется следующими параметрами:

• соотношение физических и гемагглютинирующих единиц вируса составляет около 4×108 вирионов на 1 ГАЕ (рН 6.8) — в один эритроцит может связать до 4×105 вирионов;

• сорбция вируса на эритроцитах эффективно происходит при рН 5.6−7.6- элюция вируса, сорбированного на эритроцитах происходит при рН>7.8- константа диссоциации комплекса вирус эритроцит при рН 7.2 составляет 3,29×10″ б±1,51×10−6,.

• вирус ГБК можно выявить в РГА если его концентрация в образце составляет не менее 400 млн вирионов на 1 мл;

• сорбция вируса на эритроцитах происходит за 1−2 минуты при перемешивании смеси вирус — эритроциты;

• элюция вируса с эритроцитов при рН выше 8 происходит за 4−8 минутв после элюирования сорбированного вируса с эритроцитов рецептор, расположенный на их поверхности, остается интактным и эритроциты могут адсорбировать на себе новые порции вируса.

7. Вирус геморрагической болезни кроликов обладает морфологическим полиморфизмом, выражающимся в формировании капсидов шести форм. в Первая — «стандартный» вирион — икосаэдрическая частица с палочкообразными выростами на поверхности.

• Вторая, отличающаяся при просмотре под электронным микроскопом от «стандартного» вириона наличием электронноплотного ободка, окружающего сердцевину, включает нуклеиновую кислоту в меньшем, чем стандартный вирион количестве. Третья — «пустая» частица — вирусный капсид не содержащий генетического материала. в Четвертая — «голая» частица, отличающаяся от стандартной отсутствием выростов на поверхности. в Пятая — «вирусный кор» — обособившийся в результате «разламывания» капсида — сердцевина «стандартного» вириона.

• Шестая — «малая форма» — фрагменты капсида — агрегат капсоме-ров — частица диаметром около 20 нм с характерными выростами на поверхности.

8. Морфологический полиморфизм вируса геморрагической болезни кроликов обусловливает наличие полиморфизма вирионов по плавучей плотности и константе седиментации, но не по физико-химическим свойствам поверхности вирионов, которые у основных форм вирионов не отличаются.

При фракционировании вируса при зонально-скоростном и изопикниче-ском центрифугировании он разделяется на две фракции, первая из которых представлена стандартными вирионами, вторая — пустыми капсидами. Эти фракции перекрываются растянутой зоной в которой локализуются вирионы, содержащие нуклеиновую кислоту в меньшем, чем полный размер генома, количестве.

9. В состав вирионов вируса геморрагической болезни кроликов входит 5 полипептидов с молекулярными массами 60, 54, 42, 37.5 и 16 кДа. Эти полипептиды взаимодействуют с антителами из сывороток реконвалесцентных кроликов, и антигенно не родственны друг другу — иммунизация их очищенными препаратами серонегативных кроликов вызывает у последних синтез антител, которые взаимодействуют с соотвествующим полипептидом и не взаимодействуют с остальными, причем Ур54, Ур37 и УрбО экспонированы на поверхности вирионов.

10. Вирус геморрагической болезни кроликов, проникает в клетки различных органов зараженных животных, репродуцируется в цитоплазме клеток печени, головного мозга, тимуса и легких. При репродукции образует пара-кристаллические и околомембранные цитоплазматические скопления. Выход вирусного потомства составляет до 14 000 вирионов на одну зараженную клетку. Специфический механизм экзоцитоза и специфическое цито-патогенное действие отсутствуют, причем основными изменениями в зараженных клетках являются гиперплазия аппарата Гольджи и митохондрий, а также пикноз клетки.

11. Заражение вирусом геморрагической болезни кроликов штамм Воронеж-87 взрослых серонегативных животных вызывает у них болезнь, характеризующуюся высокой — около 100% летальностью (гибель большинства зараженных животных наступает через 18−72 часа), основным патогенетическим механизмом которой является тромбогеморрагический синдром, приводящий к повышению фибринолитической активности сыворотки крови, что является причиной фибринолиза микрослоя фибрина на стенках кровеносных сосудов. Последний приводит к увеличению их проницаемости, выражающейся в образовании кровоизлияний различных типов в ряде органов и тканей. Непосредственной причиной смерти при острой форме болезни является остановка дыхания вследствие повреждения дыхательного центра, вызванного субдуральными кровоизлияниями в продолговатом мозге, которые представляют собой патогномонический признак данной формы ВГБК, регистрируемый у всех животных, погибших от острой формы болезни. Профилактическое или лечебное введение гепарина, предотвращающего тромбообразование в сосудах, смягчает течение вирусной геморрагической болезни и значительное — до трех-четырех раз — снижение смертности.

12. Установлено, что УрбО является протективным антигеном вируса геморрагической болезни кроликов. Иммунизация животных очищенным УрбО вызывает иммунологический ответ, характеризующийся образованием ви-руснейтрализующих антител и активацией антителонезависимых эффекторов иммунитета, которые играют основную роль в защите животных в поздние сроки после иммунизации.

Введение

животным экзогенных ви-русспецифических антител в малых количествах может вызывать эффект антителозависимого усиления инфекции.

13. С использованием приготовленной панели моноклональных антител к УрбО: в разработаны диагностические тест — системы для выявления вируса и противовирусных антител, характеризующиеся более высокой специфичностью и чувствительностью по сравнению с ранее применяемымипоказано, что антигенная структура данного бежа характеризуется наличием не менее, чем шести антигенных детерминант в составе трех антигенных регионовустановлена антигенная гомогенность изолятов вируса геморрагической болезни кроликов, циркулировавших в России в 1986 -1996 годах, и их антигенное тождество с эталонным штаммом Воронеж-87, используемым для приготовления вакцин против этой болезни.

8. Практические предложения.

В практике проведения НИР могут быть использованы разработанные или адаптированные в отношении вируса ГБК методические приемы: экстракция вируса из зараженных тканей с использованием трипсинового про-теолиза, очистка вируса, вирусных полипептидов.

В ходе работы охарактеризован по физико-химическим свойствам, ан-тигенности и ряду других параметров штамм В-87 вируса ГБК, принятый референс-штаммом данного вируса для России (авторское свидетельство на изобретение № 294 945 с приоритетом от 09.03.88).

Получен и охарактеризован клон гибридных клеток, продуцирующих моноклональные антитела к УрбО вируса ГБК (линия клеток депонирована и запатентована, патент № 2 007 452 с приоритетом от 16.01.92.).

На основе этих моноклональных антител разработаны и испытаны усовершенствованные диагностические наборы для выявления вирусных антигенов и противовирусных антител (составлены методические указания по диагностике вирусной геморрагической болезни кроликов иммуноферментным методом с использованием моноклональных антител, утвержденные 30.10.1992 г.), наборы производятся во ВНИИВВиМ.

Разработан новый химиотерапевтический препарат для профилактики и лечения бактериозов и бактериальных осложнений после вирусных заболеваний Абактан (комплект НТД рассмотрен и одобрен на Ветфармбиосовете ВГНКИ, проводится патентование) бактериозов и болезней, вызываемых РНК-содержащими вирусами Абактан Р (положительное решение о выдаче патента № 97 104 338/13 с приоритетом от 20.03.97, комплект НТД рассмотрен и одобрен на Ветфармбиосовете ВГНКИ), новый химиотерапевтический препарат для лечения бактериозов и болезней, вызываемых ДНК — содержащими вирусами, Абактан Д (положительное решение о выдаче патента на изобретение № 97 104 650/13 (4 921) от 19.01.1998 г. с приоритетом от 25.03.1997 г., комплект НТД рассмотрен и одобрен на Ветфармбиосовете ВГНКИ), новый дезинфектант для ветеринарии Теотропин (комплект НТД рассмотрен и одобрен на Ветфармбиосовете ВГНКИ, производится патентование), а так же методы их применении для ветеринарных целей. Все четыре препарата выпускают во ВНИИВВиМ.

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