����JFIFXX�����    $.' ",#(7),01444'9=82<.342  2!!22222222222222222222222222222222222222222222222222����"��4�� ���,�PG"Z_�4�˷����kjز�Z�,F+��_z�,�© �����zh6�٨�ic�fu���#ډb���_�N�?��wQ���5-�~�I���8����TK<5o�Iv-�����k�_U_�����~b�M��d����Ӝ�U�Hh��?]��E�w��Q���k�{��_}qFW7HTՑ��Y��F�?_�'ϔ��_�Ջt��=||I ��6�έ"�����D���/[�k�9���Y�8ds|\���Ҿp6�Ҵ���]��.����6�z<�v��@]�i%��$j��~�g��J>��no����pM[me�i$[����s�o�ᘨ�˸ nɜG-�ĨU�ycP�3.DB�li�;��hj���x7Z^�N�h������N3u{�:j�x�힞��#M&��jL P@_���� P��&��o8������9�����@Sz6�t7#O�ߋ �s}Yf�T���lmr����Z)'N��k�۞p����w\�Tȯ?�8`�O��i{wﭹW�[�r�� ��Q4F�׊���3m&L�=��h3����z~��#�\�l :�F,j@�� ʱ�wQT����8�"kJO���6�֚l����}���R�>ډK���]��y����&����p�}b��;N�1�m�r$�|��7�>e�@B�TM*-iH��g�D�)� E�m�|�ؘbҗ�a��Ҿ����t4���o���G��*oCN�rP���Q��@z,|?W[0�����:�n,jWiE��W��$~/�hp\��?��{(�0���+�Y8rΟ�+����>S-S����VN;�}�s?.����� w�9��˟<���Mq4�Wv'��{)0�1mB��V����W[�����8�/<� �%���wT^�5���b��)iM� pg�N�&ݝ��VO~�q���u���9� ����!��J27����$O-���! �:�%H��� ـ����y�ΠM=t{!S�� oK8������t<����è:a������[�����ա�H���~��w��Qz`�po�^ ����Q��n� �,uu�C�$ ^���,������8�#��:�6��e�|~���!�3�3.�\0��q��o�4`.|� ����y�Q�`~;�d�ׯ,��O�Zw�������`73�v�܋�<���Ȏ�� ـ4k��5�K�a�u�=9Yd��$>x�A�&�� j0� ���vF��� Y�|�y��� ~�6�@c��1vOp�Ig����4��l�OD���L����� R���c���j�_�uX6��3?nk��Wy�f;^*B� ��@�~a�`��Eu������+���6�L��.ü>��}y���}_�O�6�͐�:�YrG�X��kG�����l^w���~㒶sy��Iu�!� W ��X��N�7BV��O��!X�2����wvG�R�f�T#�����t�/?���%8�^�W�aT��G�cL�M���I��(J����1~�8�?aT ���]����AS�E��(��*E}� 2��#I/�׍qz��^t�̔���b�Yz4x���t�){ OH��+(E��A&�N�������XT��o��"�XC��'���)}�J�z�p� ��~5�}�^����+�6����w��c��Q�|Lp�d�H��}�(�.|����k��c4^�"�����Z?ȕ ��a<�L�!039C� �Eu�C�F�Ew�ç ;�n?�*o���B�8�bʝ���'#Rqf���M}7����]����s2tcS{�\icTx;�\��7K���P���ʇ Z O-��~��c>"��?�������P��E��O�8��@�8��G��Q�g�a�Վ���󁶠�䧘��_%#r�>�1�z�a��eb��qcPѵ��n���#L��� =��׀t� L�7�`��V���A{�C:�g���e@�w1 Xp3�c3�ġ����p��M"'-�@n4���fG��B3�DJ�8[Jo�ߐ���gK)ƛ��$���� ���8�3�����+���� �����6�ʻ���� ���S�kI�*KZlT _`���?��K����QK�d����B`�s}�>���`��*�>��,*@J�d�oF*����弝��O}�k��s��]��y�ߘ��c1G�V���<=�7��7����6�q�PT��tXԀ�!9*4�4Tހ3XΛex�46���Y��D ����� �BdemDa����\�_l,��G�/���֌7���Y�](�xTt^%�GE�����4�}bT���ڹ�����;Y)���B�Q��u��>J/J �⮶.�XԄ��j�ݳ�+E��d ��r�5�_D�1 ��o�� �B�x�΢�#���<��W�����8���R6�@g�M�.��� dr�D��>(otU��@x=��~v���2� ӣ�d�oBd��3�eO�6�㣷�����ݜ6��6Y��Qz`��S��{���\P�~z m5{J/L��1������<�e�ͅPu�b�]�ϔ���'������f�b� Zpw��c`"��i���BD@:)ִ�:�]��hv�E�w���T�l��P���"Ju�}��وV J��G6��. J/�Qgl߭�e�����@�z�Zev2u�)]կ�����7x���s�M�-<ɯ�c��r�v�����@��$�ޮ}lk���a���'����>x��O\�ZFu>�����ck#��&:��`�$�ai�>2Δ����l���oF[h��lE�ܺ�Πk:)���`�� $[6�����9�����kOw�\|���8}������ބ:��񶐕��I�A1/�=�2[�,�!��.}gN#�u����b��� ~��݊��}34q����d�E��Lc��$��"�[q�U�硬g^��%B �z���r�pJ�ru%v\h1Y�ne`ǥ:g���pQM~�^�Xi� ��`S�:V29.�P���V�?B�k�� AEvw%�_�9C�Q����wKekPؠ�\�;Io d�{ ߞo�c1eP����\� `����E=���@K<�Y���eڼ�J���w����{av�F�'�M�@/J��+9p���|]�����Iw &`��8���&M�hg��[�{��Xj��%��Ӓ�$��(����ʹN���<>�I���RY���K2�NPlL�ɀ)��&e����B+ь����( � �JTx���_?EZ� }@ 6�U���뙢ط�z��dWI�n` D����噥�[��uV��"�G&Ú����2g�}&m��?ċ�"����Om#��������� ��{�ON��"S�X��Ne��ysQ���@Fn��Vg���dX�~nj�]J�<�K]:��FW��b�������62�=��5f����JKw��bf�X�55��~J �%^����:�-�QIE��P��v�nZum� z � ~ə ���� ���ة����;�f��\v���g�8�1��f24;�V���ǔ�)����9���1\��c��v�/'Ƞ�w�������$�4�R-��t���� e�6�/�ġ �̕Ecy�J���u�B���<�W�ַ~�w[B1L۲�-JS΂�{���΃������A��20�c#��@ 0!1@AP"#2Q`$3V�%45a6�FRUq��� ����^7ׅ,$n�������+��F�`��2X'��0vM��p�L=������5��8������u�p~���.�`r�����\���O��,ư�0oS ��_�M�����l���4�kv\JSd���x���SW�<��Ae�IX����������$I���w�:S���y���›R��9�Q[���,�5�;�@]�%���u�@ *ro�lbI �� ��+���%m:�͇ZV�����u�̉����θau<�fc�.����{�4Ա� �Q����*�Sm��8\ujqs]{kN���)qO�y�_*dJ�b�7���yQqI&9�ԌK!�M}�R�;������S�T���1���i[U�ɵz�]��U)V�S6���3$K{�ߊ<�(� E]Զ[ǼENg�����'�\?#)Dkf��J���o��v���'�%ƞ�&K�u�!��b�35LX�Ϸ��63$K�a�;�9>,R��W��3�3� d�JeTYE.Mϧ��-�o�j3+y��y^�c�������VO�9NV\nd�1 ��!͕_)a�v;����թ�M�lWR1��)El��P;��yوÏ�u 3�k�5Pr6<�⒲l�!˞*��u־�n�!�l:����UNW ��%��Chx8vL'��X�@��*��)���̮��ˍ��� ���D-M�+J�U�kvK����+�x8��cY������?�Ԡ��~3mo��|�u@[XeY�C�\Kp�x8�oC�C�&����N�~3-H���� ��MX�s�u<`���~"WL��$8ξ��3���a�)|:@�m�\���^�`�@ҷ)�5p+��6���p�%i)P M���ngc�����#0Aruz���RL+xSS?���ʮ}()#�t��mˇ!��0}}y����<�e� �-ή�Ԩ��X������ MF���ԙ~l L.3���}�V뽺�v�����멬��Nl�)�2����^�Iq��a��M��qG��T�����c3#������3U�Ǎ���}��לS�|qa��ڃ�+���-��2�f����/��bz��ڐ�� �ݼ[2�ç����k�X�2�* �Z�d���J�G����M*9W���s{��w���T��x��y,�in�O�v��]���n����P�$�JB@=4�OTI�n��e�22a\����q�d���%�$��(���:���: /*�K[PR�fr\nڙdN���F�n�$�4�[�� U�zƶ����� �mʋ���,�ao�u 3�z� �x��Kn����\[��VFmbE;�_U��&V�Gg�]L�۪&#n%�$ɯ�dG���D�TI=�%+AB�Ru#��b4�1�»x�cs�YzڙJG��f��Il��d�eF'T� iA��T���uC�$����Y��H?����[!G`}���ͪ� �纤Hv\������j�Ex�K���!���OiƸ�Yj�+u-<���'q����uN�*�r\��+�]���<�wOZ.fp�ێ��,-*)V?j-kÊ#�`�r��dV����(�ݽBk�����G�ƛk�QmUڗe��Z���f}|����8�8��a���i��3'J�����~G_�^���d�8w������ R�`(�~�.��u���l�s+g�bv���W���lGc}��u���afE~1�Ue������Z�0�8�=e�� f@/�jqEKQQ�J��oN��J���W5~M>$6�Lt�;$ʳ{���^��6�{����v6���ķܰg�V�cnn �~z�x�«�,2�u�?cE+Ș�H؎�%�Za�)���X>uW�Tz�Nyo����s���FQƤ��$��*�&�LLXL)�1�" L��eO��ɟ�9=���:t��Z���c��Ž���Y?�ӭV�wv�~,Y��r�ۗ�|�y��GaF�����C�����.�+� ���v1���fήJ�����]�S��T��B��n5sW}y�$��~z�'�c ��8 ��� ,! �p��VN�S��N�N�q��y8z˱�A��4��*��'������2n<�s���^ǧ˭P�Jޮɏ�U�G�L�J�*#��<�V��t7�8����TĜ>��i}K%,���)[��z�21z ?�N�i�n1?T�I�R#��m-�����������������1����lA�`��fT5+��ܐ�c�q՝��ʐ��,���3�f2U�եmab��#ŠdQ�y>\��)�SLY����w#��.���ʑ�f��� ,"+�w�~�N�'�c�O�3F�������N<���)j��&��,-� �љ���֊�_�zS���TǦ����w�>��?�������n��U仆�V���e�����0���$�C�d���rP �m�׈e�Xm�Vu� �L��.�bֹ��� �[Դaզ���*��\y�8�Է:�Ez\�0�Kq�C b��̘��cө���Q��=0Y��s�N��S.���3.���O�o:���#���v7�[#߫ ��5�܎�L���Er4���9n��COWlG�^��0k�%<���ZB���aB_���������'=��{i�v�l�$�uC���mƎҝ{�c㱼�y]���W�i ��ߧc��m�H� m�"�"�����;Y�ߝ�Z�Ǔ�����:S#��|}�y�,/k�Ld� TA�(�AI$+I3��;Y*���Z��}|��ӧO��d�v��..#:n��f>�>���ȶI�TX��� 8��y����"d�R�|�)0���=���n4��6ⲑ�+��r<�O�܂~zh�z����7ܓ�HH�Ga롏���nCo�>������a ���~]���R���̲c?�6(�q�;5%� |�uj�~z8R=X��I�V=�|{v�Gj\gc��q����z�؋%M�ߍ����1y��#��@f^���^�>N�����#x#۹��6�Y~�?�dfPO��{��P�4��V��u1E1J �*|���%���JN��`eWu�zk M6���q t[�� ��g�G���v��WIG��u_ft����5�j�"�Y�:T��ɐ���*�;� e5���4����q$C��2d�}���� _S�L#m�Yp��O�.�C�;��c����Hi#֩%+) �Ӎ��ƲV���SYź��g |���tj��3�8���r|���V��1#;.SQ�A[���S������#���`n�+���$��$I �P\[�@�s��(�ED�z���P��])8�G#��0B��[ى��X�II�q<��9�~[Z멜�Z�⊔IWU&A>�P~�#��dp<�?����7���c��'~���5 ��+$���lx@�M�dm��n<=e�dyX��?{�|Aef ,|n3�<~z�ƃ�uۧ�����P��Y,�ӥQ�*g�#먙R�\���;T��i,��[9Qi歉����c>]9�� ��"�c��P�� �Md?٥��If�ت�u��k��/����F��9�c*9��Ǎ:�ØF���z�n*�@|I�ށ9����N3{'��[�'ͬ�Ҳ4��#}��!�V� Fu��,�,mTIk���v C�7v���B�6k�T9��1�*l� '~��ƞF��lU��'�M ����][ΩũJ_�{�i�I�n��$���L�� j��O�dx�����kza۪��#�E��Cl����x˘�o�����V���ɞ�ljr��)�/,�߬h�L��#��^��L�ф�,íMƁe�̩�NB�L�����iL����q�}��(��q��6IçJ$�W�E$��:������=#����(�K�B����zђ <��K(�N�۫K�w��^O{!����)�H���>x�������lx�?>Պ�+�>�W���,Ly!_�D���Ō�l���Q�!�[ �S����J��1��Ɛ�Y}��b,+�Lo�x�ɓ)����=�y�oh�@�꥟/��I��ѭ=��P�y9��� �ۍYӘ�e+�p�Jnϱ?V\SO%�(�t� ���=?MR�[Ș�����d�/ ��n�l��B�7j� ��!�;ӥ�/�[-���A�>�dN�sLj ��,ɪv��=1c�.SQ�O3�U���ƀ�ܽ�E����������̻��9G�ϷD�7(�}��Ävӌ\�y�_0[w ���<΍>����a_��[0+�L��F.�޺��f�>oN�T����q;���y\��bՃ��y�jH�<|q-eɏ�_?_9+P���Hp$�����[ux�K w�Mw��N�ی'$Y2�=��q���KB��P��~������Yul:�[<����F1�2�O���5=d����]Y�sw:���Ϯ���E��j,_Q��X��z`H1,#II ��d�wr��P˂@�ZJV����y$�\y�{}��^~���[:N����ߌ�U�������O��d�����ؾe��${p>G��3c���Ė�lʌ�� ת��[��`ϱ�-W����dg�I��ig2��� ��}s ��ؤ(%#sS@���~���3�X�nRG�~\jc3�v��ӍL��M[JB�T��s3}��j�Nʖ��W����;7��ç?=X�F=-�=����q�ߚ���#���='�c��7���ڑW�I(O+=:uxq�������������e2�zi+�kuG�R��������0�&e�n���iT^J����~\jy���p'dtG��s����O��3����9* �b#Ɋ�� p������[Bws�T�>d4�ۧs���nv�n���U���_�~,�v����ƜJ1��s�� �QIz��)�(lv8M���U=�;����56��G���s#�K���MP�=��LvyGd��}�VwWBF�'�à �?MH�U�g2�� ����!�p�7Q��j��ڴ����=��j�u��� Jn�A s���uM������e��Ɔ�Ҕ�!)'��8Ϣ�ٔ��ޝ(��Vp���צ֖d=�IC�J�Ǡ{q������kԭ�߸���i��@K����u�|�p=..�*+����x�����z[Aqġ#s2a�Ɗ���RR�)*HRsi�~�a &f��M��P����-K�L@��Z��Xy�'x�{}��Zm+���:�)�) IJ�-i�u���� ���ܒH��'�L(7�y�GӜq���� j��� 6ߌg1�g�o���,kر���tY�?W,���p���e���f�OQS��!K�۟cҒA�|ս�j�>��=⬒��˧L[�� �߿2JaB~R��u�:��Q�] �0H~���]�7��Ƽ�I���(}��cq '�ήET���q�?f�ab���ӥvr� �)o��-Q��_'����ᴎo��K������;��V���o��%���~OK ����*��b�f:���-ťIR��`B�5!RB@���ï�� �u �̯e\�_U�_������� g�ES��3�������QT��a����x����U<~�c?�*�#]�MW,[8O�a�x��]�1bC|踤�P��lw5V%�)�{t�<��d��5���0i�XSU��m:��Z�┵�i�"��1�^B�-��P�hJ��&)O��*�D��c�W��vM��)����}���P��ܗ-q����\mmζZ-l@�}��a��E�6��F�@��&Sg@���ݚ�M����� ȹ 4����#p�\H����dYDo�H���"��\��..R�B�H�z_�/5˘����6��KhJR��P�mƶi�m���3�,#c�co��q�a)*Pt����R�m�k�7x�D�E�\Y�閣_X�<���~�)���c[[�BP����6�Yq���S��0����%_����;��Àv�~�| VS؇ ��'O0��F0��\���U�-�d@�����7�SJ*z��3n��y��P����O���������m�~�P�3|Y��ʉr#�C�<�G~�.,! ���bqx���h~0=��!ǫ�jy����l�O,�[B��~��|9��ٱ����Xly�#�i�B��g%�S��������tˋ���e���ې��\[d�t)��.+u�|1 ������#�~Oj����hS�%��i.�~X���I�H�m��0n���c�1uE�q��cF�RF�o���7� �O�ꮧ� ���ۛ{��ʛi5�rw?׌#Qn�TW��~?y$��m\�\o����%W� ?=>S�N@�� �Ʈ���R����N�)�r"C�:��:����� �����#��qb��Y�. �6[��2K����2u�Ǧ�HYR��Q�MV��� �G�$��Q+.>�����nNH��q�^��� ����q��mM��V��D�+�-�#*�U�̒ ���p욳��u:�������IB���m���PV@O���r[b= �� ��1U�E��_Nm�yKbN�O���U�}�the�`�|6֮P>�\2�P�V���I�D�i�P�O;�9�r�mAHG�W�S]��J*�_�G��+kP�2����Ka�Z���H�'K�x�W�MZ%�O�YD�Rc+o��?�q��Ghm��d�S�oh�\�D�|:W������UA�Qc yT�q������~^�H��/��#p�CZ���T�I�1�ӏT����4��"�ČZ�����}��`w�#�*,ʹ�� ��0�i��課�Om�*�da��^gJ݅{���l�e9uF#T�ֲ��̲�ٞC"�q���ߍ ոޑ�o#�XZTp����@ o�8��(jd��xw�]�,f���`~�|,s��^����f�1���t��|��m�򸄭/ctr��5s��7�9Q�4�H1꠲BB@l9@���C�����+�wp�xu�£Yc�9��?`@#�o�mH�s2��)�=��2�.�l����jg�9$�Y�S�%*L������R�Y������7Z���,*=�䷘$�������arm�o�ϰ���UW.|�r�uf����IGw�t����Zwo��~5 ��YյhO+=8fF�)�W�7�L9lM�̘·Y���֘YLf�큹�pRF���99.A �"wz��=E\Z���'a� 2��Ǚ�#;�'}�G���*��l��^"q��+2FQ� hj��kŦ��${���ޮ-�T�٭cf�|�3#~�RJ����t��$b�(R��(����r���dx� >U b�&9,>���%E\� Ά�e�$��'�q't��*�א���ެ�b��-|d���SB�O�O��$�R+�H�)�܎�K��1m`;�J�2�Y~9��O�g8=vqD`K[�F)k�[���1m޼c��n���]s�k�z$@��)!I �x՝"v��9=�ZA=`Ɠi �:�E��)`7��vI��}d�YI�_ �o�:ob���o ���3Q��&D&�2=�� �Ά��;>�h����y.*ⅥS������Ӭ�+q&����j|UƧ����}���J0��WW< ۋS�)jQR�j���Ư��rN)�Gű�4Ѷ(�S)Ǣ�8��i��W52���No˓� ۍ%�5brOn�L�;�n��\G����=�^U�dI���8$�&���h��'���+�(������cȁ߫k�l��S^���cƗjԌE�ꭔ��gF���Ȓ��@���}O���*;e�v�WV���YJ\�]X'5��ղ�k�F��b 6R�o՜m��i N�i����>J����?��lPm�U��}>_Z&�KK��q�r��I�D�Չ~�q�3fL�:S�e>���E���-G���{L�6p�e,8��������QI��h��a�Xa��U�A'���ʂ���s�+טIjP�-��y�8ۈZ?J$��W�P� ��R�s�]��|�l(�ԓ��sƊi��o(��S0��Y� 8�T97.�����WiL��c�~�dxc�E|�2!�X�K�Ƙਫ਼�$((�6�~|d9u+�qd�^3�89��Y�6L�.I�����?���iI�q���9�)O/뚅����O���X��X�V��ZF[�یgQ�L��K1���RҖr@v�#��X�l��F���Нy�S�8�7�kF!A��sM���^rkp�jP�DyS$N���q��nxҍ!U�f�!eh�i�2�m���`�Y�I�9r�6� �TF���C}/�y�^���Η���5d�'��9A-��J��>{�_l+�`��A���[�'��յ�ϛ#w:݅�%��X�}�&�PSt�Q�"�-��\縵�/����$Ɨh�Xb�*�y��BS����;W�ջ_mc�����vt?2}1�;qS�d�d~u:2k5�2�R�~�z+|HE!)�Ǟl��7`��0�<�,�2*���Hl-��x�^����'_TV�gZA�'j� ^�2Ϊ��N7t�����?w�� �x1��f��Iz�C-Ȗ��K�^q�;���-W�DvT�7��8�Z�������� hK�(P:��Q- �8�n�Z���܃e貾�<�1�YT<�,�����"�6{/ �?�͟��|1�:�#g��W�>$����d��J��d�B��=��jf[��%rE^��il:��B���x���Sּ�1հ��,�=��*�7 fcG��#q� �eh?��2�7�����,�!7x��6�n�LC�4x��},Geǝ�tC.��vS �F�43��zz\��;QYC,6����~;RYS/6���|2���5���v��T��i����������mlv��������&� �nRh^ejR�LG�f���? �ۉҬܦƩ��|��Ȱ����>3����!v��i�ʯ�>�v��オ�X3e���_1z�Kȗ\<������!�8���V��]��?b�k41�Re��T�q��mz��TiOʦ�Z��Xq���L������q"+���2ۨ��8}�&N7XU7Ap�d�X��~�׿��&4e�o�F��� �H����O���č�c�� 懴�6���͉��+)��v;j��ݷ�� �UV�� i��� j���Y9GdÒJ1��詞�����V?h��l����l�cGs�ځ�������y�Ac�����\V3�? �� ܙg�>qH�S,�E�W�[�㺨�uch�⍸�O�}���a��>�q�6�n6����N6�q������N ! 1AQaq�0@����"2BRb�#Pr���3C`��Scst���$4D���%Td�� ?���N����a��3��m���C���w��������xA�m�q�m���m������$����4n淿t'��C"w��zU=D�\R+w�p+Y�T�&�պ@��ƃ��3ޯ?�Aﶂ��aŘ���@-�����Q�=���9D��ռ�ѻ@��M�V��P��܅�G5�f�Y<�u=,EC)�<�Fy'�"�&�չ�X~f��l�KԆV��?�� �W�N����=(� �;���{�r����ٌ�Y���h{�١������jW����P���Tc�����X�K�r��}���w�R��%��?���E��m�� �Y�q|����\lEE4���r���}�lsI�Y������f�$�=�d�yO����p�����yBj8jU�o�/�S��?�U��*������ˍ�0������u�q�m [�?f����a�� )Q�>����6#������� ?����0UQ����,IX���(6ڵ[�DI�MNލ�c&���υ�j\��X�R|,4��� j������T�hA�e��^���d���b<����n�� �즇�=!���3�^�`j�h�ȓr��jẕ�c�,ٞX����-����a�ﶔ���#�$��]w�O��Ӫ�1y%��L�Y<�wg#�ǝ�̗`�x�xa�t�w��»1���o7o5��>�m뭛C���Uƃߜ}�C���y1Xνm�F8�jI���]����H���ۺиE@I�i;r�8ӭ����V�F�Շ| ��&?�3|x�B�MuS�Ge�=Ӕ�#BE5G�����Y!z��_e��q�р/W>|-�Ci߇�t�1ޯќd�R3�u��g�=0 5��[?�#͏��q�cf���H��{ ?u�=?�?ǯ���}Z��z���hmΔ�BFTW�����<�q�(v� ��!��z���iW]*�J�V�z��gX֧A�q�&��/w���u�gYӘa���; �i=����g:��?2�dž6�ى�k�4�>�Pxs����}������G�9��3 ���)gG�R<>r h�$��'nc�h�P��Bj��J�ҧH� -��N1���N��?��~��}-q!=��_2hc�M��l�vY%UE�@|�v����M2�.Y[|y�"Eï��K�ZF,�ɯ?,q�?v�M 80jx�"�;�9vk�����+ ֧�� �ȺU��?�%�vcV��mA�6��Qg^M����A}�3�nl� QRN�l8�kkn�'�����(��M�7m9و�q���%ޟ���*h$Zk"��$�9��: �?U8�Sl��,,|ɒ��xH(ѷ����Gn�/Q�4�P��G�%��Ա8�N��!� �&�7�;���eKM7�4��9R/%����l�c>�x;������>��C�:�����t��h?aKX�bhe�ᜋ^�$�Iհ �hr7%F$�E��Fd���t��5���+�(M6�t����Ü�UU|zW�=a�Ts�Tg������dqP�Q����b'�m���1{|Y����X�N��b �P~��F^F:����k6�"�j!�� �I�r�`��1&�-$�Bevk:y���#yw��I0��x��=D�4��tU���P�ZH��ڠ底taP��6����b>�xa����Q�#� WeF��ŮNj�p�J* mQ�N����*I�-*�ȩ�F�g�3 �5��V�ʊ�ɮ�a��5F���O@{���NX��?����H�]3��1�Ri_u��������ѕ�� ����0��� F��~��:60�p�͈�S��qX#a�5>���`�o&+�<2�D����: �������ڝ�$�nP���*)�N�|y�Ej�F�5ټ�e���ihy�Z �>���k�bH�a�v��h�-#���!�Po=@k̆IEN��@��}Ll?j�O������߭�ʞ���Q|A07x���wt!xf���I2?Z��<ץ�T���cU�j��]��陎Ltl �}5�ϓ��$�,��O�mˊ�;�@O��jE��j(�ا,��LX���LO���Ц�90�O �.����a��nA���7������j4 ��W��_ٓ���zW�jcB������y՗+EM�)d���N�g6�y1_x��p�$Lv:��9�"z��p���ʙ$��^��JԼ*�ϭ����o���=x�Lj�6�J��u82�A�H�3$�ٕ@�=Vv�]�'�qEz�;I˼��)��=��ɯ���x �/�W(V���p�����$ �m�������u�����񶤑Oqˎ�T����r��㠚x�sr�GC��byp�G��1ߠ�w e�8�$⿄����/�M{*}��W�]˷.�CK\�ުx���/$�WPw���r� |i���&�}�{�X� �>��$-��l���?-z���g����lΆ���(F���h�vS*���b���߲ڡn,|)mrH[���a�3�ר�[1��3o_�U�3�TC�$��(�=�)0�kgP���� ��u�^=��4 �WYCҸ:��vQ�ר�X�à��tk�m,�t*��^�,�}D*� �"(�I��9R����>`�`��[~Q]�#af��i6l��8���6�:,s�s�N6�j"�A4���IuQ��6E,�GnH��zS�HO�uk�5$�I�4��ؤ�Q9�@��C����wp�BGv[]�u�Ov���0I4���\��y�����Q�Ѹ��~>Z��8�T��a��q�ޣ;z��a���/��S��I:�ܫ_�|������>=Z����8:�S��U�I�J��"IY���8%b8���H��:�QO�6�;7�I�S��J��ҌAά3��>c���E+&jf$eC+�z�;��V����� �r���ʺ������my�e���aQ�f&��6�ND��.:��NT�vm�<- u���ǝ\MvZY�N�NT��-A�>jr!S��n�O 1�3�Ns�%�3D@���`������ܟ 1�^c<���� �a�ɽ�̲�Xë#�w�|y�cW�=�9I*H8�p�^(4���՗�k��arOcW�tO�\�ƍR��8����'�K���I�Q�����?5�>[�}��yU�ײ -h��=��% q�ThG�2�)���"ו3]�!kB��*p�FDl�A���,�eEi�H�f�Ps�����5�H:�Փ~�H�0Dت�D�I����h�F3�������c��2���E��9�H��5�zԑ�ʚ�i�X�=:m�xg�hd(�v����׊�9iS��O��d@0ڽ���:�p�5�h-��t�&���X�q�ӕ,��ie�|���7A�2���O%P��E��htj��Y1��w�Ѓ!����  ���� ࢽ��My�7�\�a�@�ţ�J �4�Ȼ�F�@o�̒?4�wx��)��]�P��~�����u�����5�����7X ��9��^ܩ�U;Iꭆ 5 �������eK2�7(�{|��Y׎ �V��\"���Z�1� Z�����}��(�Ǝ"�1S���_�vE30>���p;� ΝD��%x�W�?W?v����o�^V�i�d��r[��/&>�~`�9Wh��y�;���R��� ;;ɮT��?����r$�g1�K����A��C��c��K��l:�'��3 c�ﳯ*"t8�~l��)���m��+U,z��`(�>yJ�?����h>��]��v��ЍG*�{`��;y]��I�T� ;c��NU�fo¾h���/$���|NS���1�S�"�H��V���T���4��uhǜ�]�v;���5�͠x��'C\�SBpl���h}�N����� A�Bx���%��ޭ�l��/����T��w�ʽ]D�=����K���ž�r㻠l4�S�O?=�k �M:� ��c�C�a�#ha���)�ѐxc�s���gP�iG��{+���x���Q���I= �� z��ԫ+ �8"�k�ñ�j=|����c ��y��CF��/��*9ж�h{ �?4�o� ��k�m�Q�N�x��;�Y��4膚�a�w?�6�>e]�����Q�r�:����g�,i"�����ԩA�*M�<�G��b�if��l^M��5� �Ҩ�{����6J��ZJ�����P�*�����Y���ݛu�_4�9�I8�7���������,^ToR���m4�H��?�N�S�ѕw��/S��甍�@�9H�S�T��t�ƻ���ʒU��*{Xs�@����f�����֒Li�K{H�w^���������Ϥm�tq���s� ���ք��f:��o~s��g�r��ט� �S�ѱC�e]�x���a��) ���(b-$(�j>�7q�B?ӕ�F��hV25r[7 Y� }L�R��}����*sg+��x�r�2�U=�*'WS��ZDW]�WǞ�<��叓���{�$�9Ou4��y�90-�1�'*D`�c�^o?(�9��u���ݐ��'PI&� f�Jݮ�������:wS����jfP1F:X �H�9dԯ���˝[�_54 �}*;@�ܨ�� ð�yn�T���?�ןd�#���4rG�ͨ��H�1�|-#���Mr�S3��G�3�����)�.᧏3v�z֑��r����$G"�`j �1t��x0<Ɔ�Wh6�y�6��,œ�Ga��gA����y��b��)��h�D��ß�_�m��ü �gG;��e�v��ݝ�nQ� ��C����-�*��o���y�a��M��I�>�<���]obD��"�:���G�A��-\%LT�8���c�)��+y76���o�Q�#*{�(F�⽕�y����=���rW�\p���۩�c���A���^e6��K������ʐ�cVf5$�'->���ՉN"���F�"�UQ@�f��Gb~��#�&�M=��8�ט�JNu9��D��[̤�s�o�~������ G��9T�tW^g5y$b��Y'��س�Ǵ�=��U-2 #�MC�t(�i� �lj�@Q 5�̣i�*�O����s�x�K�f��}\��M{E�V�{�υ��Ƈ�����);�H����I��fe�Lȣr�2��>��W�I�Ȃ6������i��k�� �5�YOxȺ����>��Y�f5'��|��H+��98pj�n�.O�y�������jY��~��i�w'������l�;�s�2��Y��:'lg�ꥴ)o#'Sa�a�K��Z� �m��}�`169�n���"���x��I ��*+� }F<��cГ���F�P�������ֹ*�PqX�x۩��,� ��N�� �4<-����%����:��7����W���u�`����� $�?�I��&����o��o��`v�>��P��"��l���4��5'�Z�gE���8���?��[�X�7(��.Q�-��*���ތL@̲����v��.5���[��=�t\+�CNܛ��,g�SQnH����}*F�G16���&:�t��4ُ"A��̣��$�b �|����#rs��a�����T�� ]�<�j��BS�('$�ɻ� �wP;�/�n��?�ݜ��x�F��yUn�~mL*-�������Xf�wd^�a�}��f�,=t�׵i�.2/wpN�Ep8�OР���•��R�FJ� 55TZ��T �ɭ�<��]��/�0�r�@�f��V��V����Nz�G��^���7hZi����k��3�,kN�e|�vg�1{9]_i��X5y7� 8e]�U����'�-2,���e"����]ot�I��Y_��n�(JҼ��1�O ]bXc���Nu�No��pS���Q_���_�?i�~�x h5d'�(qw52] ��'ޤ�q��o1�R!���`ywy�A4u���h<קy���\[~�4�\ X�Wt/� 6�����n�F�a8��f���z �3$�t(���q��q�x��^�XWeN'p<-v�!�{�(>ӽDP7��ո0�y)�e$ٕv�Ih'Q�EA�m*�H��RI��=:��� ���4牢) �%_iN�ݧ�l]� �Nt���G��H�L��� ɱ�g<���1V�,�J~�ٹ�"K��Q�� 9�HS�9�?@��k����r�;we݁�]I�!{ �@�G�[�"��`���J:�n]�{�cA�E����V��ʆ���#��U9�6����j�#Y�m\��q�e4h�B�7��C�������d<�?J����1g:ٳ���=Y���D�p�ц� ׈ǔ��1�]26؜oS�'��9�V�FVu�P�h�9�xc�oq�X��p�o�5��Ա5$�9W�V(�[Ak�aY錎qf;�'�[�|���b�6�Ck��)��#a#a˙��8���=äh�4��2��C��4tm^ �n'c���]GQ$[Wҿ��i���vN�{Fu ��1�gx��1┷���N�m��{j-,��x�� Ūm�ЧS�[�s���Gna���䑴�� x�p 8<������97�Q���ϴ�v�aϚG��Rt�Һ׈�f^\r��WH�JU�7Z���y)�vg=����n��4�_)y��D'y�6�]�c�5̪�\� �PF�k����&�c;��cq�$~T�7j ���nç]�<�g ":�to�t}�159�<�/�8������m�b�K#g'I'.W�����6��I/��>v��\�MN��g���m�A�yQL�4u�Lj�j9��#44�t��l^�}L����n��R��!��t��±]��r��h6ٍ>�yҏ�N��fU�� ���� Fm@�8}�/u��jb9������he:A�y�ծw��GpΧh�5����l}�3p468��)U��d��c����;Us/�֔�YX�1�O2��uq�s��`hwg�r~�{ R��mhN��؎*q 42�*th��>�#���E����#��Hv�O����q�}�����6�e��\�,Wk�#���X��b>��p}�դ��3���T5��†��6��[��@�P�y*n��|'f�֧>�lư΂�̺����SU�'*�q�p�_S�����M�� '��c�6�����m�� ySʨ;M��r���Ƌ�m�Kxo,���Gm�P��A�G�:��i��w�9�}M(�^�V��$ǒ�ѽ�9���|���� �a����J�SQ�a���r�B;����}���ٻ֢�2�%U���c�#�g���N�a�ݕ�'�v�[�OY'��3L�3�;,p�]@�S��{ls��X�'���c�jw�k'a�.��}�}&�� �dP�*�bK=ɍ!����;3n�gΊU�ߴmt�'*{,=SzfD� A��ko~�G�aoq�_mi}#�m�������P�Xhύ����mxǍ�΂���巿zf��Q���c���|kc�����?���W��Y�$���_Lv����l߶��c���`?����l�j�ݲˏ!V��6����U�Ђ(A���4y)H���p�Z_�x��>���e��R��$�/�`^'3qˏ�-&Q�=?��CFVR �D�fV�9��{�8g�������n�h�(P"��6�[�D���< E�����~0<@�`�G�6����Hг�cc�� �c�K.5��D��d�B���`?�XQ��2��ٿyqo&+�1^� DW�0�ꊩ���G�#��Q�nL3��c���������/��x ��1�1[y�x�პCW��C�c�UĨ80�m�e�4.{�m��u���I=��f�����0QRls9���f���������9���~f�����Ǩ��a�"@�8���ȁ�Q����#c�ic������G��$���G���r/$W�(��W���V�"��m�7�[m�A�m����bo��D� j����۳� l���^�k�h׽����� ��#� iXn�v��eT�k�a�^Y�4�BN��ĕ��0 !01@Q"2AaPq3BR������?���@4�Q�����T3,���㺠�W�[=JK�Ϟ���2�r^7��vc�:�9 �E�ߴ�w�S#d���Ix��u��:��Hp��9E!�� V 2;73|F��9Y���*ʬ�F��D����u&���y؟��^EA��A��(ɩ���^��GV:ݜDy�`��Jr29ܾ�㝉��[���E;Fzx��YG��U�e�Y�C���� ����v-tx����I�sם�Ę�q��Eb�+P\ :>�i�C'�;�����k|z�رn�y]�#ǿb��Q��������w�����(�r|ӹs��[�D��2v-%��@;�8<a���[\o[ϧw��I!��*0�krs)�[�J9^��ʜ��p1)� "��/_>��o��<1����A�E�y^�C��`�x1'ܣn�p��s`l���fQ��):�l����b>�Me�jH^?�kl3(�z:���1ŠK&?Q�~�{�ٺ�h�y���/�[��V�|6��}�KbX����mn[-��7�5q�94�������dm���c^���h� X��5��<�eޘ>G���-�}�دB�ޟ� ��|�rt�M��V+�]�c?�-#ڛ��^ǂ}���Lkr���O��u�>�-D�ry� D?:ޞ�U��ǜ�7�V��?瓮�"�#���r��չģVR;�n���/_� ؉v�ݶe5d�b9��/O��009�G���5n�W����JpA�*�r9�>�1��.[t���s�F���nQ� V 77R�]�ɫ8����_0<՜�IF�u(v��4��F�k�3��E)��N:��yڮe��P�`�1}�$WS��J�SQ�N�j�ٺ��޵�#l���ј(�5=��5�lǏmoW�v-�1����v,W�mn��߀$x�<����v�j(����c]��@#��1������Ǔ���o'��u+����;G�#�޸��v-lη��/(`i⣍Pm^���ԯ̾9Z��F��������n��1��� ��]�[��)�'������:�֪�W��FC����� �B9،!?���]��V��A�Վ�M��b�w��G F>_DȬ0¤�#�QR�[V��kz���m�w�"��9ZG�7'[��=�Q����j8R?�zf�\a�=��O�U����*oB�A�|G���2�54 �p��.w7� �� ��&������ξxGHp� B%��$g�����t�Џ򤵍z���HN�u�Я�-�'4��0��;_��3 !01"@AQa2Pq#3BR������?��ʩca��en��^��8���<�u#��m*08r��y�N"�<�Ѳ0��@\�p��� �����Kv�D��J8�Fҽ� �f�Y��-m�ybX�NP����}�!*8t(�OqѢ��Q�wW�K��ZD��Δ^e��!� ��B�K��p~�����e*l}z#9ң�k���q#�Ft�o��S�R����-�w�!�S���Ӥß|M�l޶V��!eˈ�8Y���c�ЮM2��tk���� ������J�fS����Ö*i/2�����n]�k�\���|4yX�8��U�P.���Ы[���l��@"�t�<������5�lF���vU�����W��W��;�b�cД^6[#7@vU�xgZv��F�6��Q,K�v��� �+Ъ��n��Ǣ��Ft���8��0��c�@�!�Zq s�v�t�;#](B��-�nῃ~���3g������5�J�%���O������n�kB�ĺ�.r��+���#�N$?�q�/�s�6��p��a����a��J/��M�8��6�ܰ"�*������ɗud"\w���aT(����[��F��U՛����RT�b���n�*��6���O��SJ�.�ij<�v�MT��R\c��5l�sZB>F��<7�;EA��{��E���Ö��1U/�#��d1�a�n.1ě����0�ʾR�h��|�R��Ao�3�m3 ��%�� ���28Q� ��y��φ���H�To�7�lW>����#i`�q���c����a��� �m,B�-j����݋�'mR1Ήt�>��V��p���s�0IbI�C.���1R�ea�����]H�6����������4B>��o��](��$B���m�����a�!=��?�B� K�Ǿ+�Ծ"�n���K��*��+��[T#�{E�J�S����Q�����s�5�:�U�\wĐ�f�3����܆&�)����I���Ԇw��E T�lrTf6Q|R�h:��[K�� �z��c֧�G�C��%\��_�a�84��HcO�bi��ؖV��7H �)*ģK~Xhչ0��4?�0��� �E<���}3���#���u�?�� ��|g�S�6ꊤ�|�I#Hڛ� �ա��w�X��9��7���Ŀ%�SL��y6č��|�F�a 8���b��$�sק�h���b9RAu7�˨p�Č�_\*w��묦��F ����4D~�f����|(�"m���NK��i�S�>�$d7SlA��/�²����SL��|6N�}���S�˯���g��]6��; �#�.��<���q'Q�1|KQ$�����񛩶"�$r�b:���N8�w@��8$�� �AjfG|~�9F ���Y��ʺ��Bwؒ������M:I岎�G��`s�YV5����6��A �b:�W���G�q%l�����F��H���7�������Fsv7��k�� 403WebShell
403Webshell
Server IP : 104.21.45.146  /  Your IP : 172.69.214.218
Web Server : Apache/2.4.52 (Ubuntu)
System : Linux ip-172-31-19-221 6.8.0-1029-aws #31~22.04.1-Ubuntu SMP Thu Apr 24 21:16:18 UTC 2025 x86_64
User : www-data ( 33)
PHP Version : 8.1.28
Disable Function : NONE
MySQL : OFF  |  cURL : ON  |  WGET : ON  |  Perl : ON  |  Python : OFF  |  Sudo : ON  |  Pkexec : ON
Directory :  /efsdata/scitechnol.com/httpdocs/submission/uploads/

Upload File :
current_dir [ Writeable ] document_root [ Writeable ]

 

Command :


[ Back ]     

Current File : /efsdata/scitechnol.com/httpdocs/submission/uploads/1642-1-Manuscript-2014-02-24.doc
��ࡱ�>��	}����|����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������[�	��$�bjbj7�7�	4�U�\U�\G�������>	>	��������������4��(���������	((((((($�*�H-@/(�������/(������(�!�!�!������	(�!�	(�!�!:�%,�&�����P0B�U1������ v)&
�'�(0�(3&R�-!��-�&�-��&p���!�����/(/(�!����(�������������������������������������������������������������������������-���������>	>|:	Development and in-house validation of avian group-D rotavirus specific RT-PCR assay 

Kattoor Jobin Jose1, Naveen Kumar1, Kuldeep Sharma1, Shubhnkar Sircar1, Neeraj Singh1, Munish Batra2, and Yashpal Singh Malik1*


1Division of Biological Standardization, Indian Veterinary Research Institute, Izatnagar 243122, Bareilly, Uttar Pradesh, India.
2Department of Veterinary Pathology, GB Pant University of Agriculture and Technology, Udham Singh Nagar, Uttarakhand, India.













*Corresponding author: Tel) +91-581-2302777, Fax) +91-581- 2301757, 
       E-mail:  HYPERLINK "mailto:malikyps@ivri.res.in" malikyps@ivri.res.in
Abstract

The group D rotavirus (RVD) is one of the evolving viral causes of acute gastroenteritis in avian species world over. Despite the evolving epidemiology, the frequency of avian group D rotavirus (RVD) infection among Indian poultry has yet to be well characterised. Paucity of epidemiological studies with unavailability of sensitive and rapid detection methods for group D rotaviruses in poultry led to design of the present study. This study describes the development and in-house validation of a RT-PCR assay targeting the conserved group specific region of VP6 gene of RVD. The detection limit of RT-PCR assay developed in this study (2.95x10-7ng/�L=1.49�103 copy numbers) was superior to that reported previously (5x10-4 ng/�L) from Brazil. The confirmation of the RT-PCR amplicons was done by sequencing and product analysis. The findings emphasise that the developed assay has the potential to serve as an invaluable test in the epidemiological studies which will further strengthen the diagnostic tools in growing poultry sector of India. 

Keywords:  Avian group-D rotavirus, RT-PCR, VP6 gene, India 












Introduction 
Rotavirus is amongst the leading causes of acute gastroenteritis affecting several mammalian and avian species [1-3]. In poultry, it leads to runting and stunting syndrome (RSS) and causes colossal economic loss to poultry rearers [4]. Rotavirus belongs to the family Reoviridae and is a non-enveloped, 11 segmented, double stranded RNA genome virus [3]. The group specific antigenic determinants located within the inner capsid protein (VP6) is employed to classify the virus in to seven groups (A-G). Rotaviruses are also classified in to various electropherotypes based on their genomic segments migration pattern in polyacrylamide gel. Among 7 groups, rotavirus type A strains are predominant and affect both mammals and birds, while type D, F, and G are reported only in birds. For diagnosis, ribonucleic acid-polyacrylamide gel electrophoresis (RNA-PAGE) is the one which is more commonly employed for avian rotavirus infection confirmation [4,5], but suffers with the problem of sensitivity which is inferior to other molecular methods available. Presence of avian rotaviruses has been documented from India only on few occasions [6-8]. Though, reverse transcription-polymerase chain reaction (RT-PCR) based diagnostic assays have been employed for the detection for group A avian rotavirus, group D rotavirus detection is still based on classical polyacrylamide gel electrophoresis [8, 9]. Paucity of epidemiological studies with unavailability of sensitive and rapid detection methods for group D rotaviruses in poultry led to design of the present study. In this study, targeting group specific VP6 gene, a rapid RT-PCR assay for detection of group D rotavirus in poultry was developed and in-house validated.   
Materials and Methods
Group-D rotavirus samples
The sequence confirmed clinical samples obtained from diarrhoeic and post mortem enteritis cases of chicken from Uttarakhand state of India were used for development of the assay. Suspensions (10% w/v) of faecal samples in phosphate buffer saline (PBS) (pH 7.4; Sigma-Aldrich, St. Louis, USA) were centrifuged at 2000 g for 20 min to remove coarse particulate matter and the upper aqueous layer was then filtered through 0.22 �m syringe filters and stored at -20p C until required.


Viral RNA extraction and cDNA synthesis
	Total RNA was extracted from 500 �l of 10% faecal suspension in PBS using an equal volume of TriReagent-LS (Sigma-Aldrich, St. Louis, USA). The RNA was eluted with Nuclease Free Water (NFW) in a final volume of 25 �l. The isolated RNA was assessed qualitatively and quantitatively using Nanodrop Spectrophotometer (ND-1000, Thermo-Scientific, USA) and stored at -70p C until further use. Reverse-transcription for cDNA synthesis from viral RNA was performed using 1.0 �l (100 ng/�l) random hexamer (Fermentas, Lithuania), 100 ng of viral RNA, and 2 �L of dimethyl sulphoxide (DMSO) were added to PCR tube containing sufficient volume of NFW, followed by incubation of the reaction mixture at 70�C for 5 min to melt secondary structures within the template. The mixture was immediately snap chilled on ice followed by the addition of 4 �L of 5X RT buffer, 2 �L of 10 mM dNTPs (Fermentas, Lithuania), 40 U RNase Inhibitor (Ambion, USA), and 200 U MMLV-RT (Promega, USA) and kept at 37�C for 60 minutes. The enzyme was denatured at 80p C for 3 min at the end of the incubation step to inactivate residual MMLV-RT. The cDNA thus obtained was used for PCR and the remaining was kept at �20�C until further use.
Designing of avian group D primers
To design an assay that can detect emerging group D avian rotaviruses, 12 individual sequences of the VP6 gene of chicken RVD strains from different geographical locations within India and the rest of the world were retrieved from the NCBI nucleotide database ( HYPERLINK "http://www.ncbi.nlm.nih.gov/nuccore" http://www.ncbi.nlm.nih.gov/nuccore). The sequences were assembled using the ClustalW program of Lasergene 6.0 software (DNASTAR Inc, USA) for the alignment of all the retrieved sequences. Through VP6 gene nucleotide sequences alignment analysis, the conserved regions of VP6 gene of group D rotavirus (GenBank accession number: JN034685) were used for generating sense primer (5�-GCTATACAT TTCGCTGCATTTG-3�) and anti-sense primer (5�-TGGCCAATAGTGTGTGGCAGCT-3�) using GeneTool Lite software (BioTools Inc., Edmonton, Canada). All primers were custom synthesized (Integrated DNA Technologies Inc., India).
Optimization of the Reverse transcription-PCR assay
The plasmid construct of partial avian RVD VP6 gene (185bp) was used as standard for the optimization of PCR reaction conditions. In brief, the avian group D rotavirus positive diarrhoeic faecal sample (UKD-34) retrieved from Enteric Virus Laboratory, Indian Veterinary Research Institute, India was processed for viral ribonucleic acid (RNA) extraction using TriReagent-LS (Sigma, St. Louis, USA) followed by cDNA synthesis as described above. The partial length VP6 gene of avian group D rotavirus was amplified using forward and reverse primers (mentioned above). Various components of the RT-PCR reaction mixture included 2.5 �L 10X PCR buffer mix, 0.5 �L 25mM MgCl2, 0.5 �L 10mM dNTPs, 2.0 �L of forward and reverse primers (10pmol/�L each), 2.0 �L cDNA, 0.25 �L Taq polymerase (5 U/�L) made up to final volume of 25.0 �L using nuclease free water. The PCR amplicon was visualized in ethidium bromide stained 1.5% agarose gel and documented using Transilluminator-UV�300 (UVP Inc., Upland, USA). 
Cloning and sequencing
The specific RT-PCR amplicon (185 bp) was gel-purified by QIAquick Gel Extraction kit (Qiagen, Germany) and cloned into pGEM�-T Easy Cloning vector (Promega Corporation, USA). The clones were screened for the presence of insert by colony PCR and restriction digestion. The positive clones were further confirmed by sequencing using automated sequencer ABIPRISM�3100 (AME Bioscience, Toroed, Norway) at Scigenome Labs Pvt. Ltd. Kochin, Kerala. The plasmid was extracted from positive clones using GeneJET� Plasmid Miniprep Kit (Fermentas, Lithuania) for further use in RT-PCR assay. 
Evaluation of the assay
For calculation of sensitivity, ten-fold dilutions (10-1 to 10-10) of VP6 gene plasmid construct were used and RT-PCR reaction was performed as mentioned earlier. The PCR amplified products were resolved in 1.5% agarose gel and visualized in Transilluminator-UV�300 (UVP Inc., Upland, USA). The specificity of the primers was checked using RNA extracted from enteric viruses originating from different species viz. picobirnavirus, bovine group A rotaviruses, porcine group A rotaviruses, human group A rotaviruses, and avian group A rotaviruses. The assay was validated on 20 diarrhoeic poultry samples collected from the field with clinical diarrhoeic history. 



Results 
RT-PCR assay optimization
The optimum PCR conditions of initial denaturation at 95�C for 5 min, followed by 35 cyclic denaturation at 94�C for 10 sec, annealing at 53�C for 30 sec and extension at 68�C for 30 sec with final extension at 68�C for 10 min, yielded expected size amplicon of 185 bp. The PCR products were electrophoresed in 1.5% agarose gel and were visualized in Transilluminator-UV�300 (UVP Inc., Upland, USA) and documented.  
Evaluation of RT-PCR assay
The sensitivity of RT-PCR assay for detection of group D rotavirus was found to be 1.49�103 copy numbers. The assay was found 100% specific in amplification of only avian group D rotavirus, as no amplification was observed when the assay was checked with other enteric viruses. This assay was also initially evaluated on 20 clinical samples obtained from the cases of diarrhoea/enteritis, where 15% (3/20) samples were detected positive for group D rotavirus. The specific PCR amplicons (185 bp) in all the positive samples were visualized in 1.5% ethidium bromide stained agarose gel (Fig. 1) and the obtained sequences of selected isolates were also confirmed through BLAST analysis with high sequence similarity with existing group D rotavirus isolates. 
Discussion 
Rotavirus infection in poultry is of great concern throughout the world. The epidemiological reports confirm that new rotavirus groups are evolving in poultry (Otto et al., 2012) and might inflict the growing poultry sector in the coming years in the country. The diversity and epidemiology in avian group A rotaviruses throughout the world have been studied by several researchers [10-12], but epidemiological studies are barely accessible for other groups of rotaviruses like D, F and G [13,14]. Currently, insufficient detection methodologies are available for avian rotaviruses, predominantly for the new and emerging viruses. Amid the existing assays, RNA-PAGE is the one which is more frequently employed for recognizing the avian rotavirus infections throughout the world [4,9]. A timely identification of rotavirus is, therefore, critical for control of disease and to minimize losses to the poultry sector. In avian species, the rotavirus clinical diagnosis mainly relies on PAGE, and certain serological tests like latex-agglutination tests. Of late, RT-PCR has been proved to be a powerful, sensitive and robust tool for rotavirus detection, particularly for group A rotavirus infection [10], which is more predominant and affects a wide number of mammalian species including human beings. Literature search divulge that group D, F and G rotaviruses are documented only in avian species, while group D has till date been testified only in chicken [8-9, 14-16]. 
In view of non-availability of nucleic acid based sensitive and specific techniques for easy and rapid detection of Indian isolates of avian group D rotavirus, a RT-PCR assay was developed and evaluated. The primers designed targeting the conserved regions of VP6 gene of avian group D rotavirus and reaction mixture conditions were optimized. The sensitivity of this assay was found superior to the one reproted by Bezerra et al. [17], in which detection limit was up to 5x10-4 ng/�L dilution, whereas in the current study detection was 2.95x10-7 ng/�L dilution which is equilent of 1.49x103 copy numbers. Bezerra et al. [17] had not shown the limit in terms of copy numbers detected. In another study, Otto et al. [14] described detection of 2.65x102 copies in the real-time PCR assay, however, such assays are still expensive and are not affordable in developing countries as diagnstoic assays. The present RT-PCR assay was found 100% specific in amplification of only avian group D rotavirus when checked with other enteric viruses. After going through optimization, sensitivity and specific barrier checks, this RT-PCR assay was evaluated on 20 clinical samples obtained from the cases of diarrhoea/enteritis. The avian group D rotaviruses detected in the clinical samples by RT-PCR assay (Fig. 1) were further confirmed by sequencing and NCBI blast analysis with other group D avian rotaviruses. The RVD positive samples from our previous study [18] based on 742bp VP6 gene amplification for validation also yielded positive amplification using 185bp primer set.   
Overall, the results confirmed importance of VP6 gene specific RT-PCR assay developed in this study for detection of avian group D rotavirus which is a rapid and more sensitive assay than the available in-use assays. This assay would enable precise detection of RVD infection in poultry and could therefore be invaluable in field diagnosis. As the assay is rapid and reliable, most probably it will more widely integrate into routine molecular techniques and in future has the potential to serve as an irreplaceable test in the epidemiological studies which will further strengthen the diagnostic tools in poultry sector of India. This appears to be the first report to develop RT-PCR based detection method for group D avian rotavirus from India.

ACKNOWLEDGMENTS 
	The authors are thankful to the Director, Indian Veterinary Research Institute (IVRI) for infrastructural support to carry out the research work. The authors are also thankful to Indian Council of Agricultural Research (ICAR), New Delhi for fellowship grant to first author.   
Interest of Conflict: There is no interest of conflict over the publication. 
REFERENCES 
 HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Shukry%20S%5BAuthor%5D&cauthor=true&cauthor_uid=3023441" Shukry S,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Zaki%20AM%5BAuthor%5D&cauthor=true&cauthor_uid=3023441" Zaki AM,   HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=DuPont%20HL%5BAuthor%5D&cauthor=true&cauthor_uid=3023441" Du Pont HL,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Shoukry%20I%5BAuthor%5D&cauthor=true&cauthor_uid=3023441" Shoukry I,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=el%20Tagi%20M%5BAuthor%5D&cauthor=true&cauthor_uid=3023441" el Tagi M,  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=Hamed%20Z%5BAuthor%5D&cauthor=true&cauthor_uid=3023441" Hamed Z (1986) Detection of enteropathogens in fatal and potentially fatal diarrhea in Cairo. Egy  HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed/3023441" \o "Journal of clinical microbiology." J Clin Microbiol 6: 959-962.
Linhares AC, Gabbay YB, Freitas RB, da Rosa ES, Mascarenhas JD, Loureiro EC (1989) Longitudinal study of rotavirus infections among children from Bel�m, Brazil. Epid Infect.102: 129-145.
Estes M, Kapikian A (2007) Rotaviruses. In Fields Virology, 5th edn, vol.2, pp.1917�1974. Eds. D.M. Knipe, P.M. Howley, D.E. Griffin, R.A. Lamb, M.A. Martin, B. Roizman and S.E. Straus. Philadelphia, PA: Kluwer/Lippincott, Williams and Wilkins.
Otto PH, Liebler-Tenorio EM, Elschner M, Reetz J, Lohren U, Dille R (2006) Detection of rotaviruses and intestinal lesions in broiler chicks from flocks with runting and stunting syndrome (RSS). Avian Dis 50: 411-418.
McNulty MS, Allan GM, Stuart JC (1978) Rotavirus infection in avian species. Vet Rec 103: 319-320.
Wani SA, Bhat MA, Ishaq SM, Ashrafi MA, Buchh AS, Haq M (2003) Detection of a mammalian- like group A rotavirus in diarrhoeic chicken. Vet Microbiol 94: 13-18.
Minakshi, Prasad G, Verma S, Dahiya S (2004) Detection of avian rotaviruses from diarrhoeic poultry in India. Ind J Microbiol 44: 205-206.
Savita, Kusumakar AL, Malik YPS, Minakshi, Prasad G (2008) Detection and characterization of group A and D avian rotaviruses in India. Ind J Biotechnol 7: 554-556.
Niture GS, Kapre AG Prasad M, Zade NN (2010) Electrophoretic pattern analysis of rotaviruses of buffalo, poultry and human. J Vet Public Hlth 1: 7-10.
Pantin-Jackwood MJ, Day JM, Jackwood MW, Spackman E (2008) Enteric viruses detected by molecular methods in commercial chicken and turkey flocks in the United States between 2005 and 2006. Avian Dis 52: 235�244.
Trojnar E, Otto P, Johne R (2009) The first complete genome sequence of a chicken group A ro$'HKUVW`efijm��ʸ�iIiI'iBh.A4h�5�B*CJH*KH$OJQJ\�aJmH	@nH	@phsH	@tH	@?h.A4h	�5�B*CJKH$OJQJ\�aJmH	@nH	@phsH	@tH	@?h.A4h�5�B*CJKH$OJQJ\�aJmH	@nH	@phsH	@tH	@?h.A4h�,�5�B*CJKH$OJQJ\�aJmH	@nH	@phsH	@tH	@h.A4h�,�CJOJQJaJ"h.A4h�,�5�CJOJQJ\�aJ"h.A4hj�5�CJOJQJ\�aJ"h.A4h	�5�CJOJQJ\�aJ"h.A4h�th5�CJOJQJ\�aJVW���Z	�	�	�	�	�	�	�	�	�	�	�	�	�	�	��������������������$
�����dh^�`��a$gd.A4$dh�Z�Z-D@&M�
����a$gd.A4dh�Z�Z-D@&M�
����gd.A4dhgd.A4msxy|�����������������������Z	[	�	߿��߿��߿��߿��߿��߿߿��}^}^<h.A4h�JB*CJKH$OJQJ\�aJmH	@nH	@phsH	@tH	@?h.A4h�JB*CJH*KH$OJQJ\�aJmH	@nH	@phsH	@tH	@Bh.A4h�5�B*CJH*KH$OJQJ\�aJmH	@nH	@phsH	@tH	@?h.A4h�5�B*CJKH$OJQJ\�aJmH	@nH	@phsH	@tH	@?h.A4h	�5�B*CJKH$OJQJ\�aJmH	@nH	@phsH	@tH	@�	�	�	�	�	�	�	9
:
c
d
߿��cI*I*<jh.A4h�CJKH$OJQJU\�aJmH	@nH	@sH	@tH	@3h.A4h�CJKH$OJQJ\�aJmH	@nH	@sH	@tH	@6h.A4h�5�CJKH$OJQJ\�aJmH	@nH	@sH	@tH	@?h.A4h�]�5�B*CJKH$OJQJ\�aJmH	@nH	@phsH	@tH	@?h.A4h�5�B*CJKH$OJQJ\�aJmH	@nH	@phsH	@tH	@?h.A4h-a�5�B*CJKH$OJQJ\�aJmH	@nH	@phsH	@tH	@?h.A4h�Cw5�B*CJKH$OJQJ\�aJmH	@nH	@phsH	@tH	@
�	*
z
�
�
������������������������������������
$dha$gd.A4$dh�7$8$H$a$gd.A4$��dh`��a$gd.A4dh�7$8$H$gd.A4����dh^��`��gd.A4dhgd.A4d
x
y
z
�
�
�
�
�4Mݾ��jO4O%Oh.A4h]6CJOJQJaJ5h.A4hgIB*CJOJQJaJmH	@nH	@phsH	@tH	@5h.A4h�X�B*CJOJQJaJmH	@nH	@phsH	@tH	@8h.A4h�X�5�B*CJOJQJaJmH	@nH	@phsH	@tH	@8h.A4h}Z55�B*CJOJQJaJmH	@nH	@phsH	@tH	@3h.A4h�CJKH$OJQJ\�aJmH	@nH	@sH	@tH	@<jh.A4h�CJKH$OJQJU\�aJmH	@nH	@sH	@tH	@Ch.A4h�0J>*B*CJKH$OJQJ\�aJmH	@nH	@ph�sH	@tH	@
M]quv���������
	

��������x^xF/h.A4h	�CJOJQJ\�aJmHnH	@sHtH	@2h.A4h]6CJH*OJQJ\�aJmHnH	@sHtH	@/h.A4h]6CJOJQJ\�aJmHnH	@sHtH	@h.A4h]6CJOJQJaJh.A4h8�CJOJQJaJ5h.A4h�X�B*CJOJQJaJmH	@nH	@phsH	@tH	@5h.A4h�B*CJOJQJaJmH	@nH	@phsH	@tH	@5h.A4h8�B*CJOJQJaJmH	@nH	@phsH	@tH	@6��������ѷ��eK�05h.A4h�X�B*CJOJQJaJmH	@nH	@phsH	@tH	@2h.A4hgICJH*OJQJ\�aJmHnH	@sHtH	@2h.A4h]6CJH*OJQJ\�aJmHnH	@sHtH	@/h.A4h]6CJOJQJ\�aJmHnH	@sHtH	@h.A4h]6CJOJQJaJ h.A4h	�CJOJPJQJaJ2h.A4h	�CJH*OJQJ\�aJmH	@nH	@sH	@tH	@/h.A4h	�CJOJQJ\�aJmH	@nH	@sH	@tH	@,h.A4h	�CJOJQJaJmH	@nH	@sH	@tH	@��(DL��nv��������������享��sX�X�<7h.A4hj�5�B*CJOJPJQJ\�aJmH	@phsH	@5h.A4hj�B*CJOJQJaJmH	@nH	@phsH	@tH	@8h.A4hj�5�B*CJOJQJaJmH	@nH	@phsH	@tH	@5h.A4h]6B*CJOJQJaJmH	@nH	@phsH	@tH	@h.A4h8�CJOJQJaJh.A4h]6CJOJQJaJ5h.A4h�X�B*CJOJQJaJmH	@nH	@phsH	@tH	@5h.A4h8�B*CJOJQJaJmH	@nH	@phsH	@tH	@��������W\�����#$FIKsu}����ʻ⫝̸����}m�^���O^O@h.A4h�!�CJOJQJaJh.A4h�I5CJOJQJaJh.A4h�pCJOJQJaJh.A4h?]S6�CJOJQJaJh.A4h�d-6�CJOJQJaJh.A4h�&[CJOJQJaJh.A4h�d-CJOJQJaJh.A4h}Z55�CJOJQJaJh.A4h.A4CJOJQJaJh.A4CJOJQJaJh8�CJOJQJaJh.A4h8�CJOJQJaJh.A4hlCJOJQJaJ����������ST����;CDps���'CX^h���V\^

������ĵ���Ӧĵė��Ĉĵ�yjĵĵjĈĈ�j�yĦh.A4h�&[CJOJQJaJh.A4ht�CJOJQJaJh.A4hrm�CJOJQJaJh.A4hF�CJOJQJaJh.A4h�ACJOJQJaJh.A4h�O=CJOJQJaJh.A4h�d-CJOJQJaJh.A4h�c?CJOJQJaJh.A4h�!�CJOJQJaJh.A4h?]SCJOJQJaJ)
��������	?DOP������������������xi�x�ZK�Z�h.A4hFE�CJOJQJaJh.A4hqg�CJOJQJaJh.A4h�HACJOJQJaJh.A4h?]SCJOJQJaJh.A4h�CJOJQJaJh.A45�CJOJQJaJh.A4h�5�CJOJQJaJh.A4h�d-5�CJOJQJaJh.A4h}Z55�CJOJQJaJh.A4h8�CJOJQJaJh.A4hrm�CJOJQJaJh.A4h�d-CJOJQJaJ�������D"g"�%1&*2*y,�,(/)/*/+/4/��������������������$��dh7$8$H$`��a$gd.A4$dh7$8$H$a$gd.A4$
��dha$gd.A4
$dha$gd.A4$��dh`��a$gd.A4���� ��_f���h j ,!.!D"f"g"����ӷ����w�w����cR h.A45�CJOJPJQJ\�aJ&h.A4h�d-5�CJOJPJQJ\�aJh.A4h�*�CJOJQJaJh.A4h�HACJOJQJaJh.A4h?]SCJOJQJaJ$h.A4h�d-CJOJQJaJmHsHh.A4CJOJQJaJh.A4h�d-5�CJOJQJaJh.A45�CJOJQJaJh.A4h�d-CJOJQJaJ h.A4h�d-CJOJQJ^JaJg"�"k#l#m#�#�#�#�#�#�#�#]$�$�$�$�$%%%&%��ı���|�����g�R�C4�h.A4h�*�CJOJQJaJh.A4hqg�CJOJQJaJ)h.A4h�*�B*CJOJPJQJaJph)h.A4hgsB*CJOJPJQJaJph)jh.A4h��0JCJOJQJUaJ h.A4h�d-0JCJOJQJaJh.A4h��CJOJQJaJ%jh.A4h��CJOJQJUaJh.A4h�d-CJOJQJaJ,h.A4h�d-CJOJQJaJmH	@nH	@sH	@tH	@)h.A4h�d-B*CJOJPJQJaJph&%'%4%5%N%O%R%S%i%j%m%n%t%x%y%}%~%�%�%�%�%&0&1&�&'3'4'(����������ӯ����⯚��sf�W�H�h.A4h�&]CJOJQJaJh.A4h0RCJOJQJaJh.A45�CJOJQJaJh.A4h�d-5�CJOJQJaJ,h.A4h�d-5�B*CJOJPJQJaJph)h.A4h�*�B*CJOJPJQJaJph)h.A4h�d-B*CJOJPJQJaJphh.A4hqg�CJOJQJaJh.A4h�*�CJOJQJaJh.A4h�d-CJOJQJaJh.A4huK�CJOJQJaJ($(2(3(T(U(`(e(f(g(h(�(�(�(�(�(�(B)C)�)�)�)�)�)�)�)�)�)�)�)����ĵ�◈y��i�Z��K����K���h.A4h}.�CJOJQJaJh.A4h-a�CJOJQJaJh.A4h�*�CJH*OJQJaJh.A4h�Q�CJOJQJaJh.A4h�/'CJOJQJaJh.A4h�*�CJOJQJaJh.A4h|0LCJOJQJaJh.A4h88CJOJQJaJh.A4hFE�CJOJQJaJh.A4h?]SCJOJQJaJh.A4h�d-CJOJQJaJh.A4h*DACJOJQJaJ�)�)�)�)�)�)***1*2*�*�*=+>+?+�+�+�+L,U,V,W,[,c,q,x,�����Ӵ����x����h�Y�YJY�Y�h.A4h�HACJOJQJaJh.A4h88CJOJQJaJh.A4h}.�CJH*OJQJaJh.A4h�d-CJH*OJQJaJh.A45�CJOJQJaJh.A4h�5�CJOJQJaJh.A4h�CJOJQJaJh.A4h|0LCJOJQJaJh.A4hV%CJH*OJQJaJh.A4h}.�CJOJQJaJh.A4h-a�CJOJQJaJh.A4h�d-CJOJQJaJx,y,�,�,�,�,�,�,�,�,----�-�-�-�-�-.*.@.G.M.�.�.�.�.�.�.��̽������񞽍{�j���������[�h.A4h�CJOJQJaJ h.A4h-a�CJOJPJQJaJ#h.A4h�d-CJH*OJPJQJaJ h.A4h�d-CJOJPJQJaJh.A4h�Q�CJOJQJaJh.A4h�d-CJH*OJQJaJh.A4h�d-CJOJQJaJ h.A45�CJOJPJQJ]�aJ&h.A4h�`5�CJOJPJQJ]�aJh.A4h�*�CJOJQJaJ�.�.�.�.�.�.�.�.///%/(/+/3/4/@/M/N/R/Z/i/l/�/�/	0000[0�0�0�����������޼���|�m^m^m^m^m^mOh.A4h%HCJOJQJaJh.A4h�JBCJOJQJaJh.A4h|0LCJOJQJaJh.A4h�JB5�CJOJQJaJh.A4h�@)5�CJOJQJaJh.A4h�5�CJOJQJaJh.A4h}Z55�CJOJQJaJh.A45�CJOJQJaJ)h.A4h�B*CJOJPJQJaJph h.A4h�d-CJOJPJQJaJ h.A4h�CJOJPJQJaJ4/N/�0
144�9EA1D2DCDZE�E�EnI��������������$
&F�dh�x1$7$8$H$]�a$gd.A4$�dh�x1$7$8$H$]�a$gd.A4$
��dh�xa$gd.A4dhgd.A4$���dh�x1$7$8$H$]�`��a$gd.A4$��dh`��a$gd.A4
$dha$gd.A4�0�0�0�0�0�0�0�0	1
1*181]1a1d1f1�1�����񳣖�t�]E+�2h.A4h�@)CJH*OJQJ\�aJmH	@nH	@sH	@tH	@/h.A4h�@)CJOJQJ\�aJmH	@nH	@sH	@tH	@,h.A4h�@)CJOJQJaJmH	@nH	@sH	@tH	@ h.A4hV�CJOJPJQJaJ h.A4h�@)CJOJPJQJaJh.A45�CJOJQJaJh.A4h�JB5�CJOJQJaJh.A4h|0L5�CJOJQJaJh.A4ht�CJOJQJaJh.A4h�JBCJOJQJaJh.A4h|0LCJH*OJQJaJh.A4h|0LCJOJQJaJ�1�1�1�12 2+2/242>2K2L2M2N2�2�2�2�2�2�2�2�2�23N3O3S3Z3[3`3�3�34�������������ﰎ����}�}l[}�� h.A4h�&[CJOJPJQJaJ h.A4h%HCJOJPJQJaJ h.A4h|0LCJOJPJQJaJ h.A4h	�CJOJPJQJaJ h.A4hV�CJOJPJQJaJ h.A4h�CJOJPJQJaJh.A4h�CJOJQJaJh.A4CJOJPJQJaJ h.A4h�@)CJOJPJQJaJ h.A4h�JBCJOJPJQJaJ 444�4�4�4�4�4�4�4�4�455�5�5�5�5�5�5�5�5�5�5	66 6-6S6`6a6e6m6q6�6�6�6�6�6��о�РРРРБРРРБРРРРРЂ�s�h.A4hV�CJOJQJaJh.A4hRKlCJOJQJaJh.A4hs-CJOJQJaJh.A4h%HCJOJQJaJh.A4h-a�CJOJQJaJ"h.A4h�th6�CJOJQJ]�aJh.A4h�pCJOJQJaJh.A4h�p5�CJOJQJaJh.A4h}Z55�CJOJQJaJ&�6�6�6�6�6�677 7&7�7�8�839:9a9k9�9�9�9�9�9�9�9Z:m:�:%;8;=;G;M;u;��������������������yayay/h.A4hRKlCJOJQJ\�aJmHnH	@sHtH	@/h.A4h�pCJOJQJ\�aJmHnH	@sHtH	@h.A4h �CJOJQJaJ h.A4hRKlCJOJPJQJaJ h.A4h�c]CJOJPJQJaJh.A4hs-CJOJQJaJh.A4hRKlCJOJQJaJh.A4h�pCJOJQJaJ h.A4h�pCJOJPJQJaJ u;z;{;|;�;�;�;�;�;�;�;<<<f<v<�<�<�̴��l�l�R8�l�l�R2h.A4huK�CJH*OJQJ\�aJmHnH	@sHtH	@2h.A4h�pCJH*OJQJ\�aJmHnH	@sHtH	@/h.A4hRKlCJOJQJ\�aJmHnH	@sHtH	@/h.A4h�pCJOJQJ\�aJmHnH	@sHtH	@/h.A4h�w�CJOJQJ\�aJmHnH	@sHtH	@/h.A4h-a�CJOJQJ\�aJmHnH	@sHtH	@2h.A4huK�6�CJOJQJ\�aJmHnH	@sHtH	@2h.A4h-a�6�CJOJQJ\�aJmHnH	@sHtH	@�<�<�<�<�<�<�<�<�<====�ζ�΄jΞ�P62h.A4h�w�6�CJOJQJ\�aJmHnH	@sHtH	@2h.A4h-a�6�CJOJQJ\�aJmHnH	@sHtH	@2h.A4h��CJH*OJQJ\�aJmHnH	@sHtH	@2h.A4h�pCJH*OJQJ\�aJmHnH	@sHtH	@/h.A4h��CJOJQJ\�aJmHnH	@sHtH	@/h.A4hRKlCJOJQJ\�aJmHnH	@sHtH	@/h.A4h�pCJOJQJ\�aJmHnH	@sHtH	@2h.A4hRKlCJH*OJQJ\�aJmHnH	@sHtH	@==&=(=0=2=4=@=B=f=h=�=�=�=�=�=�=>>> >3>6>>>N>^>�>�>�>�>�Ϸ�����������kϷQ�����������2h.A4h�pCJH*OJQJ\�aJmHnH	@sHtH	@2h.A4h-a�6�CJOJQJ\�aJmHnH	@sHtH	@2h.A4huK�6�CJOJQJ\�aJmHnH	@sHtH	@/h.A4h��CJOJQJ\�aJmHnH	@sHtH	@/h.A4h�pCJOJQJ\�aJmHnH	@sHtH	@/h.A4h�w�CJOJQJ\�aJmHnH	@sHtH	@/h.A4h-a�CJOJQJ\�aJmHnH	@sHtH	@�>�>�>-?.?�?�?�?�?�?@6@\@a@z@�@�@�@DAEAlA���Ʒ�����t��c�RA�� h.A4h.A4CJOJPJQJaJ h.A4h�w�CJOJPJQJaJ h.A4hJx,CJOJPJQJaJ h.A4hs-CJOJPJQJaJh.A4h�pCJOJQJ\�aJ h.A4h	�CJOJPJQJaJ h.A4h�pCJOJPJQJaJh.A4h�pCJOJQJaJ/h.A4h�pCJOJQJ\�aJmHnH	@sHtH	@ h.A4h��CJOJPJQJaJ h.A4h �CJOJPJQJaJlArA�A�A�A�A�A�ABBKBYB�BC�C0D1D2DBDCD�DWEZE�������ԵԤ���yl\L�=�h.A4h�b�CJOJQJaJh.A4h�p5�CJOJQJaJh.A4hLEb5�CJOJQJaJh.A45�CJOJQJaJ6h.A4h�pB*CJOJQJaJfHph333q�
����h.A4hLEbCJOJQJaJ h.A4h�w�CJOJPJQJaJ h.A4h��CJOJPJQJaJh.A4CJOJPJQJaJ h.A4h�pCJOJPJQJaJh.A4h�pCJOJQJaJh.A4CJOJQJaJZEpE�E�E�E�E"F#F+F,F.F/F�F�F�F�F�F�FGG#G%G�G�G�G�GHHHHHH�H�H�H�H�H�H�H�H���н���������������������������������s(jh.A4h�w�6�CJOJQJUaJ&h.A4h�w�CJKH$OJQJ\�]�aJ#h.A4h�w�CJKH$OJQJ\�aJh.A4h�w�CJOJQJaJ%jh.A4h�w�CJOJQJUaJh.A4h�w�5�CJOJQJaJh.A4h�]�CJOJQJaJh.A4h�]�5�CJOJQJaJ'�HOIPI`IaIbIdIJJJJ)J>JCJ��ʴ���ʃq�N+Eh.A4h�w�0JCJOJQJ\�aJeh@fHq�
����r��@Eh.A4h�w�0J5�CJOJQJaJeh@fHq�
����r��@"h.A4h�w�5�CJOJQJ\�aJ"h.A4h�w�6�CJOJQJ]�aJh.A4h�w�5�CJOJQJaJh.A4h�w�CJOJQJaJ+jh.A4h�w�6�CJOJQJU]�aJh.A4h�w�CJOJQJ]�aJ(jh.A4h�w�6�CJOJQJUaJh.A4h�w�6�CJOJQJaJ
nI)JK�K[L�L�M*N�N�O_��ݙ��5�����<�=�������������������$�dh�x1$7$8$H$]�a$gd.A4$
&F�dh�x7$8$H$]�a$gd.A4$
&F�dh�x1$7$8$H$]�a$gd.A4$
&F�dh�x]�a$gd.A4CJTJcJK�K�K�K�KLLMLQL[L�L�L�L��财���qa�N:&'h.A4h�w�5�CJOJQJaJmH	@sH	@'h.A4h�w�CJOJQJ]�aJmH	@sH	@$h.A4h�w�CJOJQJaJmH	@sH	@h.A4h�w�5�CJOJQJaJh.A4h�w�6�CJOJQJaJh.A4h�w�CJOJQJaJ#h.A4h�w�5�CJOJQJ_H9aJ#h.A4h�w�CJOJQJ]�_H9aJ h.A4h�w�CJOJQJ_H9aJEh.A4h�w�0J6�CJOJQJaJeh@fHq�
����r��@-h.A4h�w�CJOJQJaJfHq�
�����L�LyM|M
NNN N*N�N�N�N�N�N~O�O�O���޾��ޛ�uc��O9*h.A4h�w�6�CJOJQJ]�aJmH	@sH	@'h.A4h�w�CJOJQJ]�aJmH	@sH	@#h.A4h�w�5�CJOJQJ_H9aJ&h.A4h�w�6�CJOJQJ]�_H9aJ#h.A4h�w�CJOJQJ]�_H9aJ h.A4h�w�CJOJQJ_H9aJ"h.A4h�w�5�CJOJQJ]�aJh.A4h�w�CJOJQJ]�aJh.A4h�w�5�CJOJQJaJh.A4h�w�CJOJQJaJ$h.A4h�w�CJOJQJaJmH	@sH	@�O�O�O�O�Q�U�_������ę˙̙ϙݙ���������+�-�����������f�����ɷ�أ����}k��أ���}��}��Z� h.A4h�w�CJOJQJ_H9aJ"h.A4h�w�6�CJOJQJ]�aJh.A4h�w�CJOJQJ]�aJ*h.A4h�w�6�CJOJQJ]�aJmH	@sH	@'h.A4h�w�CJOJQJ]�aJmH	@sH	@h.A4h�w�5�CJOJQJaJUh.A4h�w�CJOJQJaJ$h.A4h�w�CJOJQJaJmH	@sH	@'h.A4h�w�5�CJOJQJaJmH	@sH	@tavirus indicates independent evolution of mammalian and avian strains. Virology 386: 325-333.
Jindal N, Patnayak DP, Chander Y, Ziegler AF, Goyal SM (2010) Detection and molecular characterization of enteric viruses from poult enteritis syndrome in turkeys. Poul Sci 89: 217-226. 
Trojnar E, Otto P, Roth B, Reetz J, Johne R (2010) The genome segments of a group D rotavirus possess group A- like conserved termini but encode group-specific proteins. J Virol 84: 10254-10265.
Otto PH, Ahmed MU, Machnowska P, Reetz J, Roth B, Trojnar E, Johne R (2012) Detection of avian rotaviruses of groups A, D, F and G in diseased chickens and turkeys from Europe and Bangladesh. Vet Microbiol 156: 8�15.
Ahmed MS, Ahmed MU (2006) Detection of avian rotavirus-like virus in broiler chickens in Bangladesh. Bangl J Vet Med 4: 73-77.
Islam MS, Alam MM, Rahman MM, Ahmed MU (2009) Rotavirus infection in human and birds. Bangl J Vet Med 7: 314-319.
Bezerra DAM, da Silva RR, Kaiano JHL, Silvestre RVD, de Souza Oliveira D, Linhares AC, et al. (2012) Detection of avian group D rotavirus using the polymerase chain reaction for the VP6 gene. J Virol Methods 185: 189-192.
Kattoor JJ, Malik YPS, Sharma K, Kumar N, Batra M, Jindal N, Yadav AS (2013) Molecular evidences of group D rotavirus in commercial broiler chicks, India. Avian Biol Res 6: 313-316. 









FIGURE CAPTIONS
Fig. 1. Agarose gel electrophoresis of RT-PCR products of avian group-D rotavirus. Lanes, M: 1 Kb Plus DNA Ladder (Thermo scientific); 1: UKD-119; 2: UKD-126; 3: UKD-142 and 4: NTC (Non template control). 

f�g�w�{�|�����������������Ҝ.�/���Ͽ���t^�^H��8h.A4h.A46�CJOJQJaJ+h.A4h.A4B*CJH*OJQJ\�aJph+h.A4h.A4B*CJH*OJQJ\�aJph.h.A4h.A45�>*B*CJOJQJ\�aJph(h.A4h.A4B*CJOJQJ\�aJphh.A4h.A4CJOJQJaJh.A4h�w�CJOJQJaJh.A4h�w�CJOJQJ\�aJh.A4h�w�5�CJOJQJaJh.A4h�w�CJOJQJ]�aJh.A4h�w�6�CJOJQJaJ/�1�>�D�E�T�U�[���������������"�����Ĵ��f�N6N6N6N/h.A4h[CJOJQJ\�aJmH	@nH	@sH	@tH	@/h.A4h�pCJOJQJ\�aJmH	@nH	@sH	@tH	@2h.A4h�J5�CJOJQJ\�aJmH	@nH	@sH	@tH	@2h.A4h�p5�CJOJQJ\�aJmH	@nH	@sH	@tH	@2h.A4h	�5�CJOJQJ\�aJmH	@nH	@sH	@tH	@h.A4h�p5�CJOJQJaJh.A4hLEb5�CJOJQJaJh.A4CJOJQJaJh.A4h.A4CJOJQJaJh.A4h.A45�CJOJQJaJ=�>�?�@�A�B�C�D�E�U�#�$������������$��dh`��a$gd.A4$�dh]�a$gd.A4$��dh`��a$gd.A4$�dh�x1$7$8$H$]�a$gd.A4"�#�$���h.A4h�pCJOJQJaJ,h.A4h�pCJOJQJaJmH	@nH	@sH	@tH	@21�h:pV%��. ��A!��"��#��$��%�������s���������666666666vvvvvvvvv666666>666666666666666666666666666�6666666666�666666666666hH66666666666666666666666666666666666666666666666666666666666666666p62����&6FVfv������2(��&6FVfv������&6FVfv������&6FVfv������&6FVfv������&6FVfv������&6FVfv��8X�V~�������� 0@�� 0@�� 0@�� 0@�� 0@�� 0@�� 0@�� 0@�� 0@�� 0@�� 0@�� 0@�� 0@�� 0@$OJPJQJ^J_HmH	nH	sH	tH	R`�R�d-Normald�� CJPJ^J_HaJmH	sH	tH	DA`�DDefault Paragraph FontRi�R
0Table Normal�4�
l4�a�(k �(
0No List6U`��6�d-0	Hyperlink>*B*ph�R�@R	�c?0Balloon Textd��CJOJQJ^JaJZ�o�Z�c?0Balloon Text Char CJOJPJQJ^JaJmH	sH	B�O�!B�papple-converted-space*W`�1*�p`Strong5�\�0�O�A0�pcit-pub-date.X`�Q.�p@Emphasis6�]�D�`bD�w� List Paragraph
��^��m$PK!����[Content_Types].xml���N�0E�H���-J��@%�ǎǢ|�ș$�ز�U��L�TB� l,�3��;�r��Ø��J��B+$�G]��7O٭V��<a���(7��I��R�{�pgL�=��r���8�5v&����uQ�뉑8��C����X=��$␴�?6N�JC������F�B.ʹ'�.�+���Y�T���^e5�5�� ��ð�_�g -�;�����Yl�ݎ��|6^�N��`�?���[��PK!�֧�6_rels/.rels���j�0���}Q��%v/��C/�}�(h"���O�
����=������ ����C?�h�v=��Ʌ��%[xp��{۵_�Pѣ<�1�H�0���O�R�Bd���JE�4b$��q_����6L�R�7`������0̞O��,�En7�Li�b��/�S���e��е�����PK!ky���theme/theme/themeManager.xml�M
� @�}�w��7c�(Eb�ˮ��C�AǠҟ����7��՛K
Y,�
�e�.���|,���H�,l����xɴ��I�sQ}#Ր���� ֵ+�!�,�^�$j=�GW��)�E�+&
8���PK!�R%���theme/theme/theme1.xml�Y]��F}/�?�;�����l�ζ�MB��ql��Ɏ4F3ލ	��<
�i�C}�CiH�/��6�M!�wF�<c���%��d
�4:�Ι{��I��܏�s�NX�p��
���!�x�po�����p���,�
w��{e��.��;`��pC!�;�<�0���pm̒	8M&�Q���oD�B����]'F��1�!v�ҥ��tޡp.�4�I�ذP��aQ"��4q�m�0ψ��}�:qnA�����y��0�b��f�U���谤�L&�lR��J3�Tl�:�N�S�)a�)ݧߪ�����C��v�].x�y�sӗ?�@�o��E�@)��{^�x^�R|e_-4�^�+PHI|��.��r�\m3�g��}�[--��PP
Yu�)�,�j-B�X��R$H����8@���I�7E1�0\(��2��?O����4k���!���ÄLE������߼|�<zq�藓ǏO��:2��P<ѭ^���O?u�z���'_��\���g����+]�����x���7���������s�:>vn��B`2ǃ��,�!"�E3�p#9��G���Qd����;	H�
xuv� ��� ����0F[,�F᚜KsO�'3w�#�����vfS�Vbs�ؠy��X�	��p�5v��euw	1�z@�	�l,���i!b
I��jZ��2��|�9�������L$��Z�15�x��l.�(�z���m${�d��:\@�'�2�3œ�ln$�^-��@^�i?���D&��|�#�td�!��6l�ġ���B�"�&6�3�yy@��t�!�H��jp�U��*ye�Xry3�~{s:FXI
����O�5Y��[Y!}��S˪.��7b����d|n]�������6��71�.�
�t��n�/����w/�+���[�t��6���}��P�s���ںs�L�.J;�̊��i��N�	�$A��I������)�t2���L�m����x:��(}\-�i*��x����QC��Ju���Wl'�QyI@ھ
	m2�D�B���AR�4	��w¢naQ��`Բ���Ɂ
W�=0#x�B�d��T/����.3�-�F�>bY�L�%׭˓�KK��6Hh�f�P�Q=��h��)G�B�ms]_�Ԡ'C���ZѨ���ys
v��@c])h�7�Jه��i��c?FS�.���N���P$�
e�&\�Ӏ+�I� "'%QÕ���@c�!�[��pa��AV.9H��d<��Ӯ��H����VX�*��%�A�{����YrA��բ�px�SL�9"�:3�U��5�����U
��NC��(���p%�u��@;[���d��`���4�)��]�t#9M4W=�P�5�*f̰lk�<_��X-C��w�T��%��Ժ�}B�% �Y�,]�
A���̠&oʰ��Ũ�;�<��Y������۵�e=�:����z���x��T�V�=�Olpģ
/�gTp�J�� ��Ԟ$�
�E�ŭG�,!
�A�ozA�r����ye�����r���b�/ڭ�Ch,"��~�ͥ���|��E�o|}��o�.
Y�g��J^W__���__��R���V%W/7�9�ݪ��A��kW�j���Z���u��k��ҩ�*� �y���_��^����Z�k>\lc`�|,b�U�v���PK!
ѐ��'theme/theme/_rels/themeManager.xml.rels��M
�0���wooӺ�&݈Э���5
6?$Q��
�,.�a��i����c2�1h�:�q��m��@RN��;d�`��o7�g�K(M&$R(.1�r'J��ЊT���8��V�"��AȻ�H�u}��|�$�b{��P����8�g/]�QAsم(����#��L�[������PK-!����[Content_Types].xmlPK-!�֧�60_rels/.relsPK-!ky���theme/theme/themeManager.xmlPK-!�R%����theme/theme/theme1.xmlPK-!
ѐ��'�	theme/theme/_rels/themeManager.xml.relsPK]�
<?xml version="1.0" encoding="UTF-8" standalone="yes"?>
<a:clrMap xmlns:a="http://schemas.openxmlformats.org/drawingml/2006/main" bg1="lt1" tx1="dk1" bg2="lt2" tx2="dk2" accent1="accent1" accent2="accent2" accent3="accent3" accent4="accent4" accent5="accent5" accent6="accent6" hlink="hlink" folHlink="folHlink"/>G�����m�	d
M���
�g"&%(�)x,�.�0�14�6u;�<=�>lAZE�HCJ�L�Of�/�"�$�(*+-./012356789:;=>?@ABCDEFGIJKPQS�	�4/nI=�$�),4<HR9cx���6(71747�7�7�78)8*8�8�8�899$9�9�9�9U:f:GX��X���X���X����X���X����X���X���X����L�#����@�����������0�(	�
��B
�S����	?�W^_d{�����������	RVY^������
GQ	����'KMNSU^qv����GO[d������7<����������!('7=|������"�"A%J%P%R%�%�%�-�-�-�-�-�-�-�-�-�-�-....9.�.�.�.�.�.�.�.�.�.�.�.�.�.�.�.�.�.�.////)7/7�7�7�8�899�9�9�9�9�9�9X:\:]:f:t:|:�:�:�:�:�:�:�:�:;;;;8;@;o;r;�;�;�;�;�;�;-<<<A<I<M<R<V<\<`<e<�<�<a=e=j=n=s=x=}=�=�=�=�=�=�=�=>	>>>>$>o>r>u>~>�>�>�>�>�>�>???#?0?6?;?@?N?R?�?�?�?�?�?�?�?�?�@�@�@�@`AhAmAtA�A�A�A�A�A�A�ABBB,B1B5B:B�B�B�B�B�B�BC
CCC�C�CDD6D:D�D�D�D�D�D�D�D�D`EeE|E�E�E�EF!FG�	�	�
�
7<R(U(s)u)�*�*-�/�=�=�@�@G33333333�����������!�!�!�!c#d#y$z$j2j2r2x2�2�2�2�275855F;FG�
M	d�������������}Hghb�h��������������^��`���56B*CJaJph�.�����^��`���.��~�L�^�~`�L�.��N���^�N`���.�����^�`���.����L�^��`�L�.������^��`���.������^��`���.��^�L�^�^`�L�.�����^��`���CJOJQJo(.�
�����^��`����h�H.�
�p�L�^�p`�L��h�H.�
�@���^�@`����h�H.�
����^�`����h�H.�
���L�^��`�L��h�H.�
�����^��`����h�H.�
�����^��`����h�H.�
�P�L�^�P`�L��h�H.�}Hg�
M	������������FA(k								�f�								V�!l�s-%H�ST�]6V%[�`�$�/'�@)IV+�Y+�H,Jx,�d-.A4�I5}Z5�Q688�O=85?�c?�A*DA�HAh%B�JBgI�J|0L0R�~R?]S�]U�&[�&]�c]LEb�thRKl$[m�6n�p�%pgs�Cwqg�xe�}.�UR�t����X�	�j�uK�V�FE��Q���F��]��,�(5�8��M� ��b�-a��8��w��!���f�H%��*�rm�A���GG�@�F2F2F2F2|ZZZZ./�@Gp@pp @pp<@p pD@p<p|@p0@��Unknown������������G��*�Ax�	�Times New Roman5��Symbol3.��*�Cx�	�Arial7.���@�CalibriA���$B�Cambria Math5��Mangal5.��.�[`�)�Tahoma"q���hQ�"'�"'��
<
$���
<
$���������20�F�FO�q���HP	�	$P����������������������A�2!xx����my	YPS Malik����Oh��+'��0h��������	�
$0
<HPX`�myNormalYPS Malik3Microsoft Office Word@��@� ,A1�@z~�U1�
�
<����՜.��+,��D��՜.��+,��4�hp��������
���Microsoft�$�FTitle� 8@_PID_HLINKS�A@6!4+http://www.ncbi.nlm.nih.gov/pubmed/3023441Dh_http://www.ncbi.nlm.nih.gov/pubmed?term=Hamed%20Z%5BAuthor%5D&cauthor=true&cauthor_uid=3023441J!chttp://www.ncbi.nlm.nih.gov/pubmed?term=el%20Tagi%20M%5BAuthor%5D&cauthor=true&cauthor_uid=30234418ahttp://www.ncbi.nlm.nih.gov/pubmed?term=Shoukry%20I%5BAuthor%5D&cauthor=true&cauthor_uid=3023441nMahttp://www.ncbi.nlm.nih.gov/pubmed?term=DuPont%20HL%5BAuthor%5D&cauthor=true&cauthor_uid=3023441"	_http://www.ncbi.nlm.nih.gov/pubmed?term=Zaki%20AM%5BAuthor%5D&cauthor=true&cauthor_uid=3023441,S`http://www.ncbi.nlm.nih.gov/pubmed?term=Shukry%20S%5BAuthor%5D&cauthor=true&cauthor_uid=3023441,($http://www.ncbi.nlm.nih.gov/nuccore6Omailto:malikyps@ivri.res.in	

 !"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHIJKLMNOPQRST����VWXYZ[\]^_`abcdefghijk����mnopqrs����uvwxyz{��������~��������Root Entry��������	�F�XT�U1���1Table��������U�-WordDocument��������4�SummaryInformation(����lDocumentSummaryInformation8������������tCompObj������������r��������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������
����	�F Microsoft Word 97-2003 Document
MSWordDocWord.Document.8�9�q

Youez - 2016 - github.com/yon3zu
LinuXploit