Физико-химические свойства и антигенная структура вируса геморрагической болезни кроликов тема диссертации и автореферата по ВАК РФ 03.00.06, доктор биологических наук Власов, Николай Анатольевич

• Власов, Николай Анатольевич
• доктор биологических наук
• 1998

Оглавление диссертации доктор биологических наук Власов, Николай Анатольевич

3. Введение ю

3.1. Актуальность темы

3.2. Цель и задачи исследования

3.3. Научная новизна работы

3.4. Практическая значимость работы

3.5. Основные положения, выносимые на защиту

3.6. Апробация результатов

3.7. Публикация результатов исследований

3.8. Внедрение результатов исследований

3.9. Благодарности

4. Обзор литературы

4.1. Вирусная геморрагическая болезнь кроликов и ее этиологический агент

4.1,1. История выявления ВГБК, начальная стадия изучения болезни и вируса 20 *т. i.^i л Kjpui/vmnu средств диагнсстики и профилактики болезни

4.1.3. Адаптация вируса к культурам клеток

4.1.4. Разработка и испытания вакцин

4.1.5. Изучение антигенности вируса и разработка диагностических реакций

4.2. Распространение вирусной геморрагической болезни кроликов в настоящее время

4.2.1. Пути передачи вируса

4.2.2. Возможность формирования стационарных эпизоотических очагов в природе за счет проникновения вируса ГБК в популяции зайцев и кроликов

4.3. Свойства вируса ГБК

4.3.1. Структура вириона

4.3.2. Данные ультраструктурного анализа по вопросам изучения цитопатического действия вируса и поиска клеток-мишеней для вирусной репродукции in vitro

4.3.3. Физико-химические характеристики вируса

4.3.4. Нуклеиновая кислота

4.3.5. Вирусные белки

4.3.6. Таксономия вируса

4.4. Характеристика болезни, вызываемой вирусом ГБК

4.4.1. Клинические признаки болезни

4.4.2. Патологоанатомическая картина

4.4.3. Цитопатоморфологические изменения

4.4.4. Сведения о механизмах патогенеза

4.5. Краткий сравнительный обзор молекулярной биологии мелких безоболочечных вирусов

4.5.1. Рагуоутйае (СЪогйорагуоутпае)

4.5.2. Ркогпаут4ае

4.5.3. СаИстпйае

4.5.4. А$ШуМ(1ае

4.5.5. агсоут(1ае

4.5.6. Неклассифицированные мелкие безоболочечные вирусы

Заключение диссертации по теме «Вирусология», Власов, Николай Анатольевич

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°С - сохраняет инфекционную и гемагглютини-рующую активность в течение 3 часов, при 37°С - не менее 2 недель. При 20°С - его гемагглютинирующая и инфекционная активность сохраняется неизменной в течение не менее, чем 4 недель, температурах ниже 0°С - 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°С.).

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

• соотношение физических и гемагглютинирующих единиц вируса составляет около 4x108 вирионов на 1 ГАЕ (рН 6.8); в один эритроцит может связать до 4x105 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 3,29х10"б±1,51х10-6,

• 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 минут; в 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 14000 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 применяемыми; показано, что антигенная структура данного бежа характеризуется наличием не менее, чем шести антигенных детерминант в составе трех антигенных регионов; установлена 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 № 294945 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 № 2007452 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 № 97104338/13 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 № 97104650/13 (004921) 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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